w ww.e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Morphoanatomical
and
physicochemical
profile
of
Piper
callosum
:
valuable
assessment
for
its
quality
control
Rolf
J.F.
Silva
a,∗,
Ana
C.A.
de
Aguiar-Dias
b,
Kelson
do
C.F.
Faial
c,
Maria
S.
de
Mendonc¸
a
daProgramadeCapacitac¸ãoInstitucional,MuseuParaenseEmílioGoeldi,Coordenac¸ãodeCiênciasdaTerraeEcologia,Belém,PA,Brazil
bInstitutodeCiênciasBiológicas,UniversidadeFederaldoPará,Belém,PA,Brazil
cLaboratóriodeToxicologia,Sec¸ãodeMeioAmbiente,InstitutoEvandroChagas,Ananindeua,PA,Brazil
dFaculdadedeCiênciasAgrárias,UniversidadeFederaldoAmazonas,Manaus,AM,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received14April2016 Accepted13July2016
Availableonline28September2016
Keywords:
Metalcontent
Pharmacognosticalstudy Piperaceae
Qualitycontrol Rawplantmaterial Secretorystructures
a
b
s
t
r
a
c
t
PipercallosumRuiz&Pav.,Piperaceae,popularlyknownas“elixir-paregórico”and“matricá”inBrazil, isusedinfolkmedicinetotreatgonorrhea,generalpain,anddigestivedisorders,andhasrepellent, astringent,diuretic,depurative,andhaemostaticproperties.Despitethefactthatthisplantissoldasa traditionalphytotherapeuticproduct,wedidnotfindreportsonitsqualitycontrol.We,therefore, per-formedmacroscopic,microscopic,histochemical,andphysicochemicalanalysesusingstandardmethods toestablishbotanicalauthenticationandpuritydegreeparametersforleavesandstemofthisspeciesin twoforms:medicinalplantandherbaldrug.Weobservedthesize,shape,color,texture,fracturesurface andtransectioncharacteristics,leafvenationpatterns,andcallusesarevaluablediagnosticcharacters toidentifytheherbaldrugswhentheyarenotgroundorpowdered.Sincemedicinalplantsandherbal drugsdidnotdifferanatomically,thefollowingkeyanatomicalcharactersforP.callosumcanbeusedfor diagnosticpurposesofbothtypesrawplantmaterials:epicuticularwaxandcuticularflangespatterns; collenchymafeatures;fibersinthemidrib;arrangementpatternofthevascularbundlesofthemidriband petiole;shapeofthemidrib,leafmargin,petiole,andstem;occurrenceofraphides;andmorphologyof thestarchgrains.Acidlipids,essentialoils,oleoresins,steroids,tanninsandflavonoidswere histochem-icallyidentified.Totalash(leaves:11.25%;stem:5.25%),sulphatedash(leaves:68.02%;stem:12.50%), acid-insolubleash(leaves:2.82%;stem:0.27%),moisture(leaves:8.60%;stem:6.10%),lossondrying (leaves:11.08%;stem:8.58%),andpH(leaves:5.57,stem:5.28)valuesweredetermined.Theorderof analyzedmetallevelsinleafandstemherbaldrugswasAl>V>Cu>Mn>Cr>Ni.SimilarlevelsofCdand CoandlowlevelsofHgwerefound.Theresultsobtainedcanbeusedasqualitycontrolparametersfor medicinalplantsandherbaldrugsofP.callosum.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Piper callosum Ruiz & Pav., Piperaceae, popularly known as “elixir-paregórico,” “óleo-elétrico,” “ventre-livre,” “erva-de-soldado,” “panquilé,” “matricá” and “joão-brandin” in Brazil (Andradeet al.,2009),is a shrubnativetoBolivia,Brazil,Peru, andColombia.InBrazil,itoccursinAcre,Amazonas,Amapá,Pará, Rondônia, Distrito Federal, Mato Grosso, Espírito Santo, Rio de Janeiro,andParanáStates(Guimarãesetal.,2014).
InBrazilianfolkmedicine,P.callosumleavesandyoungstem areusedintheformofinfusionorpoulticetotreat dysmenor-rhea,intestinalcolic,diarrhea,nausea,toothache,rheumaticand
∗ Correspondingauthor.
E-mail:[email protected](R.J.Silva).
muscularpain,mosquitobites,andgonorrhea,andhaverepellent, astringent,haemostatic,digestive,diuretic,anddepurative proper-ties(Andradeetal.,2009).Atopen-airmarketsinnorthernBrazil, vegetativeaerialpartsofP.callosumaresoldfresh,dried,ground, andrarelypowderedorasaningredientinartisanalpreparations called“garrafadas”formedicinalpurposes.Theplantisalso culti-vatedinbackyardsandmedicinalgardens(authors’observations). Anumberofvolatileandfixedphytoconstituentshavebeen iso-latedfromP.callosum,includingalkaloidamides;terpenes,suchas hydrocarbonmonoterpenes,oxygenatedmonoterpenes, hydrocar-bonsesquiterpenes,oxygenatedsesquiterpenes,andsteroids;and phenolics,suchasoxygenatedflavonoidsandphenylpropanoids (Parmaretal.,1997;Facundoetal.,2004;Andradeetal.,2009). StudiesofessentialoilsobtainedfromP.callosumhave demon-stratedantifungal,insecticidal,andlarvicidalactivities(Andrade etal.,2009).
http://dx.doi.org/10.1016/j.bjp.2016.07.006
P.callosum,currentlybeingtradedasatraditional phytother-apeutic product, represents a promising medicinal plant for phytopharmaceuticaldevelopmentduetothe ethnopharmacolog-icalevidenceforthenumerouspopularmedicinalusesattributed tothis plantandof thepharmacologicalpotentialofits phyto-constituents.Despitethis,wedidnotfindanysystematicreports ofitsqualitycontrolparameters.Thequalityofrawplant mate-rialsrepresentsthefirststepfortheestablishmentofminimum criteriaofacceptanceandisapre-requisitefortheproductionand registrationofphytomedicines(Coutoetal.,2013;Anvisa,2014). Hence,thepresentworkaimedtoestablishparametersof botan-icalauthentication and purity degreefor the qualitycontrol of P.callosumleavesand stem asraw plantmaterialsin forms of medicinalplantandherbaldrug.
Materialsandmethods
Plantmaterial
Fertilesamples(n=14 specimens;7 specimens per sampled area) of Piper callosum Ruiz & Pav., Piperaceae, were collected from natural populations of two Brazilian states: Manaus-AM, andBelém-PA.Avoucherspecimen(MG206892)wasdepositedat theJoãoMurc¸aPires(MG)HerbariumoftheEmílioGoeldiParaense Museum.ThetaxonomicidentitywasconfirmedbyElsieFranklin Guimarães,specialistinPiperaceae(RiodeJaneiroBotanicalGarden ResearchInstitute).
Preparationoftheherbaldrugs
AerialpartsofP.callosum(leavesfromthe1stto4thnodesand stemuptothe4th internode)werewashedin70%(v/v)ethanol anddriedat40◦Cinahot-airoven(SteriliferSX1.5DTMS)until reachingaconstantweight(Silvaetal.,2016).Partoftheleafand stemherbaldrugsweregroundtoapowderinaknifemill (Mar-coniMA580).Thewholeandpowderedherbaldrugswerestored atroomtemperatureinairtight,light-resistantcontainers(WHO, 1998).
Pharmacobotanicalanalysis
Macroscopicandorganolepticcharacterizationwasperformed onthewholeandpowderedherbaldrugsusingstandardmethods (WHO, 1998;OliveiraandAkisue,2003;FarmacopeiaBrasileira, 2010).Theleafherbaldrugswererehydrated,clarified,andstained forobservationoftheleafvenation(Silvaetal.,2016).The pho-tomacrographswere obtained using a digital camera (Nikon D 3100).Thestereoscopicphotomicrographsbyreflectivelight(RL) andbydifferentialinterferencecontrast(DIC)werecapturedwitha digitalcamera(Motic2500)attachedtoastereoscopicmicroscope (MoticSMZ-168)usingMoticImagesPlus2.0software.
Microscopic characterization was performed on the herbal drugsandfreshplantmaterials.Forthelatter,leaf(fullyexpanded mature leaves from the 4th node) and stem (from the 1st to 4th internodes) sampleswereobtainedaccording toSilvaet al.
(2014),fixedinNBF-neutralbufferedformalin(Lillie,1965)and bufferedglutaraldehyde/osmiumtetroxide(Potiguaraetal.,2013), andpreserved(Johansen,1940).NBFandglutaraldehyde/osmium tetroxide-fixedsampleswereusedforlightmicroscopy(LM)and scanningelectronmicroscopy(SEM)observations,respectively.
Epidermalpeelsoftheleafbladewereobtainedthrough macer-ationinJeffrey’ssolution,stainedwithastrablue,andmounted on glass slides with glycerol jelly (Johansen, 1940). Samples wereinfiltratedandembeddedinmethacrylateresin(Historesin, Leica®
),andsectionedinarotary,auto-advancemicrotome(Leica®
RM 2245). The histological sections (transverse and longitudi-nal,1.5–3.5mthick)werestainedwithcitrate-bufferedtoluidine blue,pH4.7(O’Brienetal.,1964),andmountedonglassslideswith syntheticresin(Permount-Fisher®)forstructuralcharacterization.
Histologicalsectionsfromfreshplantmaterialsweremadebyhand withasteelrazorandusedforhistochemicalscreening(Table1). Foralltests,standardcontrolprocedureswerecarriedout simul-taneouslyusingthesameprocedures,anduntreatedsectionswere usedtoverifythenaturalcolorationoftheanalyzedtissues(white). The photomicrographsbytransmitted and polarizedlight were obtainedwithadigitalcamera(Motic2500)attachedtoan opti-calmicroscope(MoticBA310)equippedwithanepifluorescence unit.
TheSEManalysisfollowedtheproceduresdescribedbySilva etal.(2014).Samplesboiledinchloroformforonehourforpartial ortotalremovalofwaxydepositswerealsoused.ALeo1450VP scanningelectronmicroscopewasusedfortheobservationsand captureofimages.
Microscopiccharacterizationoftheherbaldrugswasperformed byLMandSEM.Thewholeherbaldrugswererehydratedand sub-mittedtotheabove-mentionedmethods,apartfromhistochemical screening.Thepowderedherbaldrugswereprocessedaccording toWHO(1998)andFarmacopeiaBrasileira(2010)forLM observa-tions.ForSEMobservations,samplesweremountedonSEMmetal stubs,followingproceduresdescribedbySilvaetal.(2014).
Pharmacognosticalanalysis
Pooledsamplesoftheherbaldrugswereusedforthe physico-chemicalanalysis.Thetotalash,acid-insolubleash,sulphatedash, pH,moisture(Azeotropicmethod) and lossondrying (INFRAT-EST) weredetermined usingstandard procedures (WHO,1998; FarmacopeiaBrasileira,2010).Theanalyticalmethodtodetermine theselectedmetals(Al,Cd,Co,Cr,Cu,Mn,Mo,Ni,Pb,Ti,V,Hgand As)followedPratsmoyaetal.(1997),andthemeasurementswere performedbyinductivelycoupledplasmaopticalemission spec-trometry(ICP-OES)usingaVarianmodelVISTA-MPXspectrometer forAl,Cd,Co,Cr,Cu,Mn,Mo,Ni,Pb,TiandV,andaThermomodel ICAP6000spectrometerforHgandAs.StandardReferenceMaterial (SRM1547:peachleaves)fromtheNationalInstituteofStandard andTechnology(NIST)wasusedforvalidationoftheapplied ana-lyticalmethodusingthesameprocedures.
Allreagentswereofanalyticalgrade.Ultrapurewater(18.2M -cmat25◦C)fromaMilli-Qsystem(MerckMillipore)wasused.All determinationswereperformedintriplicate,andtheresultswere expressedasmean±standarddeviation(mean±S.D.).
Results
Pharmacobotanicalcharacterization
Theherbaldrugsofwholeleavesarecomplete, i.e.withleaf blade,petiole,andleafsheath;ca.3.4–8.7cmlongand1.2–4.6cm wide;wrinkledorfolded;friableintexture;greenishincoloron bothfaces,somewhatbrightontheadaxialface;characteristic aro-maticodor;tastepredominantlycharacteristicaromatic,turning slightlybitter,andendingslightlyspicy.Theherbaldrugsof pow-deredleavesaredark-greenincolorandhavethesameodorand tasteasthewholeherbaldrugs(Fig.1AandQ).
Table1
HistochemicalscreeningperformedinsecretorystructuresofPipercallosum.
Compoundgroups Metabolicclasses Reagents(Authors)
Lipids Totallipids SudanblackB(Pearse,1980)
Acidandneutrallipids NileblueA(Cain,1947)
Unsaturatedlipids Osmiumtetroxide(GanterandJollés,1969,1970)
Fattyacids Copperacetate/rubeanicacid(GanterandJollés,1969,1970)
Terpene compounds
Essentialoilsandoleoresins NADIreagent(DavidandCarde,1964)
Steroids Antimonytrichloride(HardmanandSofowora,1972;Maceetal.,1974)
Sesquiterpenelactones Abrahamreaction(Caniatoetal.,1989)
Terpenoidswithcarbonylgroup 2,4-dinitrophenylhydrazine(GanterandJollés,1969,1970)
Phenoliccompounds Totalphenolics Ferrictrichloride(Johansen,1940)
Tannins Vanillin–hydrochloricacid(MaceandHowell,1974)
Flavonoids Aluminumtrichloridea(Charrière-Ladreix,1976)
Polysaccharides Neutralpolysaccharides Periodicacid–Schiff(PAS)reagent(FederandO’Brien,1968)
Pectins Rutheniumred(Johansen,1940)
Mucins Mayer’stannicacid-ferricchloridestain(PizzolatoandLillie,1973)
Acidmucopolysaccharides Alcianblue(Pearse,1980)
Alkaloids Totalalkaloids Dragendorffreagent(SvendsenandVerpoorte,1983)
aObservedunderUVlight.
A
B
K
N
P
Q
0.1cm 0.1cm 0.1cm
0.1cm 0.4cm
0.7cm
20µm 50µm
6cm
b2 c b3 b1
mg
3cm
0.1cm fs
600.0 µm
1.5cm 1.5cm
f
3cm
les
0.1cm
O
M
L
*
*
*
*
*
E
D
C
G
F
H
J
I
venation;veinsbrancheduntil6◦order;incompletemarginal ulti-matevenation;linearorcurvedsimpleveinlets;veinletsbranched x1-3;irregularareoleswithimperfectdevelopmentandrandom arrangement(Fig.1B–J).
Petiole is ca. 0.1–0.7cm long and 0.05–0.1cm wide; curved ortwisted;insertedlaterally;surfacelongitudinallystriatetothe eyeonbothfaces;concave–convexintransection,withlignified elementsin U-shaped pattern (Fig.1B, K–N).Leaf sheath is ca. 0.025–0.3cmlongand0.05–0.4cmwide;concave-convex;surface smoothontheadaxialfaceandlongitudinallystriateontheabaxial facetotheeye(Fig.1K,L,OandP).
Theherbaldrugsof wholestemhaveevident nodes;surface smoothtothetouch;surfaceglabrousandlongitudinallyfinely stri-atetotheeye;greenishincolor;characteristicaromaticodor;taste predominantlycharacteristicaromaticand endingslightlyspicy. Theherbaldrugsofpowderedstemaresomewhatfibrous,mixed incolor,rangingfromyellowishgreentograywithblackspots,and presentthesameodorandtasteasthewholeherbaldrugs(Fig.2A, BandI).
Firstand2ndinternodeseasilybroken,withsmooth-granular outerfracturesurface,andpredominantlygranularinnerfracture surface.Intransection,theyshowprimarygrowth;irregularshape; outerregionofthesectiongreenishbrownincolor;innerregion ofthesectiongreenincolor,withyellowishspots;outerandinner lignifiedelementswithnodefinedarrangement(Fig.2C–E).
Thirdand4thinternodeseasilybroken,withsmoothouter frac-turesurface,andpredominantlygranularinnerfracturesurface.In transection,theyshowinitialsecondarygrowth;circularshape; outerregionofthesection dark-greenin color;inner regionof thesectionmixedincolor,withbrownishyellowcentralarea,and greenishbrownellipticalperipheralareaswithyellowishspots sep-aratedbyyellowishstrands;innerlignifiedelementsarrangedin twoconcentriccircles(Fig.2F–H).
Freshleaves and stem and theirherbaldrugs didnot differ anatomically.Infrontalview,theanticlinalepidermalcellwallsof theleafbladearestraighttowavyontheadaxialfaceandsinuous ontheabaxialface.Thecuticleissmooth,withcontinuousplateof granularepicuticularwaxparalleltotheepidermalsurfaceonboth faces(Fig.3A–C).
Theleavesarehypostomaticandpossesstetracyticand cyclo-cytic(withfourorfivesubsidiarycells)stomata(Fig.3C–E).Sunken, sac-likeglandulartrichomesarecoatedwithsmoothcuticleand occurrandomlyspreadonbothfacesoftheleafepidermis.They arebicellularwithachalice-likelignifiedshortbasalcellencircled byepidermalcellsandasac-likesecretoryapicalcell,whichlies upontheepidermalsurface(Fig.3F).
Transectionsshowedthat theleafepidermisisuniseriate on bothfaces.Thecuticleisthickened,exceptintheleafsheath,and formsV-shapedflangesonbothfacesofthepetioleandonthe abax-ialfaceoftheleafsheath(Figs.4AandE,5Dand6C).Stomataare raisedabovetheleveloftheotherepidermalcellsandhaveguard cellswithpiriformislumenandhorn-likeouterledges (Fig.4D). Uniseriatehypodermisoccursonbothfacesoftheleafbladeand isreplacedbysclerenchymaintheleafmargin.Somehypodermal cellsontheabaxialfacecontainraphides(Fig.4A,B,E,HandI).
The mesophyll is dorsiventral with one-layered palisade parenchyma on the adaxial face and three-layered spongy parenchymaontheabaxialface.Itisrelativelyundifferentiatedin theleafmargin.Minorcollateralvascularbundlesareencircledby parenchymaticsheath.Drusesareobserved(Fig.4A,BandH).
Themidribintransectionisbiconvexwith1–2layersoffibers occurringimmediatelybeneaththeepidermisonbothfaces.The palisadeparenchymacellsbecomegraduallyshortertowardthe middleregion.Thehypodermalcells,presentonlyontheadaxial face,arecomparativelysmallerthanthoseoftheinter-veinregions. Collateralvascularbundlesinastraightlinearecentrallyembedded
inthegroundparenchymaandencircledbyparenchymaticsheath, withfibersinthexylemandphloempoles(Fig.4E).Raphidesare observedinthegroundparenchyma(Fig.4F).Theleafmarginin transectionisrevolute.Sclerenchymacells,mainlyfibers,replace themesophyllandoccupythedistalregion(Fig.4G–I).
Thepetiole infrontalview possessesepidermiscoated with smoothcuticleonbothfaces.Scatterednon-ornamental epicuticu-larwaxoccursincrustsontheadaxialfaceandinplatesparallelto theepidermalsurfaceontheabaxialface(Fig.5AandB).The peti-oleintransectionresemblesanarchinshape(Fig.5C).Continuous strataoflamellarcollenchymaarelocatedimmediatelybeneaththe epidermis(Fig.5D).Collateralvascularbundlesinanarch-shaped patternareembeddedintheperipheralgroundparenchyma.One ortwominorcollateralvascularbundlesencircledby parenchy-maticsheathoccurontheabaxialface,locatedexternallytothe otherbundles(Fig.5CandE).Prismaticcrystalsandraphidesoccur throughoutthegroundparenchyma(Fig.5F).
Transectionsofthebaseoftheleafbladeshowedthatcalluses arestructuresofthepetiole.Thedermal,fundamental,and vas-culartissuesofcallusesoriginatefromthetissuesystemsofthe petiole(Fig.5G).Theepidermisiscoatedwiththickcuticleandhas cellprotuberancesthatconsistofphenolic-containingcellsin peri-clinalandanticlinaldivisions,formingwhatsomewhatresembles meristematsomesites(Fig.5G–I).Continuousstrataoflamellar collenchymaoccurbeneaththecellprotuberancesand coalesce intotheenlargeddistalendofthecalluses,whollyoccupyingthis region (Fig. 5G and H). The vascular system consists of xylem andphloem,eitherastransversecommissuresorasinconspicuous collateralvascularbundlesembeddedinthegroundparenchyma (Fig.5J).
Theleafsheathinfrontalviewpresentsepidermiscoatedwith smoothcuticleonbothfaces(Fig.6A).Theleafsheathin transec-tionisconcave-convex(Fig.6B).Theepidermalcellsarevertically elongatedontheadaxialface(Fig.6D).Continuousstrataof lamel-larcollenchymaoccurimmediatelybeneaththeepidermisonboth faces(Fig.6C–E).Collateralvascularbundlesinanarcareembedded inthegroundparenchyma.Twoorthreeminorcollateralvascular bundlesmayoccurontheabaxialface,locatedexternallytothe otherbundles(Fig.6BandC).Prismaticcrystalsandraphidesare observedinthegroundparenchyma(Fig.6F).
Thestem epidermisinfrontalviewpresentssmoothcuticle. Aggregatecrustsofnon-ornamentalepicuticularwaxoccuron epi-dermalsurface(Fig.7A).Sac-likeglandulartrichomeslikethose described for the leaf epidermisare rarelyobserved. The stem growthispredominantlyprimaryfromthe1stto2nd internodes and inconspicuously secondary from the 3rd to 4th internodes (Fig.7B–I).
Thestemintransectionisellipticalwithwavyoutline regard-less ofthetypeofgrowth(Fig.7B).Theuniseriateepidermisis coatedwiththickcuticlethat formsV-shaped flanges(Fig.7D). Lenticelsininitialdevelopmentalstageprotrudeabovethestem surface(Fig.7C).Thecortexconsistsofcontinuousstrataof lamel-larcollenchyma,collenchymatousfibers,andgroundparenchyma (Fig.7CandD).Thecollenchymaoccursimmediatelybeneaththe epidermis,and theinner collenchymacellsaredifferentiatedas fibers(Fig.7D).Thecollateralvascularbundlesarearrangedintwo concentriccircles(Fig.7CandE).Theperipheralcircleofbundles isboundinternallyandseparatedfromthepithbyasinuouszone offibers(Fig.7C).Thephloempolemaypresentfibers(Fig.7C). Themedullarybundlesareencircledbyparenchymaticsheath,and somemaypresentafibercapnexttothexylem(Fig.7CandE). The pith is parenchymatic, in which amyloplasts with convex-biconcaveaggregatestarchgrainsoccur(Fig.7EandF).
1stN
2ndN
3rdN 4thN
infs
0.1cm 0.2cm
infs
otfs
inrs inrs otrs
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*
otfs 5thN
6cm 0.1cm
Fig.2.MacroscopicfeaturesofthestemherbaldrugsofPipercallosumRuiz&Pav.,Piperaceae.Photomacrographs(A;I).Stereoscopicphotomicrographsbydifferential interferencecontrast(CandF)andbyreflectivelight(BandD;E,GandH).A.Generalmorphologicalaspect.B.Detailofthesurface.Notelongitudinalstriae(arrows).C–E. Firstinternode.C.Fracturesurface.DandE.Transection.Noteyellowishspots(arrows,figureD)andlignifiedelements(asterisks,figureE).F–H.Fourthinternode.F.Fracture surface.GandH.Transection.G.Noteellipticalareas(arrows)andyellowishstrands(asterisks).H.Notelignifiedelements(asterisks).I.Powderedherbaldrug.Firstnode (1stN).Secondnode(2ndN).Thirdnode(3rdN).Fourthnode(4thN).Fifthnode(5thN).Innerregionofthesection(inrs).Outerregionofthesection(otrs).Innerfracturesurface (infs).Outerfracturesurface(otfs).
phloembecomeradiallyelongatedandremainorganizedas col-lateralbundlesseparatedbyparenchymaticrays.Thefiberzoneis interrupted,andthefibercapnexttothexylemisformed(Fig.7I). Theepidermal,cortical,andmedullaryfeaturesremainas previ-ouslydescribed.
Thesecretory idioblastshave a spherical toelliptical shape, andtheircell wallpresentsa trilamellarstructuralaspectwith alignifiedintermediarylayer(Fig.8K).Theyoccurinthe meso-phyll(Fig.4A,CandG),inthegroundparenchymaofthemidrib (Fig.4E),amongthesclerenchymacellssituatedbeneaththe epi-dermison theadaxial face of the leafmargin (Fig.4H),in the groundparenchymaandcollenchymaofthepetioleandleafsheath (Figs.5D,6Eand8M),inthegroundparenchymaofthecalluses (Fig.5G),andinthecorticalandmedullarygroundparenchymaof thestem(Fig.7CandE).Secretoryidioblastsarealsoobservedin thephloemandxylemparenchymaofleafandstemvascular bun-dles.Inthexylemparenchyma,theyhaveagenerallywavyoutline (Figs.5E,7CandGand8I).
Thehistochemicaltestsindicatedthatthesecretionis hetero-geneous and rich in lipids, mainlyacidlipids, in theglandular trichomes,secretoryidioblasts,and calluses(Table2,Fig.9A–F).
Essential oils, oleoresins, mixture of essentials and resins, and steroids(Table2,Fig. 9G–M) aswellas tanninsand flavonoids (Table2,Fig.9N–S)wereidentified.Theotherhistochemicaltests renderednegativeresults(Table2).
Theherbaldrugsofpowderedleavespossessleafblade frag-mentswithstraighttowavy(adaxialface)orsinuous(abaxialface) anticlinalepidermalcellwalls,sac-likeglandulartrichomes encir-cledbyepidermalcells,tetracyticandcyclocyticstomata(abaxial face),smoothcuticle,continuousplates ofgranularepicuticular wax parallel to the epidermal surface, raphides, and secretory idioblastsofacidophiliccontent.Isolatedsecretoryidioblastsof aci-dophilicoryellowish-coloredcontentandtrachearyelementswith annularwallthickeningarealsoobserved(Fig.8A–G).
epc
gt
5µm
10µm
10µm
10µm
1µm
7µm
1µm
A
C
D
F
E
B
ct
apc
ew
Fig.3. FrontalviewoftheepidermisoftheleafbladeofPipercallosumRuiz&Pav.,Piperaceae.Photomicrographs(A;C–E;insetatF).Scanningelectronmicrographs (BandF).AandB.Adaxialface.A.Generalview.B.Detailofthecuticleandepicuticularwax.C–F.Abaxialface.C–E.Tetracytic(C)andcyclocytic(D;E)stomata.F.Glandular trichome.Notebasalcell(inset).Epidermalcellsencirclingtheglandulartrichomebase(epc).Glandulartrichome(gt).Cuticle(ct).Epicuticularwax(ew).Apicalcell(apc).
Pharmacognosticalcharacterization
Thelevelsofvolatileandinorganicmatterwerecomparatively higherintheleafherbaldrugsthaninthestemherbaldrugs.The stemherbaldrugsresultedinahighreductionofthepHvalueof thedistilledwater(Table3).ThedistilledwaterusedhadapHof 7.010±0.032.
Thehighest concentrationsof mostof theexaminedmetals, exceptforCdandCr,wereobservedintheleafherbaldrugs.For theleafherbaldrugs,themetalswithhighconcentrationswereAl, V,CuandMn,inorderofincreasingconcentration.Forthestem herbaldrugs,thehighmetalconcentrationswereofAl,Cu,Vand Mn,inorderofincreasingconcentration.Alpresentedthehighest concentrationofallexaminedmetals,andHgandCothelowest concentrationsinbothleafandstemherbaldrugs.Bothleafand stemherbaldrugscontainedMo,Pb,TiandAsbelowthedetection limit.Thestemherbaldrugsdidnotcontaindetectableamountsof Hg.Theresultsareconsideredsatisfactory,withrecoveriesbeing withintherange76.48–117.20%(Table4).
Discussion
ThegeneralexternalmorphologicalfeaturesobservedinPiper callosumleafandstemherbaldrugsarein accordancewiththe morphologicaldescriptionsof thetaxon(Yuncker,1972). Exter-nalandinternalmacroscopiccharacteristics,leafvenationpatterns, andsensorycharacteristicsaredescribedhereinforfirsttimefor theP.callosumherbaldrugs.Sincethemacroscopicand organolep-ticexaminationsserveasvaluablepharmacognosticalparameters to establish the botanical identity and thedegree of purity of medicinalplantmaterialsandshouldbecarried outbeforeany further tests are undertaken (WHO, 1998; Cheng et al., 2014), theorganolepticandmorphologicalfeaturesreportedhereforthe P.callosumherbaldrugsareusefuldiagnosticcharactersfortheir qualitycontrol.
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H
Fig.4.TransectionsoftheleafbladeofPipercallosumRuiz&Pav.,Piperaceae.Photomicrographsbytransmittedlight(A,D,E,HandI)andbypolarizedlight(BandF). Scanningelectronmicrographs(C;G).A–C.Mesophyll.A.Generalview.B.Polarizedviewshowingbirefringentdruses(mesophyll)andraphides(hypodermis).Noteraphides (inset).C.Detailofthesecretoryidioblast.D.Stoma.Notelumen(asterisk)andouterledges(arrows)oftheguardcells.EandF.Midrib.E.Generalview.Notesecretory idioblasts(asterisks).F.Polarizedviewshowingbirefringentraphides.G-I.Margin.G.Generalview.H.Regionofthemesophyll.I.Distalregion.Adaxialfaceoftheepidermis (adep).Abaxialfaceoftheepidermis(abep).Epidermis(ep).Cuticle(ct).Hypodermis(hd).Substomatalchamber(stch).Palisadeparenchyma(pp).Spongyparenchyma(sp). Mesophyll(me).Undifferentiatedmesophyll(ume).Fiber(fi).Sclerenchyma(scl).Groundparenchyma(gp).Parenchymaticsheath(pas).Secretoryidioblast(sid).Vascular bundle(vb).Phloem(ph).Xylem(Xy).
Table2
ResultsofthehistochemicalscreeningperformedinsecretorystructuresofPipercallosum.
Metabolicclasses Reagents Secretoryidioblasts Callosusemergences Glandulartrichomes
Leaf Stem Petiole Leaf Stem
Totallipids SudanblackB ++ + ++ ++ +
Neutrallipids NileblueA − − − − −
Acidlipids NileblueA ++ + ++ ++ +
Unsaturatedlipids Osmiumtetroxide − − − − −
Fattyacids Copperacetate/rubeanicacid − − − − −
Essentialoils NADIreagent − − ++ − −
Oleoresins NADIreagent ++ + − − −
Mixtureofessentialsandresins NADIreagent ++ + − − −
Steroids Antimonytrichloride ++ ++ + − −
Sesquiterpenelactones Abrahamreaction − − − − −
Terpenoidswithcarbonylgroup 2,4-dinitrophenylhydrazine − − − − −
Totalphenolics Ferrictrichloride ++ − ++ ++ +
Tannins Vanillin–hydrochloricacid − − ++ − −
Flavonoids Aluminumtrichloridea ++ − − ++ −
Neutralpolysaccharides Periodicacid–Schiff(PAS)reagent − − − − −
Pectins Rutheniumred − − − − −
Mucins Mayer’stannicacid-ferricchloridestain − − − − −
Acidmucopolysaccharides Alcianblue − − − − −
Totalalkaloids Dragendorffreagent − − − − −
ew
ct
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* *
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ct
Fig.5.PetioleofPipercallosumRuiz&Pav.,Piperaceae.Photomicrographsbytransmittedlight(D,E;G–J)andbypolarizedlight(F).Scanningelectronmicrographs(A–C; insetatG).AandB.Frontalviewoftheepidermalsurface.Adaxial(A)andabaxial(B)faces.C–J.Transections.C.Generalview.Notearrangementofthevascularbundles (asterisks)andminorbundles(arrows).D.Adaxialfacedetailingepidermisandcollenchyma.Notecuticularflange(arrow).E.Vascularbundles.Notesecretoryidioblasts (arrows).F.Polarizedviewshowingbirefringentcrystals.G–J.Callosusemergence.G.Generalview.Notecellprotuberances(arrows)andsecretoryidioblast(inset).Hand I.Cellprotuberances.H.Generalview.I.Detailofthephenolic-containingcells.Notepericlinalandanticlinaldivisions(arrows).J.Vascularsystem.Cuticle(ct).Epicuticular wax(ew).Epidermis(ep).Collenchyma(co).Groundparenchyma(gp).Parenchymaticsheath(pas).Secretoryidioblast(sid).Vascularsystem(vs).Vascularbundle(vb). Transversecommissure(tc).Phloem(ph).Xylem(xy).
Table3
ResultsofphysicochemicalparametersdeterminedinherbaldrugsofPipercallosum.
Herbaldrug Parameters(mean±S.D.;n=3)
Lossondrying(INFRATEST) Moisturecontent Totalash Sulphatedash Acid-insolubleash pH
Leaf 11.08%±0.00 8.60%±0.30 11.25%±0.01 68.02%±0.03 2.82%±0.00 5.57±0.01 Stem 8.58%±0.01 6.10%±0.20 5.25%±0.01 12.50%±0.05 0.27%±0.01 5.28±0.14 SD,Standarddeviation.
nisthenumberofindependentdeterminations.
onesfor Piperaceae(Metcalfeand Chalk, 1950) and mentioned forotherPiperspecies(Marinhoetal.,2011;Gogoszetal.,2012; Periyanayagam et al., 2012; Silva et al., 2014, 2016; Machado et al., 2015; Santos et al., 2015): epidermal cells of leaf blade
ct
ct
ep
co
*
A
D
E
B
C
F
*
*
*
*
*
*
*
*
co
ep
ep
co
gp
xy
ph
vb
co
ep
sid
22µm330µm
3µm
3
µ
m
10
µ
m
10
µ
m
xy
gp
Fig.6.LeafsheathofPipercallosumRuiz&Pav.,Piperaceae.Photomicrographsbytransmittedlight(C–E)andbypolarizedlight(F).Scanningelectronmicrographs(AandB). A.Frontalviewoftheadaxialfaceoftheepidermalsurface.(B–F)Transections.B.Generalview.Notearrangementofthevascularbundles(asterisks)andminorbundles (arrows).C.Detailofthestructuralorganization.DandE.Detailoftheadaxial(D)andabaxial(E)faces.Notecuticularflange(arrow,figureE).F.Polarizedviewshowing birefringentcrystals.Epidermis(ep).Cuticle(ct).Collenchyma(co).Groundparenchyma(gp).Secretoryidioblast(sid).Vascularbundle(vb).Phloem(ph).Xylem(xy).
Table4
ResultsofdeterminationofmetalsinherbaldrugsofPipercallosum.
Herbaldrug Metalcontent(mean±S.D.mgkg−1;n=3)
Al Cd Co Cr Cu Mn Mo Ni Pb Ti V Hg As
Leaf 100.95±8.37 0.03±0.00 0.05±0.03 0.81±0.05 9.50±0.60 6.03±0.12 <LODb 0.44±0.28 <LODb <LODb 19.02±1.00 0.02±0.01 <LODb
Stem 32.75±6.68 0.03±0.00 0.01±0.00 0.88±0.14 8.52±1.08 2.42±0.35 <LODb 0.18±0.04 <LODb <LODb 3.90±0.36 <LODb <LODb
SRMa1547 233.67±4.98 0.03±0.01 0.06±0.01 1.17±0.02 3.10±0.35 74.90±3.47 <LODb 0.62±0.10 <LODb 1.10±0.28 0.40±0.29 0.03±0.01 0.06±0.02
Recovery(%) 93.84 102.96 83.85 117.20 82.86 76.48 c 89.07 c d 113.55 93.84 102.96
SD,Standarddeviation.
nisthenumberofindependentdeterminations. aStandardReferenceMaterial.
b LOD(Detectionlimit):Mo=0.001mgkg−1;Pd=0.005mgkg−1;Ti=0.002mgkg−1;Hg=0.00004mgkg−1;As=0.00012mgkg−1. c Non-satisfactoryrecovery.
d Valuenotcertified.
themidrib;dorsiventralmesophyll;lamellarcollenchyma; scler-ificationofthestemcollenchyma;structureoftheleafcollateral vascularbundles;arrangementpatternofthevascularbundlesof theleafsheath;organizationpatternofthestemvascularsystem; shapeintransectionoftheleafsheath;prismaticcrystals;druses; andpresenceofsecretoryidioblastsintheleafandstemtissues.
Concerningthedistinctivecharacteristicsthatwereobserved inP.callosum,werecordedforbothleavesandstemthefollowing: ornamentationanddispositionpatternsoftheepicuticularwax; cuticularflangespatterns;typeandpositionoftheleafandstem
collenchyma;typeofsupportingtissueinthemidrib;arrangement patternofthevascularbundlesofthemidribandpetiole;shape intransectionofthemidrib,leafmargin,petiole,andstem; occur-renceofraphides;andmorphologyofthestarchgrains.Thecrystal macropatternsdescribedforP.callosum(Silvaetal.,2014)mustbe addedtothesediagnosticmicroscopiccharacteristicsastheyare speciesspecific.
ct ew
ct
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cp 5µm
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4µm
vb xy ph
pas
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fiz
pi
cx
vb xy
fiz ph fi ep
*
mp
5µm 5µm
10µm 5µm
10µm
10µm
xy xy ph xy
pra
vb
fic ifca
fca
ph
xy
*
*
Fig.7.StemofPipercallosumRuiz&Pav.,Piperaceae.Photomicrographsbytransmittedlight(C–E;G–I)andbypolarizedlight(F).Scanningelectronmicrographs(A;B;inset
atF).(A–F)Primarygrowth.(G–I)Initialsecondarygrowth.A.Frontalviewoftheepidermalsurface.(B–I)Transections.B.Generalview.C.Primarystemstructure.Note secretoryidioblasts(arrows)andinitiallenticel(asterisk).D.Detailoftheepidermisandcortex.Notecuticularflange(arrow).EandF.Pith.E.Generalview.Notesecretory idioblasts(arrows).F.Polarizedviewshowingamyloplastswithbirefringentstarchgrains.Noteaggregatestarchgrains(inset).G.Vascularbundle.Notesecretoryidioblasts (asterisks).H.Interfascicularcambium.I.Initialsecondarystemstructure.Cuticle(ct).Epicuticularwax(ew).Epidermis(ep).Collenchyma(co).Fiber(fi).Fibercap(fic). Fiberzone(fiz).Cortex(cx).Corticalparenchyma(cp).Medullaryparenchyma(mp).Pith(pi).Parenchymaticray(pra).Parenchymaticsheath(pas).Fascicularcambium(fca). Interfascicularcambium(ifca).Vascularbundle(vb).Phloem(ph).Xylem(xy).
successfullytoseparatecloselyrelatedspecies,e.g.Smilax(Martins etal.,2013)andPiper(Gogoszetal.,2012;Horneretal.,2012;Silva etal.,2014,2016).
Weemphasizethattheconservativeanddistinctive anatomi-calcharacteristicsshouldbeconsideredtogetherforanaccurate botanicalauthentication;theformeraredesignedforgeneral taxo-nomicdelimitationandthelatterforspecifictaxonomicdiagnosis. With regard to the powdered herbal drugs, the anatomical markersofauthenticationmustbebasedonthedistinctive char-acteristicsofleafandstemofP.callosum.Fortheleafherbaldrugs, thedistinctivecharacteristicsaretheepicuticularwaxfeaturesas wellasthepresenceofraphidesintheleaffragments.Forthestem herbaldrugs,theanatomicalmarkersaretheparietaland epicutic-ularwaxfeaturesofthestemfragments,blackish-coloredparticles, andformofthestarchgrains.
Thetypes ofwallthickeningof thetrachearyelements can-notbeconsideredanatomicalmarkersforP.callosumpowdered herbal drugs because they also occur in other Piper species (Periyanayagametal.,2012;Silvaetal.,2016).
ThesmallcallositiespresentatthebaseoftheP.callosumleaf bladeconstituteanimportantdistinctivemorphologicalcharacter ofthisspeciestodistinguishitfromotherPiperspecies(Yuncker, 1972).Theanatomical featuresofthecallusesarereportedhere forthefirsttime.Fromananatomicalviewpoint,thecallusesof P.callosumcanbeconsideredascomplexglandularemergencesof petiolarorigin.Thecellprotuberancesofthecallusepidermismay
beaperidermdifferentiatedduetothearrangementofthecells indivision,suchasphellogen,andtheirphenolic(tannin)content. AccordingtoBeckman(2000),thereisstrongpositional, biochemi-calandphysiologicalevidencetosuggestthatphenolic-storingcells maybeintimatelyinvolvedinprocessesthatresultinperiderm formation.
Ascallusisageneralmorphologicaltermthatdescribes excres-centorprotuberanttissueswithoutdistinctivefeatures(Gonc¸alves and Lorenzi, 2011), we suggest that the callusesof P. callosum betermedcallosusemergences,denotingbothmorphologicaland anatomicalaspects.
adf
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Fig.8. MicroscopicfeaturesofthepowderedherbaldrugsofPipercallosumRuiz&Pav.,Piperaceae.Photomicrographsbytransmittedlight(A,B;E–H;J;K;insetatI)and bypolarizedlight(D).Scanningelectronmicrographs(C,IandL).(A–G)Leafherbaldrugs.(A–E)Leafbladefragments.D.Polarizedviewshowingbirefringentraphides. E.Notesecretoryidioblasts(asterisks).F.Isolatedsecretoryidioblasts(asterisks).G.Trachearyelements.(H–L)Stemherbaldrugs.H.Noteisolatedfibers(arrows),fiber bundles(asterisk),andblackish-coloredparticle(inset).I.Stemfragment.Noteepidermis(inset).J.Trachearyelements.K.Secretoryidioblast.Notecellwallwithtrilamellar structuralaspectandlignifiedintermediarylayer(arrow).L.Non-aggregatestarchgrains(asterisks).Adaxialface(adf).Abaxialface(abf).Glandulartrichome(gt).Glandular trichomebase(gtb).Epidermalcellsencirclingtheglandulartrichomebase(epc).Stoma(st).Cuticle(ct).Epicuticularwax(ew).Annularwallthickening(awt).Helicalwall thickening(hwt).
TheresultsachievedbythehistochemicalscreeningofP.callosum leavesand stemcorroborate theargumentsof theauthors,and thetermsecretoryidioblastsor cellsadequately describessuch structuresbecauseitdoesnotmakeinferencesfromitschemical content.
Thedifferenttherapeuticutilizations infolk medicineofthe P. callosum leaves and stem (Andrade et al., 2009) are likely relatedtothechemicalvarietyofthemetaboliccontentidentified histochemically in the idioblasts. The following pharmacologi-calpropertieshavebeenreportedforphenoliccompounds,such as tannins and flavonoids, steroids, and essential oils (Costa, 1994;Monteiroetal.,2005):antibacterial,antifungal,insecticidal, larvicidal, antispasmodic, carminative, choleretic, cholagogue, antidiarrheal, astringent, anti-inflammatory, anti-allergic, anes-thetic,analgesic,antihemorrhagic,anddiuretic.
Fromapharmacognosticalviewpoint,theP.callosumleafand stemherbaldrugspresentexcellentquality,whichisensuredby preservationintheherbaldrugsofmorphoanatomical character-isticsobservedinthefreshplantmaterials,e.g.theoccurrenceof
secretoryidioblastswithcontent.Likely,theproperconditionsof preparationoftheherbaldrugs,suchastimeandtemperatureof drying,contributedtothisfact.Plantmaterialswhensubjectedto hightemperaturescanundergobothmorphologicalandcell con-tent alterations of thetissues (Silvaet al., 2016).On theother hand,thedryingofplantmaterialsuntilconstantweightmakes itimpossibleforportionstoremainmoistduetoinefficientdrying andpreventsthedegradationofchemicalcompoundsbyexcessive drying(Hubingeretal.,2009).
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Fig.9.PhotomicrographsoftransectionsofleavesandstemofPipercallosumRuiz&Pav.,Piperaceae,showingintensepositiveresultsfromhistochemicaltests.Apicalcellof theglandulartrichomes(A,D,NandR).Secretoryidioblasts:mesophyll(K;S);groundparenchymaofthemidrib(H),petiole(B;E;J;O),callosusemergences(G),stemcortex (L),xylemandphloem(Ipe)*;collenchymaofthepetiole(M).Cellprotuberancesofthecallosusemergences(C,F,P,Q).(A–C)Totallipids.(D–F)Acidlipids.(G–J)Essentialoil (G),oleoresin(H),andmixtureofessentialsandresins(IandJ).(K–M)Steroids.(N–P)Totalphenoliccompounds.Q.Tannins.RandS.Flavonoidsdisplayingyellowishgreen fluorescenceunderUVlight.Collenchyma(co).Cuticle(ct).Secretoryidioblast(sid).Phloem(ph).Xylem(xy).*Thesuperscriptindicatesthatthexylemandphloembelong tothevascularbundlesofthepetiole(pe).
compoundsthatoccurinthecontentofitsidioblasts.Theanalysis forlossondryingdeterminesbothwaterandvolatilematter(WHO, 1998; Farmacopeia Brasileira, 2010); therefore, the difference betweenthelossondryingandmoisturevaluesmaycorrespond tovolatiles,suchasessentialoils.Thetotalandacid-insolubleash valuesintheP.callosumherbaldrugsareinaccordancewith spec-ificationsoftheBritishHerbalPharmacopoeia(1990),inwhichthe maximumlimitoftotalandacid-insolubleashis15%(w/w)and5% (w/w),respectively.TheBrazilianPharmacopeiadoesnotestablish
contributetohighamountsofash,mainlyforthesulphatedash values(Mohamadetal.,2013).
ThereductionobservedinthepHvalueofthedistillatewater revealstheoccurrenceofacidcompoundsintheP.callosumherbal drugs,whichisinagreementwiththerichnessofacidmetabolic classesidentifiedhistochemicallyinthecontentoftheidioblastsof thisspecies.
ThemetalconcentrationsfoundintheP.callosumherbaldrugs arewithinsafetybaselinelevelsforhumanconsumption(WHO, 1996,1998,2006;FarmacopeiaBrasileira,2010)andcanbeused asaqualitycriterionsincetheregulatoryagenciesdonot estab-lishpermissiblelevelsofmetalsinrawplantmaterials,exceptfor arsenic,cadmium,andlead(WHO,1998),andtheincreaseofmetal contentinplants,mainlythosethatarepotentiallytoxicortermed “heavymetals,”canindicateexternalcontaminationby environ-mentalfactorsand/orprocessingmethods(Bas¸gelandErdemo˘glu, 2006).
Conclusion
Theresultsobtainedinthepresentstudyconcerningthe macro-scopic,microscopic,andphysicochemical analysisofP.callosum leavesandstemcanbeusedassafeparametersforqualitycontrolof rawplantmaterialsinbothforms:medicinalplantandherbaldrug. Themacro-andmicroscopicdatawillcontributetothebotanical authentication,thephysicochemicaldatawillpermitthe evalua-tionofpuritydegree,andtheorganolepticcharacteristicscouldbe usedforbothbotanicalauthenticationandpuritydegree.
Authors’contributions
RJFScontributed in thecollectionand identificationofplant samples,preparation ofherbarium specimens,laboratorywork, analysisofdata,designofthestudyandthewritingofthepaper. ACAADsupervisedthelaboratorywork.KCFFcontributedto anal-ysisbyinductivelycoupledplasmaopticalemissionspectrometry. ACAADandMSMcontributedtocriticalreadingofthemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
ThefirstauthorwishestothanktheProgramadeCapacitac¸ão Institucional(MPEG/MCTI)fortheresearchscholarshipgranted.
References
Alves,M.S.M.,Mendes,P.C.,Vieira,J.G.P.,Ozela,E.F.,Barbosa,W.L.R.,Júnior,J.O.C.S., 2010.AnálisefarmacognósticadasfolhasdeArrabidaeachica(Humb.&Bonpl.) B.Verlt.,Bignoniaceae.Rev.Bras.Farmacogn.20,215–221.
Andrade,H.E.A.,Guimarães,E.F.,Maia,J.G.S.,2009.Variabilidadequímicaemóleos essenciaisdeespéciesdePiperdaAmazônia.FEC/UFPA,Belém.
ANVISA,2014. Agência Nacionalde Vigilância Sanitária,Ministério da Saúde. Resoluc¸ãodeDiretoriaColegiadan◦26de13demaiode2014.Dispõesobre
oregistrodemedicamentosfitoterápicoseoregistroeanotificac¸ãode produ-tostradicionaisfitoterápicos.DiárioOficialdaUnião,Brasília,em14demaiode 2014.
Bas¸gel,S.,Erdemo˘glu,S.B.,2006.Determinationofmineralandtraceelementsin somemedicinalherbsandtheirinfusionsconsumeinTurkey.Sci.TotalEnviron. 359,82–89.
Beckman,C.H.,2000.Phenolic-storingcells:keystoprogrammedcelldeathand peridermformationinwiltdiseaseresistanceandingeneraldefenceresponses inplants.Physiol.Mol.PlantPathol.7,101–110.
1990.BritishHerbalPharmacopoeia,1sted.BritishHerbalMedicineAssociation,
Bouthermouth.
Cain,A.J.,1947.TheuseofNileblueintheexaminationoflipids.Q.J.Microsc.Sci. 88,383–392.
Caniato,R.,Filippini,R.,Cappelletti,E.M.,Appendino,G.,1989.Detectionofperoxides inintactplantmaterial.FitoterapiaLX,549–551.
Charrière-Ladreix,Y.,1976.Répartitionintracellulairedusecrétatflavoniquede
PopulusnigraL.Planta129,167–174.
Cheng,D.,Zhang,Y.,Xin,X.,Gao,D.,2014.ComparativepharmacognosyofPyrrosia petiolosaandPyrrosiadavidii.Rev.Bras.Farmacogn.24,368–380.
Costa,A.F.,1994.Farmacognosia,II.,4aEdic¸ãoFundac¸ãoCalousteGulbenkian,Lisboa. Couto,A.G.,Kunzler,M.L.,Spaniol,B.,Magalhães,P.M.,Ortega,G.G.,Petrovick,P.R., 2013.ChemicalandtechnologicalevaluationofthePhyllanthusniruriaerialparts asafunctionofcultivationandharvestingconditions.Rev.Bras.Farmacogn.23, 36–43.
David,R.,Carde,J.P.,1964.Colorationdifférentielledesinclusionslipidiqueet ter-peniquesdespseudophyllesduPinmaritimeaumoyendureactifNadi.C.R. Acad.Sci.ParisD258,1338–1340.
Dickson,W.C.,2000.IntegrativePlantAnatomy.AcademicPress,SanDiego.
Evans,W.C.,2009.TreaseandEvans’Pharmacognosy,16thed.WBSaunders Com-pany,London.
Evert,R.F.,2006.Esau’sPlantAnatomy.Meristems,Cells,andTissuesofthePlant Body:TheirStructure,FunctionandDevelopment,3rded.JohnWiley&Sons,
Inc.,Hoboken,NewJersey.
Facundo, V.A., Morais, S.M., Filho, R.B., 2004. Flavonóides de Piper callosum
daAmazônia,http://www.sbq.org.br/anteriores/23/resumos/0765-2(accessed August2004).
FarmacopeiaBrasileira,2010.MinistériodaSaúde,vol.I.,5aEdic¸ãoAgênciaNacional
deVigilânciaSanitária,Brasília.
Feder,N.,O’Brien,T.P.,1968.Plantmicrotechnique:someprinciplesandnew meth-ods.Am.J.Bot.55,123–142.
Fonseca,F.N.,Silva,A.H.,Leal,L.K.A.M.,2010.JusticiapectoralisJacq.,Acanthaceae: preparationandcharacterisationoftheplantdrugincludingchromatographic analysisbyHPLC-PDA.Rev.Bras.Farmacogn.20,871–877.
Ganter,P.,Jollés,G.,1969.HistologieNormaleetPathologique,vol.I. Gauthier-Villars,Paris.
Ganter,P.,Jollés,G.,1970.HistologieNormaleetPathologique,vol.II. Gauthier-Villars,Paris.
Gogosz, A.M.,Boeger,M.R.T., Negrelle,R.R.B.,Bergo,C.,2012.Anatomia foliar comparativadenoveespéciesdogêneroPiper(Piperaceae).Rodriguésia63, 405–417.
Gonc¸alves,E.G.,Lorenzi,H.,2011.Morfologiavegetal:organografiaedicionário ilustradodemorfologiadasplantasvasculares,2aEdic¸ão.InstitutoPlantarum
deEstudosdaFlora,SãoPaulo.
Guimarães,E.F.,Carvalho-Silva,M., Monteiro,D.,Medeiros,E.S.,Queiroz, G.A., 2014. Piperaceae inLista de EspéciesdaFlora doBrasil.Jardim Botânico doRiodeJaneiro,http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB24217
(accessedAugust2014).
Hardman, R., Sofowora,E.A., 1972. Antimony trichloride astest reagents for steroids,especiallydiosgeninandyamogenininplanttissues.StainTechnol.47, 205–208.
Horner,H.T.,Wanke,S.,Samain,M-S.,2012.Acomparisonofleafcrystal macropat-ternsinthetwosistergeneraPiperandPeperomia(Piperaceae).Am.J.Bot.99, 983–997.
Hubinger,S.Z.,Salgado,H.R.N.,Moreira,R.R.D.,2009.Controlesfísico,físico-químico, químicoemicrobiológicodosfrutosdeDimorphandramollisBenth.Fabaceae. Rev.Bras.Farmacogn.19,690–696.
Johansen,D.A.,1940.PlantMicrotechnique.McGraw-HillBookCo.,NewYork.
Judd,W.S.,Campbell,C.S.,Kellog,E.A.,Stevens,P.F.,Donoghue,M.J.,2009. Sis-temáticaVegetal:UmEnfoqueFilogenético,3aEdic¸ão.EditoraArtmed,Porto
Alegre.
Lillie,R.D.,1965. HistopathologicTechnicandPracticalHistochemistry,3rd ed.
McGraw-HillBookCo.,NewYork.
Mace,M.E.,Howell,C.R.,1974.Histochemistryandidentificationofcondensed tan-ninprecursorinrootsofcottonseedlings.Can.J.Bot.52,2423–2426.
Mace,M.E.,Bell,A.A.,Stipanovic,R.D.,1974.Histochemistryandisolationof gossy-polandrelatedterpenoidsinrootsofcottonseedlings.Phytopathology64, 1297–1302.
Machado,N.S.O.,Pereira,F.G.,Santos,P.R.D.,Costa,C.G.,Guimarães,E.F.,2015.
Comparativeanatomy of the leaves of Piper lepturum (Kunth)C.DC. var.
lepturumandPiperlepturumvar.angustifolium(C.DC.).Yunck.Hoehnea42, 1–8.
Marinho,C.R.,Zacarob,A.A.,Ventrella,M.C.,2011.SecretorycellsinPiperumbellatum
(Piperaceae)leaves.Anewexampleforthedevelopmentofidioblasts.Flora206, 1052–1062.
Martins,A.R.,Bombo,A.B.,Soares,A.N.,Appezzato-da-Glória,B.,2013.Aerialstem andleafmorphoanatomyofsomespeciesofSmilax.Rev.Bras.Farmacogn.23, 576–584.
Metcalfe,C.R.,Chalk,L.,1950.AnatomyoftheDicotyledons,vol.II.ClarendonPress, Oxford.
Mohamad, T.A.S.T.,Naz, H., Jalal, R.S., Hussin,K., Rahman, M.R.A., Adam, A., Weber, J-F.F., 2013. Chemical and pharmacognostical characterization of twoMalaysianplants bothknownasAjisamat.Rev. Bras.Farmacogn.23, 724–730.
Monteiro,J.M.,Albuquerque,U.P.,Araújo,E.L.,Amorim,E.L.C.,2005.Taninos:uma abordagemdaquímicaàecologia.Quim.Nova28,892–896.
Oliveira,F.,Akisue,G.,2003.Farmacognosia.Atheneu,SãoPaulo.
Parmar,V.S.,Jain,S.C.,Risht,K.S.,Jain,R.,Taneja,P.,Jha,A.,Tyagi,O.M.,Prasad, A.K.,Wengel,J.,Olsen,C.E.,Boll,P.M.,1997.PhytochemistryofthegenusPiper. Phytochemistry46,597–673.
Pearse,A.G.E.,1980.HistochemistryTheoreticalandApplied:Preparativeand Opti-calTechnology,4thed.ChurchillLivingston,Edinburgh.
Periyanayagam,K.,Jagadeesan,M.,Kavimani,S.,Vetriselvan,T.,2012. Pharmacog-nosticalandphyto-physicochemicalprofileoftheleavesofPiperbetleL.var Pachaikodi(Piperaceae)-valuableassessmentofitsquality.AsianPac.J.Trop. Biomed.,S506–S510.
Pizzolato,T.D.,Lillie,R.D.,1973.Mayer’stannicacid-ferricchloridestainformucins. J.Histochem.Cytochem.21,56–64.
Potiguara,R.C.V.,Silva,R.J.F.,Kikuchi,T.Y.S.,Lucas,F.C.A.,Macedo,E.G.,2013. Estru-turasvegetaisemmicroscopiaeletrônicadevarredura.MuseuParaenseEmílio Goeldi/UniversidadedoEstadodoPará,Belém.
Pratsmoya,S.,Graneteruel,N.,Berenguernavarro,V.,Martincarratala,M.L.,1997.
Inductivelycoupled plasmaapplicationfortheclassificationof19almond cultivars using inorganic element composition. J. Agric. Food Chem. 45, 2093–2097.
Santos,V.L.P.,Franco,C.R.C.,Amano,E.,Messias-Reasond,I.J.,Budel,J.M.,2015.
AnatomicalinvestigationsofPiperamalago(jaborandi-manso)forthequality control.Rev.Bras.Farmacogn.25,85–91.
Silva, R.J.F.,Aguiar-Dias, A.C.A., Mendonc¸a, M.S.,2014. Rosetase concrescên-cias cristalinas silicificadas em Piper (Piperaceae): registros inéditos de macropadrões.ActaAmazon.44,435–446.
Silva,R.J.F.,Aguiar-Dias,A.C.A.,Faial,K.C.F.,Mendonc¸a,M.S.,2016.Caracterizac¸ão farmacognósticadePiperarboreumvar.arboreumeP.tuberculatum(Piperaceae). ActaAmazon.46,195–208.
Svendsen,A.B.,Verpoorte,R.,1983.ChromatographyofAlkaloids.ElsevierScientific PublishingCo.,NewYork.
WHO,WorldHealthOrganization,1996.TraceElementsinHumanNutritionand Health.WHO,Geneva.
WHO,WorldHealthOrganization,1998.QualityControlMethodsforMedicinal PlantMaterials.WHO,Geneva.
WHO,FAO,WorldHealthOrganizationandFoodandAgricultureOrganization,2006.
GuidelineonFoodFortificationWithMicronutrients.WHO,Geneva.
