Acessibilidade / Reportar erro

Comparative study of Passiflora taxa leaves: I. A morpho-anatomic profile

Abstract

Determining the authenticity and quality of plant raw materials used in the formulation of herbal medicines, teas and cosmetics is essential to ensure their safety and efficacy for clinical use. Some Passiflora species are officially recognized in the pharmaceutical compendia of various countries and have therapeutic uses, particularly as sedatives and anxiolytics. However, the large number of Passiflora species, coupled with the fact that most species are popularly known as passion fruit, increases the misidentification problem. The purpose of this study is to make a pharmacognostic comparison between various Passiflora species to establish a morpho-anatomical profile that could contribute to the quality control of herbal drug products that contain passion fruit. This was conducted by collecting samples of leaves from twelve Passiflora taxa (ten species and two forms of P. edulis): P. actinia, P. alata, P. amethystina, P. capsularis, P. cincinnata, P. edulisf. flavicarpa, P. edulis f. edulis, P. incarnata, P. morifolia, P. urnifolia, P. coccinea and P. setacea, from different locations and their morpho-anatomical features were analyzed using optical microscopy and scanning electron microscopy. Microscopic analysis allowed to indicate a set of characters that can help to differentiate species. These include midrib and petiole shape, midrib and petiole vascular pattern, medium vein shape, presence of trichomes, presence of blade epidermal papillae and sclerenchymatic cells adjoining the vascular bundles. These characters could be used to assist in the determination of herbal drug quality and authenticity derived from a species of Passiflora.

Keywords
Passiflora ; Morpho-anatomy; Passion fruit; Pharmacobotanical study; Quality control

Introduction

The origin of the term pharmacognosy (pharmakon: drug and gnosis: knowledge) clarifies the discipline of knowledge about drugs. According to the World Health Organization (WHO, 2000WHO, 2000. General Guidelines for Methodologies on Research and Evaluation of Tradicional Medicine. World Health Organization, 74 pp. http://whqlibdoc.who.int/hq/2000/WHO_EDM_TRM_2000.1.pdf?ua=1
http://whqlibdoc.who.int/hq/2000/WHO_EDM...
), a drug is a medicinal plant, or parts thereof, after collection processes, stabilization and drying, in its full form, fragmented or powdered. Considering that medicinal plants are chemically and microbiologically stable, they are acquired by phytotherapic industries as herbal drugs, which make their authentication difficult.

The use of Passiflora species as medicinal plants began in the seventeenth century in Europe due to their sedative property (Hoehne, 1939Hoehne, F.C., 1939. Plantas e substâncias vegetais tóxicas e medicinais: coletânea de 114 aulas primeiramente publicadas em O Estado de S. Paulo de 1934-38. Departamento de Botânica do Estado, São Paulo.). Currently, Passiflora species are widely used in folk medicine in many countries, largely as sedatives and anxiolytics (Conrado et al., 2003Conrado, D.J., Fronza, T., Paiva, R.M., Dresch, A.P., Geremias, D., Fenner, R., Viana, A.F., Rates, S.M.K., 2003. Aspectos químicos, farmacológicos e emprego terapêutico do gênero Passiflora (Maracujá). Rev. Afargs 15, 14-19.).

P. incarnata L. is included in the official pharmaceutical codes of several countries (Gosmann et al., 2011Gosmann, G., Provensi, G., Comunello, L.N., Rates, S.M.K., 2011. Composição química e aspectos farmacológicos de espécies de Passiflora L. (Passifloraceae). Rev. Bras. Bioci. 9, 88-99.) and is the most widely studied in this aspect. Consequently, in Brazil, most herbal medicines were prepared from this species. However, P. incarnata is native to the North America and cannot adapt to the Brazilian climate. For this reason, the Brazilian Pharmacopoeia 5th edition (Farmacopeia, 2010Farmacopeia Brasileira, 2010. 5th ed. Agência Nacional de Vigilância Sanitária, Brasília.) chose to make P. alataCurtis and P. edulis Sims. the constituents of herbal medicines, with one of either species in its constitution.

These data, coupled with the large number of Passiflora species (approximately 600) and the same common name (passion fruit) given to several species, have increased the probability of mistakes in species identification, or even in the adulteration of herbal drugs. In order to minimize these problems, pharmacobotanical analysis can be used. This technique is used to identify morphological and anatomical characters for differentiation between similar species. Freitas (1985)Freitas, P.C.D., Dissertação de Mestrado, 1985. Estudo farmacognóstico comparativo de espécies brasileiras do gênero Passiflora L. São Paulo. Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, 133 pp. employed pharmacobotanical analyses on a comparative study of four Passiflora species: P. alata, P. edulis, P. incarnata and P. quadrangularis, and identified some distinguishing features such as the presence, location and type of trichomes.

Considering the morphological similarities between different Passiflora species, the aim of this research was to examine, morpho-anatomically, the leaves from twelve Passiflora taxa. The revealed pharmacobotanical features can be used to identify these medicinal plants, as well as differentiate between Passiflora species, contributing to the quality control of herbal products containing passion fruit.

Material and methods

Botanical materials

Plant material (32 samples from twelve taxa) was collected from different areas of Brazil, specifically from the South (Paraná, Santa Catarina and Rio Grande do Sul), Distrito Federal and Rio Grande do Norte, in 2013 and 2014. The plant material was used to make voucher specimens that were identified by the taxonomist Daniela Cristina Imig, and a representative sample was stored in the Herbarium listed in Box 1.

Box 1
Data collection, vouchers and Herbarium of deposited Passiflora species.

Morpho-anatomical study

Passiflora taxa plant material was collected 15 cm from the apex of the plant. The leaves were fixed in FAA 70 solution (Johansen, 1940Johansen, D.A., 1940. Plant Microtechnique. Mc Graw Hill Book, New York.) and stored in 70% ethanol (Berlyn and Miksche, 1976Berlyn, G.P., Miksche, J.P., 1976. Botanical Microtechnique and Cytochemistry. Iowa State University, Ames.). The remaining leaves were dried at 50 °C and ground in an analytical mill (Quimis®G298A21). The dried, ground samples were standardized by size to ≤300 µm. The obtained powder was analyzed by light microscopy.

For permanent slides, samples were taken from the middle region of the third leaf from the apex. Technical embedding in historesin (Leica Microsystems®) was used by employing the previously determined materials. The material was trans-sectioned in the transverse plane in a rotary microtome, which produced 5 µm sections. Toluidine blue was used for staining (Feder and O’Brien, 1968Feder, N., O’Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot. 55, 123-142.).

For the frontal view of the epidermis, the leaves were subjected to clearing technique (Kraus and Arduin, 1997Kraus, J.E., Arduin, M., 1997. Manual básico de métodos em morfologia vegetal. EDUR, Rio de Janeiro.). The samples were stained with safranin and the slides were set with glycerinated gelatine (Kaiser, 1880Kaiser, E., 1880. Verfahren zur Herstellung einer tadellosen Glycerin-Gelatine. Bot. Zentral. 1880, 25-26.). Semi-permanent slides were also prepared for drug characterization in powder using chloral hydrate.

For the analysis of scanning electron microscopy (SEM), the samples were fixed in FAA 70, dehydrated in a graded ethanol series and CO2 critical point drying apparatus (Bal-Tec CPD-030), coated with gold (Balzers SCD-030) and examined using a Jeol JSM-6360LV microscope. This procedure was performed at the Electron Microscopy Center at the Federal University of Paraná (UFPR). For the morpho-anatomical description, we followed the procedure of Radford et al. (1974)Radford, A.E., Dickison, W.C., Massey, J.R., Bell, C.R., 1974. Vascular Plant Systematics. Harper & Row Publishers, New York..

Results

Morphological analysis

Morphological analysis of Passiflora leaves shows important characteristics for differentiating between the taxa (Fig. 1 and Box 2). The leaves are simple and the blade shape ranges from entire to 2-, 3-, 5-lobed or 5-partite. The leaf margin characteristics may be serrate or crenulate. The apex ranges from acute, obtuse, to acuminate. The bases are rounded, cordate, subcordate or attenuate. The dimensions of the leaf blade and petiole vary significantly between the taxa. The presence and number of glands on the petiole also vary considerably.

Fig. 1
Passiflora spp. – Morphological aspect of leaves – Adaxial side. a. P. actinia; b. P. alata; c. P. amethystina; d. P. capsularis; e. P. cincinnata; f. P. edulis f. flavicarpa; g. P. edulis f. edulis; h. P. incarnata; i. P. morifolia; j. P. urnifolia; k. P. coccinea; l. P. setacea. Ps.: the images were obtained after fixation with FAA. Bars = 2 cm.
Box 2
Morphological characteristics of Passiflora species leaves.

Anatomical analysis

Epidermis

The surface view of the blade shows different epidermal cell anticlinal walls as observed in Box 3 and in Figs. 2 and 3 for the adaxial and abaxial side, respectively.

Box 3
Epidermal cell anticlinal walls on adaxial (AD) and abaxial (AB) sides of Passiflora species.
Fig. 2
Passiflora spp. – View of the leaf surface (epidermis) – Adaxial side. a. P. actinia; b. P. alata; c. P. amethystina; d. P. capsularis; e. P. cincinnata; f. P. edulis f. flavicarpa; g1. P. edulis f. edulis sample 7. 1; g2. P. edulis f. edulis sample 7.2; h. P. incarnata; i. P. morifolia; j. P. urnifolia; k. P. coccinea;l. P. setacea. st: stomata. Bars = 20 µm.
Fig. 3
Passiflora spp. – View of the leaf surface (epidermis) – Abaxial side. a. P. actinia; b. P. alata; c. P. amethystina; d. P. capsularis; e. P. cincinnata; f. P. edulis f. flavicarpa; g1. P. edulis f. edulis sample 7.1; g2. P. edulis f. edulis sample 7.2; h. P. incarnata; i. P. morifolia; j. P. urnifolia; k. P. coccinea;l. P. setacea. st: stomata; arrows indicate papillae. Bars = 20 µm.

Regarding the presence of stomata, only P. alata and P. incarnata have amphistomatic leaves, although a few stomata can be observed on the adaxial side (Figs. 2b and 2h). All other taxa have hypostomatic leaves (Figs. 2 and 3). The stomata are located at the same level of other epidermal cells in P. actinia, P. alata, P. cincinnata and P. coccinea; at the same level or in a slight depression in P. amethystina, P. morifolia and P. urnifolia; at the same level or slightly above in P. capsularis, P. edulis f. flavicarpa, P. edulis f. edulis and P. setacea, and slightly above in P. incarnata.

P. actinia shows anomocytic stomata (Fig. 3a). P. alata, P. amethystina, P. capsularis, P. cincinnata, P. edulis f. flavicarpa, P. edulis f. edulis and on the abaxial side of P. incarnata shows anomocytic stomata (Fig. 3h), anisocytic stomata (Fig. 3c) and paracytic stomata (Fig. 3f). Anisocytic stomata can be observed on the adaxial side of P. incarnata. Although P. morifolia and P. urnifolia show anomocytic stomata (Fig. 3i, j), a few anisocytic stomata can also be observed. In P. coccinea, anomocytic (Fig. 3k), anisocytic and paracytic stomata are observed but anisocytic stomata are rare. In P. setacea (Fig. 3l), all three types are observed but paracytic stomata are uncommon.

P. actinia (Fig. 4a, b), P. alata(Fig. 4c, d) and P. amethystina (Fig. 4e, f) are glabrous on both sides of the blade. Other taxa show non-glandular trichomes (Fig. 5). P. cincinnata has evidence of non-glandular trichomes (Fig. 5b) only on the adaxial side. P. capsularis (Fig. 5a), P. edulis f. flavicarpa(Fig. 5c), P. edulis f. edulis (Fig. 5d, e), P. incarnata (Fig. 5f), P. morifolia (Fig. 5g), P. urnifolia (Fig. 5h), P. coccinea(Fig. 5i) and P. setacea (Figs. 4g, h and 5j) are pubescent on both sides.

Fig. 4
Passiflora spp. Cross-sections of midrib and petiole of glabrous species P. actinia, a–b; P. alata, c–d; P. amethystina, e–f; and densely pubescent P. setacea, g–h. Bars (µm) = 100 (a), 200 (b, c, e–g), 500 (d, h).
Fig. 5
Passiflora spp. – Non-glandular trichomes. a. P. capsularis; b. P. cincinnata; c. P. edulis f. flavicarpa; d. P. edulis f. edulis sample 7.1; e. P. edulis f. edulis sample 7.2; f. P. incarnata; g. P. morifolia; h. P. urnifolia; i. P. coccinea; j. P. setacea. t1: trichome type 1; t2: trichome type 2; arrow indicate cells organized in a rosette. Bars (µm) = 20 (c, h), 50 (a, b, d, e, g, i), 100 (f), 200 (j).

P. capsularis presents two types of non-glandular trichomes distributed throughout the leaf blade; however, there are more trichomes on the abaxial side. The first type is conical, straight (Fig. 5a, right side) and commonly bicellular, but it can be unicellular or multicellular; the base cell is wider and shows a region in half sphere; the cuticle is striate. The second type is cylindrical (Fig. 5a, left side) and unicellular; the cell wall is thin and the apex is rounded; the width is constant and covered by a striate cuticle; they are smaller than the other trichome previously described.

P. cincinnata shows conical, slightly curved (Fig. 5b) and unicellular non-glandular trichomes. They are short, the cell wall is thickened and the cuticle is striate. These trichomes are located only on the larger veins of the adaxial side (Fig. 5b).

In P. edulis f. flavicarpa, a few non-glandular trichomes can be seen on both sides of the midrib. They are conical and straight (Fig. 5c), short or medium in length and the cuticle is striate.

P. edulis f. edulis (sample 7.1) has cylindrical (Fig. 5d) and unicellular or bicellular non-glandular trichomes with a acute apex. These trichomes are encountered on both sides, particularly only on the larger vein on the adaxial side and more uniformly scattered on the abaxial surface. In sample 7.2, conical non-glandular trichomes (Fig. 5e) were encountered, positioned on the veins on both sides.

P. incarnata presents cylindrical, slightly curved, non-glandular trichomes (Fig. 5f) on both leaf sides. On the adaxial face they are unicellular or bicellular, wide and with a thick cell wall. They have different lengths and a tapering apical cell. Similar trichomes can be observed on the abaxial side, but they are multicellular and longer. On both leaf sides, these trichomes are observed mainly on the larger vein.

P. morifolia presents two types of non-glandular trichomes spread uniformly on the leaf blade. The first is unicellular and uncinated (Fig. 5g). It has a thick cell wall and a striate cuticle. The second type is unicellular and cylindrical with a rounded apex (Fig. 5g). It has a thin cell wall and a striate cuticle. The uncinated, non-glandular trichome is longer than the cylindrical trichome and on the abaxial side, the cylindrical type is less common.

P. urnifolia presents cylindrical, non-glandular trichomes (Fig. 5h) with a rounded apex, thick cell wall and a striate cuticle. They are distributed throughout the leaf blade although more frequent on the larger vein.

P. coccinea presents cylindrical (Fig. 5i) and unicellular, bicellular or multicellular non-glandular trichomes. They show a tapering apex, wide cell wall and the base cell has a region in half sphere. These trichomes are distributed uniformly on the leaf blade, more frequently on the abaxial side.

P. setacea presents uniserate, filiform, non-glandular trichomes. They are bicellular, formed by a base cell with a region in half sphere, or multicellular, formed by ten or more cells. On the adaxial side, these trichomes are only dispersed on the larger vein. On the abaxial side, they are more homogeneously distributed on the leaf blade (Fig. 5j).

The adjacent epidermal cells of the trichomes may be similar to other cells or grouped in a rosette. They vary according to taxa, trichome type or blade side. In P. capsularis, the basal portion of the trichome is surrounded by cells organized in a rosette or arranged slightly above the other epidermal cells (Fig. 5a). In P. incarnata, P. morifolia, P. urnifolia (Fig. 5h) and P. coccinea, the basal portion of the trichome is surrounded by cells organized in a rosette on both sides. In P. setacea, the adjacent cells are arranged slightly above the other epidermal cells surrounding the trichomes on the adaxial side. In P. edulisf. edulis (sample 7.1), the adjacent epidermal cells of the trichomes are similar to the other cells.

Cross section of leaf blade

In cross-section, all taxa show a uniserate epidermis covered with a thin, smooth cuticle (Fig. 6). P. actinia shows papillae distributed on the abaxial side (Fig. 6a) and P. coccinea and P. setacea show some papillae on a minor vein on the abaxial side (Fig. 6k and l).

Fig. 6
Passiflora spp. – Leaf blade in cross-section at medium vein. a. P. actinia; b. P. alata; c. P. amethystina; d. P. capsularis; e. P. cincinnata; f. P. edulis f. flavicarpa; g. P. edulis f. edulis; h. P. incarnata; i. P. morifolia; j. P. urnifolia; k. P. coccinea; l. P. setacea. ep: epidermis; pp: palisade parenchyma; sp: spongy parenchyma; st: stomata; ps: parenchymatic sheath; ec: sclerenchyma; dr: druse; pa: papillae. Bars (µm) = 50 (a, c, d–l), 100 (b).

The mesophyll is dorsiventral and a stratum of palisade parenchyma appears in all taxa (Fig. 6). The layers of spongy parenchyma vary by species; 3–6 for P. amethystina(Fig. 6c), 3–4 for P. capsularis (Fig. 6d), 3–5 for P. morifolia (Fig. 6i) and P. setacea (Fig. 6l), and 7–10 for P. alata (Fig. 6b), for example. The samples of P. capsularis differed in leaf blade thickness. The sample 4.4 presented palisade parenchyma narrower than the rest of the samples of this species (Fig. 7).

Fig. 7
Passiflora capsularis. Leaf blade in cross-section - Thickness differences of the leaf blade. a. sample 4.2 (similar as all other samples of this species); b. sample 4.4. ep: epidermis; pp: palisade parenchyma; sp: spongy parenchyma; dr: druse; vb: vascular bundle. Bars = 50 µm.

Small and medium collateral vascular bundles can be detected immersed in the mesophyll and surrounded by the parenchymatic sheath (Fig. 6). P. actinia, P. alata, P. amethystina, P. capsularis, P. cincinnata, P. edulis f. flavicarpa, P. edulis f. edulis, P. incarnata, P. urnifolia and P. setacea show druses in the parenchymatic sheath, however, P. coccinea presents druses only in the palisade parenchyma. No druses appeared in the leaf blade of P. morifolia.

In P. actinia sclerenchymatic fibers caps are adjoined to the phloem and xylem (Fig. 6a), however, they do not occur in all bundles. P. alata (Fig. 6b) and P. coccinea (Fig. 6k) showed fiber caps adjoined only to the phloem, but they are not found in all bundles. P. urnifolia showed sclerenchymatic cells at different stages of lignification located in the center of the vascular bundle (Fig. 6j). No sclerenchyma appeared in the rest of the taxa.

In some taxa, the medium vein makes projections or depressions on both sides of the leaf blades. A convexity is observed on the abaxial side of P. capsularis (Fig. 6d), P. cincinnata (Fig. 6e), P. incarnata (Fig. 6h), P. morifolia(Fig. 6i), P. urnifolia (Fig. 6j), P. coccinea (Fig. 6k), and on both sides of P. edulis f. edulis (Fig. 6g). Only P. setacea presents a convexity on the abaxial side and a concavity on the adaxial side on the medium vein (Fig. 6l).

Cross section of midrib

In the cross-section, the midrib shape shows a different standard for Passiflora taxa as observed in Fig. 8 and summarized in Box 4.

Box 4
Midrib shape in cross-section of Passifloraspecies.
Fig. 8
Midrib in cross-section. a. P. actinia; b. P. alata; c. P. amethystina; d. P. capsularis; e. P. cincinnata; f. P. edulis f. flavicarpa; g. P. edulis f. edulis; h. P. incarnata; i. P. morifolia; j. P. urnifolia; k. P. coccinea; l. P. setacea. ep: epidermis; co: chollenchyma; ec: sclerenchyma; tr: non-glandular trichome. Bars (µm) = 200 (a, c, d, e, h, i, j), 500 (b, f, g, k, l).

Beneath the epidermis of the adaxial side, the number of angular collenchyma strata vary from 7 to 8 in P. morifolia (Fig. 8i), 7 to 9 in P. cincinnata (Fig. 8e) and P. edulis f. flavicarpa (Fig. 8f), 7 to 14 in P. edulis f. edulis (Fig. 8g) and P. incarnata (Fig. 8h). On the abaxial side, 2–5 angular collenchyma strata are present in all the taxa. Druses are dispersed in the ground parenchyma in all taxa (Fig. 8).

Sclerenchymatic tissue is represented by cells or caps. Perivascular caps adjoin the phloem and free cells near the xylem in P. actinia (Fig. 8a). P. alata shows sclerenchymatic cells adjoining the phloem (Fig. 8b). P. urnifolia shows perivascular caps adjoining the phloem (Fig. 8j). P. coccineashows perivascular caps adjacent to the phloem of the dorsal sheath bundle (Fig. 8k).

The vascular system is represented by collateral vascular bundles; midrib vascular patterns are summarized in Box 5.

Box 5
Midrib vascular patterns of Passifloraspecies.

Cross section of petiole

The petiole of the Passiflora taxa has different shapes in cross-section. They vary from flat-convex to concave-convex, as observed in Fig. 9 and summarized in Box 6.

Fig. 9
Petiole in cross-section. a. P. actinia; b. P. alata; c. P. amethystina; d. P. capsularis; e. P. cincinnata; f. P. edulis f. flavicarpa; g. P. edulis f. edulis; h. P. incarnata; i. P. morifolia; j. P. urnifolia; k. P. coccinea; l. P. setacea. ep: epidermis; co: chollenchyma; ph: phloem; xy: xylem; tr: trichome. Bars (µm) = 200 (a, c), 500 (b, d–l).
Box 6
Petiole shape of Passiflora species.

The epidermis shows the same characteristics as described for the leaf blade. Trichomes can be observed in all taxa, except in P. actinia, P. alata and P. amethystina.

The vascular system is represented by collateral vascular bundles and the petiole vascular patterns are summarized in Box 7. Beneath the epidermis, 2–4 continuous angular collenchyma strata and druses are present scattered in the ground parenchyma (Fig. 9).

Box 7
Petiole vascular patterns of Passifloraspecies.

The main anatomical characteristics are summarized in Box 8.

Box 8
Anatomical features of differentiation between Passiflora species.

Microscopic analysis of powder

The microscopic analysis of powder identified some characteristics previously described in the anatomical analyses (Fig. 10).

Fig. 10
Microscopic analysis of powder. a. P. actinia, papillae in frontal view of epidermis abaxial side; b. P. amethystina, sinuous anticlinal epidermal cell wall; c. P. edulis f. edulis, epidermis and angular chollenchyma; d. P. morifolia, uncinate non-glandular trichome; e. P. morifolia, cylindrical non-glandular trichome with rounded apex; f. P. urnifolia, cylindrical non-glandular trichome with rounded apex; g. P. setacea, slightly wavy anticlinal epidermal cell wall; h. P. setacea, filiform non-glandular trichome on the vein; i. P. incarnata, cylindrical non-glandular trichome with acute apex. ep: epidermis; st: stomata; co: chollenchyma; tr: non-glandular trichome; arrow: papillae. Bars (µm) = 20 (a, b, e), 50 (c), 100 (d, f, g, i), 200 (h).

Passiflora taxa powder show druses, leaf blade fragments of the uniserate epidermis on both sides, a palisade parenchyma stratum, some strata of spongy parenchyma, collenchyma (Fig. 10c), epidermal cell walls as described for each species (Fig. 10b and g), non-glandular trichomes (Fig. 10df and hi), stomata (Fig. 10b) and vascular tissue fragments.

In non-glabrous taxa, non-glandular trichomes are observed, except for P. edulis f. flavicarpa that has few trichomes. In addition, P. actinia show not only stomata but also numerous papillae on the abaxial side (Fig. 10a).

Discussion

Determining the authenticity and quality of plant raw materials used in the formulation of herbal medicines, teas and cosmetics are essential to ensure its security and efficacy for clinical use. Misidentification and tampering increases the probability of adverse events, such as the hepatotoxicity cases reported by Pittler and Ernst (2003)Pittler, M.H., Ernst, E., 2003. Hepatotoxic events associated with herbal medicinal products. Aliment. Pharm. Therap. 18, 451-471.. Several raw materials are intentionally adulterated by other species that are morphologically similar, cheaper in price, or more readily available (Ma et al., 2002Ma, X.Q., Shi, Q., Duan, J.A., Dong, T.T.X., Tsim, K.W.K., 2002. Chemical analysis of Radix Astragali (Huangqi) in China: a comparison with its adulterants and seasonal variations. J. Agric. Food Chem. 50, 4861-4866.; Zhao et al., 2006Zhao, Z., Hu, Y., Liang, Z., Yuen, J.P., Jiang, Z., Leung, K.S., 2006. Authentication is fundamental for standardization of Chinese medicines. Planta Med. 72, 865-874.; Zhang et al., 2007Zhang, Y.B., Shaw, P.C., Sze, C.W., Wang, Z.T., Tong, Y., 2007. Molecular authentication of Chinese herbal materials. J. Food Drug Anal. 15, 1-9.).

P. edulis has been widely cultivated in Brazil and Moraes et al. (1997)Moraes, M.L.L., Vilegas, J.H.Y., Lanças, F.M., 1997. Supercritical fluid extraction of glycosylated flavonoids from Passifloraleaves. Phytochem. Anal. 8, 257-260. reported that leaves and stems have been used as an adulterant for P. alata, which was included in the first three editions of the Brazilian Pharmacopoeia and was official in the country at that time. Moreover, in Brazil, several Passiflora species are popularly known as ‘maracujá’, which reinforces the importance of identification and differentiating between them (Beraldo and Kato, 2010Beraldo, J., Kato, E.T.M., 2010. Morfoanatomia de folhas e caules de Passiflora edulis Sims, Passifloraceae. Rev. Bras. Farmacogn. 20, 233-239.).

Morphological characteristics are very important for correct species identification. However, several medicinal species are sold fragmented or powdered. Thus, the anatomical features can greatly contribute, plus to morphology, by indicating the characteristics for botanical identification.

In this research, similar morpho-anatomical characteristics were encountered in all Passiflora taxa, such as dorsiventral mesophyll, collateral vascular bundles and the presence of druses. However, the following structures might be emphasized as useful to distinguish species: midrib and petiole shape in cross-section, midrib and petiole vascular patterns, outline of medium veins in cross-section, sclerenchymatic cells adjoining the vascular bundle, presence and type of non-glandular trichomes and presence of papillae in the epidermis of the leaf blade.

The importance of standardizing medicinal plant cultivation is highlighted by a difference found between the samples of P. capsularis. The mesophyll thickness of sample 4.4 was narrower than the one of other samples, due to environmental influences. The sample 4.4 was collected from an Atlantic Forest where it had grown in the shadow of trees. The other samples were collected from an open space with direct sun exposure. Pires (2008)Pires, M.V., (Dissertação de Mestrado) 2008. Respostas morfo-fisiológicas de espécies ornamentais de Passiflora ao sombreamento. Ilhéus. Programa de Pós-Graduação em Produção Vegetal, Universidade Estadual de Santa Cruz, 99 pp.studied the behavior of some Passiflora species in relation to shading and found that P. morifolia, growing in full sun with 25% of shade, showed a thicker mesophyll formed by longer palisade cells and bigger intercellular spaces when compared with others growing in 50–75% of shade.

Crochemore et al. (2003)Crochemore, M.L., Molinari, H.B., Stenzel, N.M.C., 2003. Caracterização agromorfológica do maracujazeiro (Passifloraspp). Rev. Bras. Frutic. 25, 5-10. studied the diversity of Passiflora species in relation to morphological characteristics. They reported a great variability between the analyzed samples of P. edulis f. edulis in coincidence with the morpho-anatomical variability found in this study for the same form. Amorim et al. (2014)Amorim, J.S., Souza, M.M., Viana, A.J.C., Corrêa, R.X., Araújo, I.S., Ahnert, D., 2014. Cytogenetic, molecular and morphological characterization of Passiflora capsularis L. and Passiflora rubra L. Plant Syst. Evol. 300, 1147-1162. interpreted that the intra and interspecific variability within the Passiflora genus was associated with an ability to adapt to different environments. In addition, Viana et al. (2003)Viana, A.P., Pereira, T.N.S., Pereira, M.G., Souza, M.M., Maldonado, J.F.M., do Amaral Júnior, A.T., 2003. Diversidade genética entre genótipos comerciais de maracujazeiro amarelo (Passiflora edulis f. flavicarpa) e entre espécies de passifloras nativas determinada por marcadores RAPD. Rev. Bras. Frutic. 25, 489-493. suggested that another reason was the easiness of Passiflora species sexual reproduction.

It is important to highlight the variability found in this study in P. edulis f. flavicarpa and P. edulis f. edulis, especially regarding to the presence and type of trichomes. However, the literature discusses this topic only at the species level and not at the form level. Besides, the reports are inconsistents (Pereira et al., 2009Pereira, C.S., Kurita, H., Veja, R., Jiménez, M., Molinas, C., Benítez, F., 2009. Evaluación de la morfo-anatomía foliar de Passiflora alata Curtis y Passiflora edulis Sims. Steviana 1, 38-45.; Beraldo and Kato, 2010Beraldo, J., Kato, E.T.M., 2010. Morfoanatomia de folhas e caules de Passiflora edulis Sims, Passifloraceae. Rev. Bras. Farmacogn. 20, 233-239.; Farmacopeia, 2010Farmacopeia Brasileira, 2010. 5th ed. Agência Nacional de Vigilância Sanitária, Brasília.; Barbosa et al., 2013Barbosa, N.C.S., Barbosa, C.J., Leite, K.R.B., da Silva, L.B., 2013. Anatomia das superfícies foliares de espécies de Passiflora(Passifloraceae) como subsídio para estudos agronômicos. 64 (Congresso Nacional de Botânica. Belo Horizonte, Brasil).; Leite et al., 2013Leite, S.S.M.S., França, E.S., Coffani-Nunes, J.V., 2013. Morfoanatomia de folha e pecíolo e sua aplicação taxonômica em Passiflora (Passifloraceae). Congresso Nacional de Botânica. Belo Horizonte, Brasil, 64 pp.; Farias, 2014Farias, V., (Dissertação de mestrado) 2014. Anatomia foliar de Passiflora L. (Passifloraceae): aspectos taxonômicos e evolutivos. Curitiba. Programa de pós-graduação em Botânica, Universidade Federal do Paraná, 63 pp.; Cervi, 1997Cervi, A.C., 1997. Passifloraceae do Brasil. Estudo do gênero Passiflora L., subgênero Passiflora. Fontqueria 45, 1-92.).

In this study, only P. alata and P. incarnatashowed amphistomatic leaves. However, they are considered as hypostomatic by Farmacopeia (2010)Farmacopeia Brasileira, 2010. 5th ed. Agência Nacional de Vigilância Sanitária, Brasília., Pereira et al. (2009)Pereira, C.S., Kurita, H., Veja, R., Jiménez, M., Molinas, C., Benítez, F., 2009. Evaluación de la morfo-anatomía foliar de Passiflora alata Curtis y Passiflora edulis Sims. Steviana 1, 38-45., Farias (2014)Farias, V., (Dissertação de mestrado) 2014. Anatomia foliar de Passiflora L. (Passifloraceae): aspectos taxonômicos e evolutivos. Curitiba. Programa de pós-graduação em Botânica, Universidade Federal do Paraná, 63 pp. and WHO (2007)WHO, 2007. WHO monographs on selected medicinal plants, vol. 3. WHO Pres., Spain.. This probably happened because of the low density of stomata present on the adaxial side for both species.

Conclusion

The morphological characteristics of Passiflora spp. leaves support the differentiation of species; however, when used in herbal drugs, fragmented or powdered, anatomical characteristics provide additional useful data for differentiating them.

Acknowledgments

The authors thank Vera L. P. dos Santos and Celia R. C. Franco for their valuable work on the electronic microscopy slides preparation; an acknowledgment is also due to Dr. Fabio G. Faleiro, Embrapa Cerrados, Planaltina-DF for collecting some Passiflora species. CAMS thanks CNPq for the research schorlarship.

References

  • Amorim, J.S., Souza, M.M., Viana, A.J.C., Corrêa, R.X., Araújo, I.S., Ahnert, D., 2014. Cytogenetic, molecular and morphological characterization of Passiflora capsularis L. and Passiflora rubra L. Plant Syst. Evol. 300, 1147-1162.
  • Barbosa, N.C.S., Barbosa, C.J., Leite, K.R.B., da Silva, L.B., 2013. Anatomia das superfícies foliares de espécies de Passiflora(Passifloraceae) como subsídio para estudos agronômicos. 64 (Congresso Nacional de Botânica. Belo Horizonte, Brasil).
  • Beraldo, J., Kato, E.T.M., 2010. Morfoanatomia de folhas e caules de Passiflora edulis Sims, Passifloraceae. Rev. Bras. Farmacogn. 20, 233-239.
  • Berlyn, G.P., Miksche, J.P., 1976. Botanical Microtechnique and Cytochemistry. Iowa State University, Ames.
  • Cervi, A.C., 1997. Passifloraceae do Brasil. Estudo do gênero Passiflora L., subgênero Passiflora Fontqueria 45, 1-92.
  • Conrado, D.J., Fronza, T., Paiva, R.M., Dresch, A.P., Geremias, D., Fenner, R., Viana, A.F., Rates, S.M.K., 2003. Aspectos químicos, farmacológicos e emprego terapêutico do gênero Passiflora (Maracujá). Rev. Afargs 15, 14-19.
  • Crochemore, M.L., Molinari, H.B., Stenzel, N.M.C., 2003. Caracterização agromorfológica do maracujazeiro (Passifloraspp). Rev. Bras. Frutic. 25, 5-10.
  • Farias, V., (Dissertação de mestrado) 2014. Anatomia foliar de Passiflora L. (Passifloraceae): aspectos taxonômicos e evolutivos. Curitiba. Programa de pós-graduação em Botânica, Universidade Federal do Paraná, 63 pp.
  • Farmacopeia Brasileira, 2010. 5th ed. Agência Nacional de Vigilância Sanitária, Brasília.
  • Feder, N., O’Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot. 55, 123-142.
  • Freitas, P.C.D., Dissertação de Mestrado, 1985. Estudo farmacognóstico comparativo de espécies brasileiras do gênero Passiflora L. São Paulo. Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, 133 pp.
  • Gosmann, G., Provensi, G., Comunello, L.N., Rates, S.M.K., 2011. Composição química e aspectos farmacológicos de espécies de Passiflora L. (Passifloraceae). Rev. Bras. Bioci. 9, 88-99.
  • Hoehne, F.C., 1939. Plantas e substâncias vegetais tóxicas e medicinais: coletânea de 114 aulas primeiramente publicadas em O Estado de S. Paulo de 1934-38. Departamento de Botânica do Estado, São Paulo.
  • Johansen, D.A., 1940. Plant Microtechnique. Mc Graw Hill Book, New York.
  • Kaiser, E., 1880. Verfahren zur Herstellung einer tadellosen Glycerin-Gelatine. Bot. Zentral. 1880, 25-26.
  • Kraus, J.E., Arduin, M., 1997. Manual básico de métodos em morfologia vegetal. EDUR, Rio de Janeiro.
  • Leite, S.S.M.S., França, E.S., Coffani-Nunes, J.V., 2013. Morfoanatomia de folha e pecíolo e sua aplicação taxonômica em Passiflora (Passifloraceae). Congresso Nacional de Botânica. Belo Horizonte, Brasil, 64 pp.
  • Ma, X.Q., Shi, Q., Duan, J.A., Dong, T.T.X., Tsim, K.W.K., 2002. Chemical analysis of Radix Astragali (Huangqi) in China: a comparison with its adulterants and seasonal variations. J. Agric. Food Chem. 50, 4861-4866.
  • Moraes, M.L.L., Vilegas, J.H.Y., Lanças, F.M., 1997. Supercritical fluid extraction of glycosylated flavonoids from Passifloraleaves. Phytochem. Anal. 8, 257-260.
  • Pereira, C.S., Kurita, H., Veja, R., Jiménez, M., Molinas, C., Benítez, F., 2009. Evaluación de la morfo-anatomía foliar de Passiflora alata Curtis y Passiflora edulis Sims. Steviana 1, 38-45.
  • Pires, M.V., (Dissertação de Mestrado) 2008. Respostas morfo-fisiológicas de espécies ornamentais de Passiflora ao sombreamento. Ilhéus. Programa de Pós-Graduação em Produção Vegetal, Universidade Estadual de Santa Cruz, 99 pp.
  • Pittler, M.H., Ernst, E., 2003. Hepatotoxic events associated with herbal medicinal products. Aliment. Pharm. Therap. 18, 451-471.
  • Radford, A.E., Dickison, W.C., Massey, J.R., Bell, C.R., 1974. Vascular Plant Systematics. Harper & Row Publishers, New York.
  • Viana, A.P., Pereira, T.N.S., Pereira, M.G., Souza, M.M., Maldonado, J.F.M., do Amaral Júnior, A.T., 2003. Diversidade genética entre genótipos comerciais de maracujazeiro amarelo (Passiflora edulis f. flavicarpa) e entre espécies de passifloras nativas determinada por marcadores RAPD. Rev. Bras. Frutic. 25, 489-493.
  • WHO, 2000. General Guidelines for Methodologies on Research and Evaluation of Tradicional Medicine. World Health Organization, 74 pp. http://whqlibdoc.who.int/hq/2000/WHO_EDM_TRM_2000.1.pdf?ua=1
    » http://whqlibdoc.who.int/hq/2000/WHO_EDM_TRM_2000.1.pdf?ua=1
  • WHO, 2007. WHO monographs on selected medicinal plants, vol. 3. WHO Pres., Spain.
  • Zhang, Y.B., Shaw, P.C., Sze, C.W., Wang, Z.T., Tong, Y., 2007. Molecular authentication of Chinese herbal materials. J. Food Drug Anal. 15, 1-9.
  • Zhao, Z., Hu, Y., Liang, Z., Yuen, J.P., Jiang, Z., Leung, K.S., 2006. Authentication is fundamental for standardization of Chinese medicines. Planta Med. 72, 865-874.

Publication Dates

  • Publication in this collection
    Jul-Aug 2015

History

  • Received
    05 June 2015
  • Accepted
    15 July 2015
Sociedade Brasileira de Farmacognosia Universidade Federal do Paraná, Laboratório de Farmacognosia, Rua Pref. Lothario Meissner, 632 - Jd. Botânico, 80210-170, Curitiba, PR, Brasil, Tel/FAX (41) 3360-4062 - Curitiba - PR - Brazil
E-mail: revista@sbfgnosia.org.br