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Anatomical investigations of Piper amalago (jaborandi-manso) for the quality control

Abstract

Piper amalago L., Piperaceae, popularly known as jaborandi-manso, is a shrub that spans a height of 2–7 m. It can be found in the regions of Southern America downward up to the south of Brazil. Traditionally it is used to treat digestive problems, heart problems, and burns. This study aims to conduct an anatomical investigation and analysis of the leaves and stems of P. amalago through electron scanning and optical micro techniques. The analysis showed that P. amalago has a hypostomatic leaf, with a subepidermal layer on its surface. There are grandular trichomes that resemble sacs, conic non-glandular trichomes, dorsiventral mesophyll, and a plano-convex midrib having a single vascular bundle in the center. The petiole is short with irregularly shaped and adaxially grooved. The stem is circular in shape and contains two circles of vascular bundles and a sclerenchymatic sheath in the perimedular region. These anatomical features of the Piper amalago's leaves and stems make it easy to pick it out among other species of the Piper genus. This is helpful when conducting quality control process.

Keywords:
Anatomy; Quality control; Jaborandi-manso; Piper amalago ; Piperaceae

Introduction

According to Figueiredo and Sazima (2000)Figueiredo, R.A., Sazima, M., 2000. Pollination biology of Piperaceae species in southeastern Brazil. Ann. Bot. 85, 455–460. and Machado (2007)Machado, N.S.O., (Tese de Doutorado) 2007. Estudo da anatomia foliar de espécies do gênero Piper L. (Piperaceae) no estado do Rio de Janeiro. Rio de Janeiro. Universidade Federal do Rio de Janeiro, 103 pp., the Piperaceae family is made of approximately 3000 species covering tropical and subtropical areas. In Brazil alone, the Piperaceae consists of five genus and about 500 species mainly found in the Atlantic Forest (Souza, 2005Souza, V.C., 2005. Botânica sistemática. Plantarum, Nova Odessa (SP).; Souza and Lorenzi, 2005Souza, V.C., Lorenzi, H., 2005. Botânica Sistemática: guia ilustrado para identificação das famílias de angiospermas da flora brasileira, baseado em APG II. Nova Oddessa, São Paulo. Instituto Plantarum, 230–231.). It has several species that were popular for their medicinal use and have been studied for chemical composition and biological activities (Parmar et al., 1997Parmar, V.S., Jain, S.C., Bisht, S.K., Jain, R., Taneja, P., Tyagi, O.D., 1997. Phytochemistry of the genus Piper. Phytochemistry 46, 591–673.; Jaramillo and Manos, 2001Jaramillo, M.A., Manos, P.S., 2001. Filogenia e padrões de diversidade floral no gênero Piper (Piperaceae). Am. J. Bot 88, 706–716.; Silva and Bastos, 2007Silva, D.M.H., Bastos, C.N., 2007. Atividade antifúngica de óleos essenciais de espécies de Piper sobre Crinipellis perniciosa Phytophthora palmivora e Phytophthora capsici. Fitopatol. Bras. 32, 143–145.; Facundo et al., 2008Facundo, V.A., Pollli, A.R., Rodrigues, R.V., Militão, J.S.L.T., Stabelli, R.G., Cardoso, C.T., 2008. Constituintes químicos fixos e voláteis dos talos e frutos de Piper tuberculatum Jacq e das raízes de P. hispidum H. B. K. Acta Amaz 38, 743–748.; Baldoqui et al., 2009Baldoqui, D.C., Bolzani, V.S., Furlan, M., Kato, M.J., Marques, M.O.M., 2009. Flavonas lignanas e terpeno de Piper umbellata (Piperaceae). Quim. Nova. 32, 1107–1109.).

Piper is one of the important genus of Piperaceae and comprises about 2000 species found all over the world (Joly, 2002Joly, A.B., 2002. Botânica: introdução à taxonomia vegetal. Nacional, São Paulo (SP).; Silva et al., 2007Silva, W.C., Ribeiro, J.D.A., Menezes de Souza, H.E., Correa, R.D.S., 2007. Insecticidal activity of Piper aduncum L. (Piperaceae) on Aetalion sp (Hemiptera: Aetalionidae), plague of economic importance in Amazon. Acta Amazonica 37, 293–298.). Ethno botanical studies reported the use of Piper in traditional medicine by several communities as an antioxidant, antimicrobial and antileishmanial (Regasini et al., 2009aRegasini, L.O., Cotinguiba, F., Passerini, G.D., Bolzani, V.S., Cicarelli, R.M., Kato, M.J., Furlan, M., 2009a. Trypanocidal activity of Piper arboreum and Piper tuberculatum (Piperaceae). Rev. Bras. Farmacogn. 19, 199–203.,bRegasini, L.O., Cotinguiba, F., Morandim, A.A., Kato, M.J., Scorzoni, L., Mendes-Giannini, M.J., Bolzani, V.S., Furlan, M., 2009b. Antimicrobial activity of Piper arboreum and Piper tuberculatum (Piperaceae) against opportunistic yeasts. Afr. J. Biotechnol. 17, 2866–2870.; Carrara et al., 2012Carrara, V.S., Serra, L.Z., Cardozo-Filho, L., Cunha-Júnior, E.F., Torres-Santos, E.C., Cortez, D.A.G., 2012. HPLC analysis of supercritical carbon dioxide and compressed propane extracts from Piper amalago L. with antileishmanial activity. Molecules 17, 15–33., 2013Carrara, V.S., Cunha-Júnior, E.F., Torres-Santos, E.C., Corrêa, A.G., Monteiro, J.L., Demarchi, I.G., Lonardoni, M.V.C., Cortez, D.A.G., 2013. Antileishmanial activity of amides from Piper amalago and synthetic analogs. Rev. Bras. Farmacogn. 23, 447–454.; Chander et al., 2014).

Piper amalago L. is popularly known in Brazil by the terms jaborandi-manso, jaborandi-falso and jaborandinhandi. It is a perennial plant which may attain 2–7 m height (Guimarães and Valente, 2001Guimarães, E.F., Valente, C., 2001. Piperaceae – Piper. In: Reitz, R. (Ed.), Flora ilustrada catarinense. Herbário Barbosa Rodrigues, Itajaí.). Pharmacological studies have demonstrated anti-inflammatory (Carrara et al., 2012Carrara, V.S., Serra, L.Z., Cardozo-Filho, L., Cunha-Júnior, E.F., Torres-Santos, E.C., Cortez, D.A.G., 2012. HPLC analysis of supercritical carbon dioxide and compressed propane extracts from Piper amalago L. with antileishmanial activity. Molecules 17, 15–33.), antimicrobial, cicatrizing (Guimarães and Valente, 2001Guimarães, E.F., Valente, C., 2001. Piperaceae – Piper. In: Reitz, R. (Ed.), Flora ilustrada catarinense. Herbário Barbosa Rodrigues, Itajaí.), and antileishmanial properties (Carrara et al., 2013Carrara, V.S., Cunha-Júnior, E.F., Torres-Santos, E.C., Corrêa, A.G., Monteiro, J.L., Demarchi, I.G., Lonardoni, M.V.C., Cortez, D.A.G., 2013. Antileishmanial activity of amides from Piper amalago and synthetic analogs. Rev. Bras. Farmacogn. 23, 447–454.). Additionally, it has been suggested that it has antioxidant properties since it contains vitexin and lupeol which have been isolated through phytochemical analysis (Rovani et al., 2013Rovani, G., Santos, V.L.P., Miguel, O.G., Budel, J.M., Campos, R., 2013. Investigação fitoquímica e antioxidante de partes vegetativas aéreas de Piper amalago L. Cad. Esc. Saúde Unibrasil 2, 164–177.).

The serious problem of the herbal drugs replaced with alternative plants or tampered are common in Brazil. However, there is no requirement for the consumer market to publish official monographs for quality control (Kato et al., 2012Kato, E.T.M., Bacchi, E.M., Hernandes, L.S., 2012. Farmacobotânica e atividade antiúlcera de plantas medicinais brasileiras. In: Lopes, N.P., Souza, G.H.B., Mello, J.C.P. (Eds.), Farmacognosia: coletânea científica. UFOP, Ouro Preto, pp. 177–196.). In that respect, recently several works have been devoted to study morpho-anatomy of the plants (Budel and Duarte, 2008Budel, J.M., Duarte, M.R., 2008. Estudo farmacobotânico de folha e caule de Baccharis uncinella DC. Lat. Am. J. Pharm 27, 740–746., 2010Budel, J.M., Duarte, M.R., 2010. Macro and microscopic characters of the aerial vegetative organs of carqueja: Baccharis usterii Heering. Braz. Arch. Biol. Technol. 53, 123–131.; Oliveira et al., 2011Oliveira, A.M.A., Santos, V.L.P., Franco, C.R.C., Farago, P.V., Duarte, M.R., Budel, J.M., 2011. Comparative morpho-anatomical study of Baccharis curitybensis Heering ex Malme and Baccharis spicata (Lam.) Baill. Lat. Am. J. Pharm. 30, 1560–1566.; Squena et al., 2012Squena, A.P., Santos, V.L.P., Franco, C.R.C., Budel, J.M., 2012. Análise morfoanatômica de partes vegetativas aéreas de Pereskia aculeata Mill., Cactaceae. Cad. Esc. Saúde Unibrasil 8, 189–207.; Youssef et al., 2013Youssef, J., Döll-Boscardin, P.M., Farago, P.V., Duarte, M.R., Budel, J.M., 2013. Gochnatia polymorpha: macro-and microscopic identification of leaf and stem for pharmacognostic quality control. Rev. Bras. Farmacogn. 23, 585–591.; Camilotti et al., 2014Camilotti, J.G., Biu, C.C., Farago, P., Santos, V.L.P., Franco, C.R.C., Budel, J.M., 2014. Anatomical characters of leave and stem of Calea serrata Less Asteraceae. Braz. Arch. Biol. Techn. 57, 867–873.; Pereira et al., 2014aPereira, C.B., Farago, P., Budel, J.M., Padilha de Paula, J., Folquitto, D.G., Miguel, O.G., Miguel, M.D., 2014a. Contribution to the pharmacognostic study of Carquejas: Baccharis milleflora DC., Asteraceae. Lat. Am. J. Pharm. 33, 841–847.,bPereira, C.B., Miguel, M.D., Folquitto, D.G., Miguel, O.G., Farago, P.V., Paula, J.P., Santos, V.L.P., Franco, C.R.C., Budel, J.M., 2014b. Architecture of the aerial vegetative organs and scanning electron micrographs of Dioscorea bulbifera L for the quality control. Lat. Am. J. Pharm. 33, 1100–1105.; Amorin et al., 2014Amorin, M., Paula, J.P., Silva, R.Z., Farago, P.V., Budel, J.M., 2014. Pharmacobotanical study of the leaf and stem of Mikania lanuginosa for its quality control. Rev. Bras. Farmacogn. 24, 531–537.; Folquitto et al., 2014Folquitto, D.G., Budel, J.M., Pereira, C.B., Brojan, L.E.F., Folquitto, G.G., Miguel, M.D., Silva, R., Miguel, Z.O.G., 2014. Analytical micrography and preliminary phytochemistry of the leaves and Stems of Lobelia exaltata Pohl (Campanulaceae). Lat.Am. J. Pharm. 33, 245–250.; Jasinski et al., 2014Jasinski, V.C.G., Silva, R.Z., Pontarolo, R., Budel, J.M., Campos, F.R., 2014. Morpho-anatomical characteristics of Baccharis glaziovii in support of its pharmacobotany. Rev. Bras. Farmacogn. 24, 506–515.).

There is a close structural similarity between the species of Piper (Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.) and there has not been any previous study of microscopic characters of P. amalago. Thus, this paper therefore seeks to study the microscopic characteristics of aerial vegetative organs of P. amalago for quality control purposes of phytotherapy industry.

Materials and methods

Plant material

Aerial vegetative parts of Piper amalago L., Piperaceae, were collected at Curitiba, Paraná (24°18′ S and 49°37′ W) in July 2012. The vegetal material was identified by a taxonomist and compared with the voucher specimens deposited in the Municipal Botanical Museum of Curitiba, under register number 71947.

Anatomical analyses

Five specimens of P. amalago were used. Their leaves and stems were cut at about 5 cm from the apex. These were then put in containers containing FAA 70 solution (Johansen, 1940Johansen, D.A., 1940. Plant Microtechnique. MacGraw Hill Book, New York (NY).), and stored in 70% ethanol (Berlyn and Miksche, 1976Berlyn, G.P., Miksche, J., 1976. Botanical Microtechnique and Cytochemistry. lowa State University, Eames.).

The plants were segmented by use of hand while others were dehydrated, implanted in glycol-methacrylate (Leica historesin) then sectioned with the microtome Leica RM-2145. Toluidine blue was used to stain the transverse and longitudinal sections (O'Brien et al., 1964O'Brien, T.P., Feder, N., McCully, M.E., 1964. Polychromatic staining of plant cell walls by toluidine blue O. Protop 59, 368–373.). Some sections were stained using a mixture of astra blue and basic fuchsine (Roeser, 1972Roeser, K.R., 1972. Die Nadel der Schwarzkiefer-Massenprodukt und Kunstwerk der Natur. Mikrokosmos 6, 33–36.).

Micro chemical tests

The following standard solutions were used in the micro chemical tests: methylene blue to test for mucilage (Oliveira et al., 2005Oliveira, F., Akisue, G., Akisue, M.K., 2005. Farmacognosia. Atheneu, São Paulo.); hydrochloric phloroglucin to reveal traces of lignin (Sass, 1951Sass, J.E., 1951. Botanical Microtechnique, 2nd ed. Iowa State College, Ames.); Sudan III for testing lipophilic compounds (Foster, 1949Foster, A.S., 1949. Practical Plant Anatomy, 2nd ed. D. Van Nostrand, Princeton.); ferric chloride to test for phenolic substances (Johansen, 1940Johansen, D.A., 1940. Plant Microtechnique. MacGraw Hill Book, New York (NY).); sulphuric acid to find out the chemical composition of the crystals (Oliveira et al., 2005Oliveira, F., Akisue, G., Akisue, M.K., 2005. Farmacognosia. Atheneu, São Paulo.) and iodine-iodide to test for starch (Berlyn and Miksche, 1976Berlyn, G.P., Miksche, J., 1976. Botanical Microtechnique and Cytochemistry. lowa State University, Eames.).

Some photomicrographs were captured by a light microscope Olympus CX 31equipped using a control unit of C 7070. The semi-permanent, permanent and microchemical test slides were then analyzed in the Laboratory of Pharmacognosy at the State University of Ponta Grossa for a detailed description of the leaf and stem tissues.

Scanning electron microscopy

During the scanning electron analysis (SEM), the material was desiccated in a graded ethanolic series and carbon (IV) oxide critical point apparatus called Balzers CPD-030. Finally they were coated with gold by Balzers Sputtering SCD-030. The scanning microscope Jeol JSM-6360LV was used to capture electron micrographs (Souza, 1998Souza, W., 1998. Técnicas básicas de microscopia eletrônica aplicada as Ciências Biológicas. Sociedade Brasileira de Microscopia Eletrônica, Rio de Janeiro (RJ).). The results were forwarded to the Federal University of Paraná's Electron Microscopy Centre.

Results and discussion

Piper amalago L., Piperaceae, (Fig. 1A and B), from a frontal view of the blade, shows straight epidermal cell anticlinal walls with relatively thin on both sides (Figs. 1C–E and 2C). On the epidermis' abaxial face (Figs. 1D, E and 2A) has tetracytic stomata (Figs. 1D, E and 2C). Hypostomatic leaves are common in Piperaceae (Metcalfe and Chalk, 1950Metcalfe, C.R., Chalk, L., 1950. Anatomy of Dicotyledons: Leaves, Stem, and Woods in Relation to Taxonomy With Notes on Economic Uses, vol. 1. Clarendon, Oxford.), as described for P. crassinervium H.B.& K. (Albiero et al., 2005aAlbiero, A.L.M., Paoll, A.A.S., Souza, L.A., Mourão, K.S.M., 2005a. Morfoanatomia dos órgãos vegetativos de Piper crassinervium H.B. & K. (Piperaceae). Acta Bot. Bras. 19, 305–312.), P. hispidum Sw. (Albiero et al., 2006Albiero, A.L.M., Paoli, A.A.S., Souza, L.A., Mourão, K.S.M., 2006. Morfoanatomia dos órgãos vegetativos de Piper hispidum Sw (Piperaceae). Rev. Bras. Farmacogn 16, 379–391.), P. aduncum Vell., P. cernuum Vell., P. dilatatum Rich, P. gaudichaudianum Kunth, P. betle L. (Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.), P. glabratum Kunth, P. lindbergii C.DC., P. solmsianum C. DC. and, P. umbellatum Jaqc. (Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.). Amphistomatic leaves were however found in P. hispidinervum C. DC. (Nascimento and Vilhena-Potiguara, 1999Nascimento, M.E., Vilhena-Potiguara, C.R., 1999. Aspectos anatômicos dos órgãos vegetativos d de Piper hispidinervium C. DC. (Piperaceae) e suas estruturas secretoras. Bol. Mus. Para. Emílio Goeldi ser. Bot. 15, 39–104.) and in P. sarmentosum Roxb. (Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.).

Fig. 1
Piper amalago L. (Piperaceae). A. Aspect of aerial parts and habit. B. Detail of leaves and inflorescence. C–E – Frontal view of leaves. C. Adaxial side of epidermis. D. Abaxial side of epidermis showing stomata. E. Abaxial surface, showing detail of the stomata. st: stomata. Bar = 5cm (A), 2 cm (B), 100 μm (E).
Fig. 2
Piper amalago L. (Piperaceae). Leaves. A. Blade organization showing dorsiventral mesophyll. B. Blade organization showing dorsiventral mesophyll. C. Adaxial side of epidermis, showing stomata and glandular trichomes. D. Midrib crossed by collateral vascular bundle. E. Midrib and blade organization. co: collenchyma, ep: epidermis, fp: fundamental parenchyma, gt: glandular trichome, oi: oily cell, nt: non-glandular trichome, ph: phloem, pp: palisade parenchyma, se: subepidermal layer, sp: spongy parenchyma, nt: non-glandular trichome, vb: vascular bundles, xy: xylem. Bar = 50 μm (A), 200 μm (D).

Several species of Piper have tetracytic stomata (Albiero et al., 2006; Gogosz et al., 2012Albiero, A.L.M., Paoli, A.A.S., Souza, L.A., Mourão, K.S.M., 2006. Morfoanatomia dos órgãos vegetativos de Piper hispidum Sw (Piperaceae). Rev. Bras. Farmacogn 16, 379–391.). However, P. arboreum Aubl. has evidenced staurocytic (Souza et al., 2009Souza, L.A., Albiero, A.L.M., Almeida, O.J.G., Lopes, W.A.L., Mourão, K.S.M., Moscheta, I.S., 2009. Estudo morfo-anatômico da folha e do caule de Piper arboreum Aubl (Piperaceae). Lat. Am. J. Pharm. 28, 103–107.), P. crassinervium has presented both the ciclocytic and tetracytic type (Albiero et al., 2005aAlbiero, A.L.M., Paoll, A.A.S., Souza, L.A., Mourão, K.S.M., 2005a. Morfoanatomia dos órgãos vegetativos de Piper crassinervium H.B. & K. (Piperaceae). Acta Bot. Bras. 19, 305–312.), P. betle has showed anisocytic, anomocytic, tetracytic, polycytic, paracytic, amphicyclic and P. sarmentosum has presented anisocytic, anomocytic, actinocytic, cyclocytic and tetracytic type (Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.).

In transection, the epidermis of P. amalago is uniseriate and is covered by a thin cuticle. The stomata are located slightly above the other epidermal cells (Fig. 2A). A subepidermal layer was observed on both surfaces of the leaf (Fig. 2A and B). Multiple epidermis have been observed in species of Piper (Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.; Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.).

Variants are also present, such as, P. aduncum (Vianna and Akisue, 1997Vianna, W.O., Akisue, G., 1997. Caracterização morfológica de Piper aduncum L. Lecta 15, 11–62.) has only one sub-epidermal layer on the adaxial side; P. crassinervium (Albiero et al., 2005aAlbiero, A.L.M., Paoll, A.A.S., Souza, L.A., Mourão, K.S.M., 2005a. Morfoanatomia dos órgãos vegetativos de Piper crassinervium H.B. & K. (Piperaceae). Acta Bot. Bras. 19, 305–312.) has up to 3 sub-epidermal layers on the adaxial side; P. gaudichaudianum (Albiero et al., 2005bAlbiero, A.L.M., Souza, L.A., Mourão, K.S.M., Almeida, O.J.G., Lopes, W.A.L., 2005b. Morfo-anatomia do caule e da folha de Piper gaudichaudianum Kuntze (Piper-aceae). Acta Farm. Bom 24, 550–554.) has 1–2 layers on the abaxial and adaxial surfaces; P. hispidum (Albiero et al., 2006Albiero, A.L.M., Paoli, A.A.S., Souza, L.A., Mourão, K.S.M., 2006. Morfoanatomia dos órgãos vegetativos de Piper hispidum Sw (Piperaceae). Rev. Bras. Farmacogn 16, 379–391.) has 3–4 layers on the abaxial side and, P. arboreum (Souza et al., 2009Souza, L.A., Albiero, A.L.M., Almeida, O.J.G., Lopes, W.A.L., Mourão, K.S.M., Moscheta, I.S., 2009. Estudo morfo-anatômico da folha e do caule de Piper arboreum Aubl (Piperaceae). Lat. Am. J. Pharm. 28, 103–107.) has 2–4 layers on both sides of the blade.

A hypodermis was observed by Metcalfe and Chalk (1979)Metcalfe, C.R., Chalk, L., 1950. Anatomy of Dicotyledons: Leaves, Stem, and Woods in Relation to Taxonomy With Notes on Economic Uses, vol. 1. Clarendon, Oxford. in species of Piperaceae, however, Takemori et al. (2003)Takemori, N.K., Bona, C., Alquini, Y., 2003. Anatomia comparada das folhas de espécies de Peperomia (Piperaceae) – I Ontogênese do tecido aquífero e dos estômatos. Acta Bot. Bras. 17, 387–394. argued that the multiple epidermis is constituted by the first layers leaf blade cells of several species of Peperomia. This is because the cells develop from periclinal division of protodermis. To investigate the exact origin of the sub-epidermal layer in P. amalago requires application of ontogenetic studies.

The sac-like multicellular glandular trichomes found in P. amalago (Fig. 2B, C and E) are made of a short pedicel and an elongated apical cell. Similar trichome was witnessed in P. regnellii (Miq.) C. DC. var. regnellii (Silva and Machado, 1999Silva, E.M.J., Machado, S.R., 1999. Estrutura e desenvolvimento dos tricomassecretores em folhas de Piper regnellii (Miq) C. DC. var. regnellii (Piperaceae). Rev. Bras. Bot. 22, 117–124.), P. regnelli (Pessini et al., 2003Pessini, G.L., Holetz, F.B., Sanches, N.R., Cortez, D.A.G., Filho, B.D., Nakamura, C.V., 2003. Avaliação da atividade antibacteriana e antifúngica de extratos de plantas utilizados na medicina popular. Rev. Bras. Farmacogn. 13, 21–24.), P. mikanianum (Kunth) Steud. (Duarte and Siebenrock, 2010Duarte, M.R., Siebenrock, M.C.N., 2010. Caracteres Anatômicos de Folha e Caule de Piper mikanianum (Kunth) Steud., Piperaceae. Lat. Am. J. Pharm. 29, 45–51.), and P. sarmentosum Roxb. (Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.).

Glandular trichomes have the capability to secrete or store large quantities of specialized metabolites that defend the aerial parts of the plant against pathogens and herbivores (Tissier, 2012Tissier, A., 2012. Glandular trichomes: what comes after expressed sequence tags? Plant J. 70, 51–68.). The secretion has been showing biological activity and it has aroused the interest of pharmaceutical, pesticide, flavor and fragrance industries (Duke, 1994Duke, S.O., 1994. Glandular trichomes: a focal point of chemical and structural interactions. Int. J. Plant Sci. 155, 617–620.).

The conic trichomes are non-glandular. They are multicellular, uniseriate, consist five cells and a slightly acute apex (Figs. 2E and 3B). Trichomes that were found in several species of Piper, ranging from short to long all portrayed some similarity (Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.; Pessini et al., 2003Pessini, G.L., Holetz, F.B., Sanches, N.R., Cortez, D.A.G., Filho, B.D., Nakamura, C.V., 2003. Avaliação da atividade antibacteriana e antifúngica de extratos de plantas utilizados na medicina popular. Rev. Bras. Farmacogn. 13, 21–24.; Albiero et al., 2006Albiero, A.L.M., Paoli, A.A.S., Souza, L.A., Mourão, K.S.M., 2006. Morfoanatomia dos órgãos vegetativos de Piper hispidum Sw (Piperaceae). Rev. Bras. Farmacogn 16, 379–391.; Duarte and Siebenrock, 2010Duarte, M.R., Siebenrock, M.C.N., 2010. Caracteres Anatômicos de Folha e Caule de Piper mikanianum (Kunth) Steud., Piperaceae. Lat. Am. J. Pharm. 29, 45–51.; Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.). These non-glandular trichomes have a variety of functions. They act as a means to prevent herbivores from feeding on the vegetable species, prevent excessive sunlight, controls leaf temperature as well as reduce water loss according to Wagner (1991)Wagner, G.J., 1991. Secreting glandular trichomes: more than just hairs. Plant Physiol. 96, 675–679. and Duke (1994)Duke, S.O., 1994. Glandular trichomes: a focal point of chemical and structural interactions. Int. J. Plant Sci. 155, 617–620..

Fig. 3
Piper amalago L. (Piperaceae). Petiole. A. General aspect. B. Detail of the non-glandular trichomes. C. Petiole showing several collateral vascular bundles. D. Detail of prismatic crystals of calcium oxalate. co: collenchyma, cr: crystal of calcium oxalate, cu: cuticle, ep: epidermis, nt: non-glandular trichome, vb: vascular bundle. Bar = 200 μm (A, C).

In P. amalago, the mesophyll is dorsiventral and formed of a layer of palisade and 2–3 layers of spongy parenchyma (Fig. 2A, B, D, and E). This description is consistent with that applied to the genus, however the number of layers of palisade and spongy parenchyma may vary (Albiero et al., 2005bAlbiero, A.L.M., Souza, L.A., Mourão, K.S.M., Almeida, O.J.G., Lopes, W.A.L., 2005b. Morfo-anatomia do caule e da folha de Piper gaudichaudianum Kuntze (Piper-aceae). Acta Farm. Bom 24, 550–554., 2006Albiero, A.L.M., Paoli, A.A.S., Souza, L.A., Mourão, K.S.M., 2006. Morfoanatomia dos órgãos vegetativos de Piper hispidum Sw (Piperaceae). Rev. Bras. Farmacogn 16, 379–391.; Santiago et al., 2001Santiago, E.J.A., Pinto, J.E.B.P., Castro, E.M., Lameira, A.O., Conceição, H.E.O., Gavilanes, M.L., 2001. Aspectos da anatomia foliar da pimenta-longa (Piper hispidinervium C DC.) sob diferentes condições de luminosidade. Ciênc. Agrotec. 25, 1035–1042.; Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.; Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.). A parenchymatous endoderm surrounds small collateral vascular bundles which are immersed in the spongy parenchyma (Fig. 2B). The mesophyll contains cells with essential oil (Fig. 2A) and phenolic compounds (Fig. 5A). Castro et al. (1997)Castro, M.M., Leitão-Filho, H.F., Monteiro, W.R., 1997. Utilização de estruturas secretoras na identificação dos gêneros de Asteraceae de uma vegetação de cerrado. Rev. Bras. Bot. 20, 163–174. says that these cells with essential oils are idioblasts with additional secretory functions. Piper has a variety of cells containing oil contents (Albiero et al., 2005aAlbiero, A.L.M., Paoll, A.A.S., Souza, L.A., Mourão, K.S.M., 2005a. Morfoanatomia dos órgãos vegetativos de Piper crassinervium H.B. & K. (Piperaceae). Acta Bot. Bras. 19, 305–312.).

Fig. 4
Piper amalago L. (Piperaceae). Stem in cross section. A. General aspect. B. Detail of the previous figure. C. Detail of the sclerenchymatous ring, showing fibers. D. Epidermis and cortex area. E. Pith showing starch grains. F. Detail of the starch grains. cu: cuticle, ep: epidermis, co: collenchyma, fi: fibers, nt: non-glandular trichome, ph: phloem, pi: pith, sg: starch grains, xy: xylem. Bar = 200 μm (A, B), 50 μm (C, D, E).
Fig. 5
Piper amalago L. (Piperaceae). Leaf and stem in cross section. Microchemical analysis. A. Mesophyll in reaction using ferric chloride. B. Midrib showing xylem in reaction using hydrochloric phloroglucin. C. Stem showing xylem and fibers in reaction using hydrochloric phloroglucin. D. Stem demonstrating cuticle in reaction using Sudan III. E. Stem showing endoderm with starch grains using iodine-iodide. cu: cuticle, fi: fibers, me: mesophyll, sg: starch grain, xy: xylem. Bar = 200 μm (A–C), 20 μm (D), 10 μm (E).

The cross section shows that the midrib shape is a plano-convex one and is particularly more convex on its abaxial face as seen in Fig. 2D and E. This feature was evident in P. sarmentosum (Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.). A concave-convex shape or almost concave-convex shape has been seen in several species of Piper (Albiero et al., 2005aAlbiero, A.L.M., Paoll, A.A.S., Souza, L.A., Mourão, K.S.M., 2005a. Morfoanatomia dos órgãos vegetativos de Piper crassinervium H.B. & K. (Piperaceae). Acta Bot. Bras. 19, 305–312., 2005bAlbiero, A.L.M., Souza, L.A., Mourão, K.S.M., Almeida, O.J.G., Lopes, W.A.L., 2005b. Morfo-anatomia do caule e da folha de Piper gaudichaudianum Kuntze (Piper-aceae). Acta Farm. Bom 24, 550–554., 2006Albiero, A.L.M., Paoli, A.A.S., Souza, L.A., Mourão, K.S.M., 2006. Morfoanatomia dos órgãos vegetativos de Piper hispidum Sw (Piperaceae). Rev. Bras. Farmacogn 16, 379–391.; Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.). In addition, a biconvex shape has also been found in P. regnelli (Pessini et al., 2003Pessini, G.L., Holetz, F.B., Sanches, N.R., Cortez, D.A.G., Filho, B.D., Nakamura, C.V., 2003. Avaliação da atividade antibacteriana e antifúngica de extratos de plantas utilizados na medicina popular. Rev. Bras. Farmacogn. 13, 21–24.), P. mikanianum (Duarte and Siebenrock, 2010Duarte, M.R., Siebenrock, M.C.N., 2010. Caracteres Anatômicos de Folha e Caule de Piper mikanianum (Kunth) Steud., Piperaceae. Lat. Am. J. Pharm. 29, 45–51.), P. umbellatum (Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.) and, P. solmsianum (Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.).

A slightly thickened and striated cuticle covers the single-layered epidermis. Close to three layers of angular collenchyma can be observed on both sides. The vascular system is represented by a single collateral vascular bundle in the ground parenchyma (Fig. 2D and E). A similar pattern is also evident in P. glabratum and P. solmsianum (Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.). However, P. gaudichaudianum (Albiero et al., 2005bAlbiero, A.L.M., Souza, L.A., Mourão, K.S.M., Almeida, O.J.G., Lopes, W.A.L., 2005b. Morfo-anatomia do caule e da folha de Piper gaudichaudianum Kuntze (Piper-aceae). Acta Farm. Bom 24, 550–554.), P. diospyrifolium (Souza et al., 2004Souza, L.A., Moscheta, I.S., Oliveira, J.H.G., 2004. Comparative morphology and anatomy of the leaf and stem of Peperomia dahlstedtii, Ottonia martiana and Piper diospyrifolium (Piperaceae). Gayana Bot. 6, 6–17.), P. crassinervium (Albiero et al., 2005aAlbiero, A.L.M., Paoll, A.A.S., Souza, L.A., Mourão, K.S.M., 2005a. Morfoanatomia dos órgãos vegetativos de Piper crassinervium H.B. & K. (Piperaceae). Acta Bot. Bras. 19, 305–312.), P. hispidum (Albiero et al., 2006Albiero, A.L.M., Paoli, A.A.S., Souza, L.A., Mourão, K.S.M., 2006. Morfoanatomia dos órgãos vegetativos de Piper hispidum Sw (Piperaceae). Rev. Bras. Farmacogn 16, 379–391.), P. regnelli (Pessini et al., 2003Pessini, G.L., Holetz, F.B., Sanches, N.R., Cortez, D.A.G., Filho, B.D., Nakamura, C.V., 2003. Avaliação da atividade antibacteriana e antifúngica de extratos de plantas utilizados na medicina popular. Rev. Bras. Farmacogn. 13, 21–24.) and, P. mikanianum (Duarte and Siebenrock, 2010Duarte, M.R., Siebenrock, M.C.N., 2010. Caracteres Anatômicos de Folha e Caule de Piper mikanianum (Kunth) Steud., Piperaceae. Lat. Am. J. Pharm. 29, 45–51.) had a variable number of vascular bundles in the form of an open arc. A microchemical test using phloroglucin exposes lignin in fibers and in xylem as seen in Fig. 5B.

The petiole is short and is irregular in shape. It is also adaxially grooved (Fig. 3A and C). The epidermis has characteristics similar to those reported for the leaf blade (Fig. 3B and C). Multicellular uniseriate non-glandular trichomes are also present in the petiole (Fig. 3B). This has about eleven vascular bundles of varying sizes, arranged in a circle that is opposite the discontinuous angular collenchyma, which consists of 10–12 layers of cells (Fig. 3A and C). Although in some Piper species, the pattern of the petiole is quite similar to that found in this study. It was observed that the number of vascular bundles is not fixed but varies (Souza et al., 2004Souza, L.A., Moscheta, I.S., Oliveira, J.H.G., 2004. Comparative morphology and anatomy of the leaf and stem of Peperomia dahlstedtii, Ottonia martiana and Piper diospyrifolium (Piperaceae). Gayana Bot. 6, 6–17.; Albiero et al., 2005aAlbiero, A.L.M., Paoll, A.A.S., Souza, L.A., Mourão, K.S.M., 2005a. Morfoanatomia dos órgãos vegetativos de Piper crassinervium H.B. & K. (Piperaceae). Acta Bot. Bras. 19, 305–312.; Souza et al., 2009Souza, L.A., Albiero, A.L.M., Almeida, O.J.G., Lopes, W.A.L., Mourão, K.S.M., Moscheta, I.S., 2009. Estudo morfo-anatômico da folha e do caule de Piper arboreum Aubl (Piperaceae). Lat. Am. J. Pharm. 28, 103–107.; Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.).

The petiole contains several prismatic crystals of calcium oxalate as in (Fig. 3D). Plants contain Calcium oxalate crystals in a variety of shapes and sizes. They are usually described as being prismatics, raphides, styloids, crystal sands and druses. They are responsible for some of the very essential roles such as removal of excess Ca2+, detoxification of heavy metals and provide mechanical protection (Franceschi and Horner-Junior, 1980Franceschi, V.R., Horner-Junior, H.T., 1980. Calcium oxalate crystals in plants. Bot. Rev. 46, 361–427.; Nakata, 2003Nakata, P.A., 2003. Advances in our understanding of calcium oxalate crystal formation and function in plants. Plant Sci. 164, 901–909.).

In an incipient secondary structure, the stem is circular in shape (Fig. 4A). The epidermis is uniseriate and covered by a moderately thick cuticle (Fig. 4B) that reacted with Sudan III (Fig. 5D). As already mentioned for the leaf, non-glandular trichomes can be observed (Fig. 4D). There are some layers of angular collenchyma (Fig. 4A, B, and D). The endoderm containing some starch grains observed in Fig. 5E. Piper mikanianum (Duarte and Siebenrock, 2010Duarte, M.R., Siebenrock, M.C.N., 2010. Caracteres Anatômicos de Folha e Caule de Piper mikanianum (Kunth) Steud., Piperaceae. Lat. Am. J. Pharm. 29, 45–51.) and P. diospyrifolium Kunth (Souza et al., 2004Souza, L.A., Moscheta, I.S., Oliveira, J.H.G., 2004. Comparative morphology and anatomy of the leaf and stem of Peperomia dahlstedtii, Ottonia martiana and Piper diospyrifolium (Piperaceae). Gayana Bot. 6, 6–17.) displayed identical characteristics.

Vascular bundles are arranged in two circles within the vascular cylinder. The outer circle shows several vascular bundles and there may be perivascular fiber caps adjoining the phloem (Fig. 4AC). About nive vascular bundles called medullary bundles are located in the inner circle (Fig. 4A and B). The vascular bundles show evidence of intrafascicular cambia (Fig. 4B). In the perimedular region, there is a sinuous sclerenchymatic sheath formed by several layers of lignified cells (Fig. 4AC). These cells were seen to react with the phloroglucin (Fig. 5C) during the microchemical test. The pith occupies much of the stem's volume and is composed of parenchyma cells. Here starch grains can be found (Fig. 4E and F). Oily cells and prismatic crystals of calcium oxalate are also found in the stem.

The arrangement of vascular bundles of P. amalago is in agreement with the description by Cronquist (1981)Cronquist, A., 1981. An Integrated System of Classification of Flowering Plants. Columbia University Press, New York.. Cronquist (1981)Cronquist, A., 1981. An Integrated System of Classification of Flowering Plants. Columbia University Press, New York., in his research found that, as a result of the growth exchange-rate, the outer circle of bundles gives rise to solid cylinders of phloem and xylem, while in the outer circle, the bundles remain individualized.

The arrangement of two concentric circles bundles separated by a sclerenchymatous ring is also typical of Piper, and was observed in P. nigrum (L.), P. colubrinum Link (Ravindran and Remarshree, 1998Ravindran, P.N., Remarshree, A.B., 1998. Anatomy of Piper colubrinum link. J. Spices Aromat. Crops 7, 11–123.), P. hispidum (Albiero et al., 2006Albiero, A.L.M., Paoli, A.A.S., Souza, L.A., Mourão, K.S.M., 2006. Morfoanatomia dos órgãos vegetativos de Piper hispidum Sw (Piperaceae). Rev. Bras. Farmacogn 16, 379–391.), P. gaudichaudianum (Albiero et al., 2005bAlbiero, A.L.M., Souza, L.A., Mourão, K.S.M., Almeida, O.J.G., Lopes, W.A.L., 2005b. Morfo-anatomia do caule e da folha de Piper gaudichaudianum Kuntze (Piper-aceae). Acta Farm. Bom 24, 550–554.), P. crassinervium (Albiero et al., 2005aAlbiero, A.L.M., Paoll, A.A.S., Souza, L.A., Mourão, K.S.M., 2005a. Morfoanatomia dos órgãos vegetativos de Piper crassinervium H.B. & K. (Piperaceae). Acta Bot. Bras. 19, 305–312.), P. diospyrifolium (Souza et al., 2004Souza, L.A., Moscheta, I.S., Oliveira, J.H.G., 2004. Comparative morphology and anatomy of the leaf and stem of Peperomia dahlstedtii, Ottonia martiana and Piper diospyrifolium (Piperaceae). Gayana Bot. 6, 6–17.), P. regnelli (Pessini et al., 2003Pessini, G.L., Holetz, F.B., Sanches, N.R., Cortez, D.A.G., Filho, B.D., Nakamura, C.V., 2003. Avaliação da atividade antibacteriana e antifúngica de extratos de plantas utilizados na medicina popular. Rev. Bras. Farmacogn. 13, 21–24.), P. arboreum (Souza et al., 2009Souza, L.A., Albiero, A.L.M., Almeida, O.J.G., Lopes, W.A.L., Mourão, K.S.M., Moscheta, I.S., 2009. Estudo morfo-anatômico da folha e do caule de Piper arboreum Aubl (Piperaceae). Lat. Am. J. Pharm. 28, 103–107.), P. mikanianum (Duarte and Siebenrock, 2010Duarte, M.R., Siebenrock, M.C.N., 2010. Caracteres Anatômicos de Folha e Caule de Piper mikanianum (Kunth) Steud., Piperaceae. Lat. Am. J. Pharm. 29, 45–51.) and P. sarmentosum (Raman et al., 2012Raman, V., Galal, A.M., Khan, I.A., 2012. An Investigation of the vegetative anatomy of piper sarmentosum and a comparison with the anatomy of Piper betle (Piperaceae). Am. J. Plant Sei. 3, 1135–1144.).

In most species of Piper, the crystals and oily cells, starch grains and phenolic compounds are located in the mesophyll, fundamental parenchyma of the midrib, petiole and, stem (Gogosz et al., 2012Gogosz, A.M., Boeger, M.R.T., Negrelle, R.R.B., Bergom, C., 2012. Anatomia foliar comparativa de nove espécies do gênero Piper (Piperaceae). Rodriguésia 63, 405–417.). According to Fahn (1988)Fahn, A., 1988. Secretory tissues in vascular plants. New Phytologis. 108, 229–257., these elements are common in Piperaceae.

Smith (2010)Smith, A.M., 2010. Starch and starch granules. Gen. Introductory Life Sci., http://dx.doi.org/10.1002/9780470015902.a0001294.pub2.
http://dx.doi.org/10.1002/9780470015902....
suggests that most plants accumulate carbon in form of starch and there is a huge difference in granule size and shape between the different organs in a plant and between species. On the other hand, the phenolic compounds found in these plants are particularly meant for protection and as antioxidants. They are responsible for adaptation and resistance to adverse environmental factors. They give plants a high resistance to microorganisms (Rocha et al., 2011Rocha, W.S., Lopes, R.M., Silva, D.B., Vieira, R.F., Silva, J.P., Agostini-Costa, T.S., 2011. Compostos fenólicos totais e taninos condensados em frutas nativas do cerrado. Rev. Bras. Frutic. 33, 1215–1221.).

Conclusion

As expected, the leaf and stem anatomy of Piper amalago were similar to other Piper species. The features observed should be evaluated as a representative of the entire species, even though several structures can be highlighted as distinguishable among the species of the genus. The main characteristics include a hypostomatic leaf, a sub-epidermal layer on the surfaces, sac-like glandular trichomes, conical non-glandular trichomes, dorsiventral mesophyll, a plano-convex midrib with a single collateral vascular bundle, short petiole with irregularly shaped and adaxially grooved. At the caulinar level that was analyzed, the shape is circular and there are collateral vascular bundles arranged in two rings (the outer one in the vascular cylinder and the inner one in the pith). In the perimedular region, a sinuous sclerenchymatic sheath is apparent.

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Publication Dates

  • Publication in this collection
    Mar-Apr 2015

History

  • Received
    27 May 2014
  • Accepted
    02 Mar 2015
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