MICROSCOPY RESEARCH AND TECHNIQUE 78:1001–1009 (2015) Botanical Features for Identification of Gymnosporia arenicola Dried Leaf GUSTAVO DA SILVA, RITA SERRANO, ELSA TEIXEIRA GOMES, AND OLGA SILVA* Research Institute for Medicines (iMed. ULisboa), Faculty of Pharmacy, Universidade De Lisboa, Av. Prof. Gama Pinto, Lisbon 1649-003, Portugal KEY WORDS Herbal medicines; histochemistry; light microscopy; scanning electron microscopy ABSTRACT Gymnosporia arenicola Jordaan (Celastraceae) is a shrub or small tree, which naturally occurs in coastal sand dunes of Southern Mozambique and South Africa. Its dried leaf is often used in traditional medicine for the treatment of infectious and inflammatory diseases. Hereby, we present results of studies carried out according to the pharmacopoeia standards for the identification of herbal drugs, in the whole, fragmented, and powdered plant material. These results were complemented with scanning electron microscopy and histochemical techniques. The leaf microscopic analysis revealed a typical dorsiventral mesophyll with a corresponding spongy parenchyma–palisade parenchyma ratio of 0.60, anomocytic and paracytic stomata, papillate cells with a diameter of 4.00 6 0.40 mm, multicellular uniseriate nonglandular trichomes with a length of 27.00 6 4.10 mm and cristalliferous idioblasts containing calcium oxalate cluster crystals with a diameter of 23.04 6 5.84 mm. The present findings demonstrate that the G. arenicola leaf has both nonglandular trichomes and hypoderm, features not previously described in the corresponding botanical section (Gymnosporia sect. Buxifoliae Jordaan). The establishment of these new botanical markers for the identification of G. arenicola leaf is essential for quality, safety and efficacy reasons. Microsc. Res. Tech. 78:1001–1009, 2015. V 2015 Wiley Periodicals, Inc. C INTRODUCTION Gymnosporia arenicola Jordaan [syn. Maytenus heterophylla (Eckl. and Zeyh.) N. Robson subsp. arenaria N. Robson] is an African medicinal plant commonly known as ‘Sand spike-thorn’ or ‘Sandpendoring’, as English and Afrikaans vernacular names, respectively (Jordaan and van Wyk, 1999). This botanical species naturally occurs in coastal sand dunes of Southern Mozambique and South Africa, partially integrated in the Maputaland Centre of Endemism (Jordaan and van Wyk, 1999). This region is recognized as center of plant biodiversity and G. arenicola is considered an endangered species, included in the Red List of South African Plants (Raimondo et al., 2009). G. arenicola is often employed in African traditional medicine against infectious and inflammatory diseases (da Silva G et al., 2011a; Hedberg et al., 1982; Hutchings et al., 1996; Jansen and Mendes, 1991; Neuwinger, 2000). Preliminary studies, of our research group, indicate anti-inflammatory activity of the leaf ethanol extract of this species and consequently confirm its traditional use as an herbal medicine (da Silva G et al., 2011b). Results like these are particularly important for the users of African health systems, due to their high dependence of herbal medicines and scarce pharmacological/toxicological investigation on some species, such as G. arenicola. In addition to the scarce pharmacological and toxicological profile of these herbal medicines, there is very limited information to identify and authenticate such species. In the absence of a pharmacopeial monograph for herbal medicines as G. arenicola, C V 2015 WILEY PERIODICALS, INC. the establishment of macro- and microanatomical botanical markers is essential for quality, safety, and efficacy proposes (Serrano et al., 2010). G. arenicola is a shrub, suffrutex, or small tree that belongs to the Celastraceae family. The taxonomical classification of this family was recently changed at the generic level. As currently circumscribed, the former genus Maytenus Mol. emend Mol., even after the reinstatement of Gymnosporia (Wight and Arn.) Hook.f., is still clearly a heterogeneous group of species. This view is also supported by other authors (Jordaan and van Wyk, 2003, 2006). The genus Gymnosporia (after the inclusion of some Maytenus species in this genus) is also a heterogeneous taxon, which requires the division into more natural groups (Jordaan and van Wyk, 2003). The complex phylogenetic relationships between members of the Celastraceae family were previously studied by other authors (Simmons et al., 2001a,b). The anatomy and leaf micromorphology of some species of the Celastraceae family was already characterized, aiming at the selection anatomical characters for the species identification (Duarte and Debur, 2005; *Correspondence to: Olga Silva, Laboratory of Pharmacognosy, Department of Pharmacological Sciences. Faculty of Pharmacy, Universidade de Lisboa. Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal; E-mail: odsilva@ff.ulisboa.pt Received 8 June 2015; accepted in revised form 3 August 2015 REVIEW EDITOR: Prof. Alberto Diaspro Contract grant sponsor: National Research Foundation (FCT) (Portugal); Contract grant number: FCT-PEst-OE/SAU/UI4013/2011. DOI 10.1002/jemt.22565 Published online 25 August 2015 in Wiley Online Library (wileyonlinelibrary.com). 1002 G. DA SILVA ET AL. Gomes, 2001; Jain et al., 2009; Joffily and Vieira, 2010; Lin and Zhang, 2010). Epidermal structure and stomatal ontogeny in some Celastraceae species were already described (Pant and Kidwai, 1965). Various microscopic parameters were used to identify Gymnosporia rothiana (Walp) Lawson, including stomatal number, stomatal index, palisade ratio, vein islet number, and vein termination number (Jain et al., 2009). The present study aims at the botanical characterization of G. arenicola dried leaf, according to the current methods described on the occidental Pharmacopoeias for the quality control and botanical identification of herbal drugs. The methodology used on this study consists of the macroscopic and microscopic analysis of the whole, fragmented and powdered plant material. For deeper botanical characterization, we have complemented the usual stereomicroscopy and light microscopy (LM) analysis by the use of scanning electron microscopy (SEM) and histochemical techniques. MATERIALS AND METHODS Plant Material Gymnosporia arenicola Jordaan was collected in November 2011 by O. Silva in Macaneta Beach (Marraquene District, Mozambique), which is inserted in a Tropical Wet/Dry region with a mean annual temperature comprised between 22 and 248C and a mean annual precipitation of 1200 mm3. The collection of all the plant material was authorized by the responsible local authorities. All the plant material was dried under controlled conditions of humidity (75 6 5%) and temperature (21 6 18C) in the dark. The drying conditions that were used in this study are the usual conditions applied for herbal medicines and consequently the morphological analysis was observed in the dried plant material and not in fresh material. The identification of the plant material was conducted by Adelia Diniz, “Jardim Bot^ anico Tropical—IICT” (Lisbon) by comparison with voucher Herbarium specimen O. Silva, s.n., 1.10.2006 of the LISC Herbarium, Institute for Tropical Scientific Research (Lisbon). Leaf samples (30) were randomly selected from 250 g of the collected raw material (minimum of 50 leaves from 10 different trees) using the samplings rules of the European Pharmacopeia for herbal drugs (EDQM, 2007). Powdered Plant Material A representative portion of the total collected raw material selected to study was powdered using an Analytical Mill A-10 water-cooled laboratory mill (Staufen, Germany) and mounted in a 60% chloral hydrate aqueous solution, according to European Pharmacopoeia (EDQM, 2007). Macroscopic Analysis Each dried leaf sample of G. arenicola was directly observed with the naked eye and using an Olympus SZ61 stereobinocular microscope coupled with an Olympus ColorView IIIu camera (Tokyo, Japan). Image analysis was performed with Cell D 2006 Olympus Software (Tokyo, Japan). Light Microscopy Each dried leaf sample of G. arenicola was previously hydrated in water. For the anatomical analysis, the leaf was sectioned freehand. Lamina transverse sections (midrib and distal part of the blade) and tangential longitudinal sections (leaf surface) were cleared and mounted in a 60% chloral hydrate aqueous solution. Microscopic analysis of the prepared leaf sections and powdered plant material was conducted on an Olympus CX40 upright microscope (Tokyo, Japan), coupled with an Olympus ColorView IIIu camera (Tokyo, Japan). Image analysis was performed with Cell D 2006 Olympus Software (Tokyo, Japan). Scanning Electron Microscopy The dried plant material was sectioned, dehydrated at 358C for 24 h and directly mounted on stubs using double-side adhesive tape. Ten samples were sputtered with a thin layer of gold in a JEOL JSM-1200 Fine Coater and observed in a JEOL JSM-T220 SEM at 15 kV, with a digital image acquisition integrated system (MA). Histochemical Tests The chromatic staining reactions were performed in leaf transverse sections before examination under LM. The histochemical tests were 10% ferric trichloride for detection of o-dihydroxyphenols (Johansen, 1940), Brady’s reagent (2,4-dinitrophenylhydrazine) for terpenoids with carbonyl group (Ganter and Jolles, 1969), Dittmar’s reagent for alkaloids (Furr and Mahlberg, 1981), Sudan red III for total lipids (Bronner, 1975), and Lugol’s solution (I2KI) for starch (Pearse, 1960). All histochemical tests were compared with the respective unstained controls. The results were observed by LM, using an Olympus CX40 upright microscope (Tokyo, Japan), coupled with an Olympus ColorView IIIu camera (Tokyo, Japan). Image analysis was performed with Cell D 2006 Olympus Software (Tokyo, Japan). Statistical Data For the determination of the macroscopic features, the observations were performed in 10 adult leaves (n 5 10), and for the microscopic measurements, 10 replicates were determined in 1 mm2 for each sample, following the pharmacopoeia recommendations (EDQM, 2007). All the macro- and microscopic results were expressed as mean 6 SD, with the exception of the determination of the spongy parenchyma–palisade parenchyma ratio and stomatal indexes. The stomatal index (SI) was determined by the following formula: SI5 100 3 number of stomata number of epidermal cells1number of stomata All the statistical data are summarized in Table 1. RESULTS The leaf of G. arenicola (Fig. 1) is simple and entire, pale green colored, with oblong or ovate to obovate lamina. The apex is round to emarginated and the Microscopy Research and Technique LEAF IDENTIFICATION OF GYMNOSPORIA ARENICOLA 1003 TABLE 1. Quantitative macro- and microscopic main features of G. arenicola leaf Statistical parameters N 5 10 Structure Dimension Mean value Standard deviation Lamina (cm) Length Width Thickness Diameter Length Width Length Width Length Width Length Width Length Width Width Length 5.8 2.6 5.30 23.04 16.24 30.13 33.54 26.63 15.25 32.25 50.30 19.88 33.14 34.83 4.00 27.00 1.0 0.2 0.67 5.84 0.77 2.58 4.53 2.65 1.11 4.83 4.96 0.76 1.91 2.30 0.40 4.10 Cuticle (mm) Crystaliferous idioblasts (mm) Upper epidermis (mm) Hypodermis (mm) Lower epidermis (mm) Palisade parenchyma (mm) Spongy parenchyma (mm) Papillate cells (mm) Trichomes (mm) base is cuneate. The margin is indurate, revolute, entire to irregularly serrulated, and the petiole is short. The leaf surface is apparently glaucous, glabrous, coriaceous, or subcoriaceous. Venation and midrib are more prominent on the lower surface of the blade. LM analysis of transversal sections of G. arenicola leaf (Fig. 2) shows an asymmetric organization with a mesophyll comprising three to five strata of palisade parenchyma (Fig. 2a) and various layers of spongy parenchyma, with a corresponding spongy parenchyma–palisade parenchyma ratio of 0.60. Uniseriate upper (Fig. 2b) and lower epidermises (Figs. 2c–2e) are coated by a smooth and thick cuticle. A clearly differentiated layer of thick-walled hypodermal cells is present below the upper epidermis, sometimes with crystalliferous idioblasts containing calcium oxalate cluster crystals (Fig. 2b). The cells of the palisade tissue are columnar, with their long axes oriented at right angles to the upper epidermis, with straight walls which are distinctly thickened and pitted (Fig. 2b). The cells of the lower epidermis are similar to those of the upper epidermis but they are usually smaller and the striations on the cuticle are sometimes less marked (Fig. 2c). The spongy parenchyma cells are irregular in shape (Fig. 2d) and surface walls of epidermal cells show short papillate cells (Fig. 2e) and occasionally multicellular uniseriate nonglandular trichomes (Fig. 2f), composed of three to four cells. Midrib transversal sections (Fig. 3) show an amphicrival vascular bundle surrounded by a discontinuous sclerenchymatic sheath, collateral with the xylem. Phloem is external to xylem. The xylem is toward the upper epidermis (adaxial surface) and the phloem is toward the lower epidermis (abaxial surface). A developed angular collenchyma is observed near both epidermis and calcium oxalate cluster crystals are frequently present in the phloem. Cristalliferous idioblasts containing with calcium oxalate cluster crystals may be seen in the hypodermis (Figs. 2a and 2b) and chlorenchyma and near the phloem (Figs. 2c and 3). The leaf surface examination (Fig. 4) by light and SEM shows a slightly sinuous cuticle in both Microscopy Research and Technique Fig. 1. G. arenicola leaf upper and lower surface view (top and bottom, respectively). The leaf is petiolated, simple and entire, with oblong or ovate to obovate shape and entire to irregularly serrulated margin. Scale bar: 1 cm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] epidermises (Figs. 4a, 4c, and 4f), a upper epidermis composed of polygonal to rectangular cells with straight walls which are distinctly pitted and some cells show peculiar shinning bodies (Fig. 4b), a random stomata distribution (Fig. 4c), and a lower epidermis with similar cells to those of the upper epidermis but they are usually smaller and the striations on the cuticle are sometimes less clearly marked (Fig. 4d). Anomocytic stomata (Fig. 4d) are surrounded by a ring of four to six subsidiary cells and are more frequent than paracytic stomata (Fig. 4e). The calculated SI is 1.25 on the upper epidermis and 6.36 on the lower epidermis. The powder of G. arenicola dried leaf presents a green color and the microscopic examination of the powdered plant material reveals the presence of the above-mentioned and illustrated structures, eventually with some evidence of physical fragmentation of the plant tissues. The microanalysis of the powder G. arenicola leaf revealed crystalliferous idioblasts with calcium oxalate cluster crystals on hypodermal cells (Fig. 5a), which were also observed on spongy parenchyma (Fig. 5b), and over the veins and insular areas (Fig. 5c). Further microscopic elements, as anomocytic and paracytic stomata, and shinning bodies in the cells of the stomatal region in the lower epidermis (Fig. 5d) were also present in the powdered material. Fragments of the lower epidermis containing short surface papillae (Fig. 5e) and fragments of vascular tissue with sclerenchyma fibers (Fig. 5f) were observed in the G. arenicola leaf powder. 1004 G. DA SILVA ET AL. Fig. 2. LM transverse sections of G. arenicola leaf. a: Cut sections of the blade showing the leaf asymmetric organization, with three layers of palisade parenchyma and 60% of the chlorenchyma corresponding to spongy parenchyma. b: A thick cuticle visible in the upper epidermis, calcium oxalate cluster crystals occurring within idioblasts in the hypodermis (arrowhead) and thickened pitted straight walls occurring in the palisade tissue. c: Calcium oxalate cluster crystals also occurring in spongy parenchyma cells (arrowhead) and a thick cuticle visible in the lower epidermis. d: Spongy tissue with irregular cells occurring near the lower epidermis and some stomata with a sub-stomatal chamber (arrow). e: Papillate cells visible in the lower epidermis (arrowhead). f: Multicelled nonglandular trichome (arrowhead) detail. Scale bars: a 5 100 mm; b– f 5 50 mm. The results of the in situ histochemical localization of alkaloids, o-dihydroxyphenols, terpenoids with carbonyl group, total lipids, and starch are shown in Figure 6. Dark brownish mesophyll showing the presence of o-dihydroxyphenols is revealed by ferric trichloride (Fig. 6b). Brady’s reagent orange staining Microscopy Research and Technique LEAF IDENTIFICATION OF GYMNOSPORIA ARENICOLA Fig. 3. LM and SEM transverse sections of G. arenicola leaf (a and b, respectively), showing details of the midrib structure. Amphicrival bundle, collateral with the xylem, which faces toward the upper surface, surrounded by a discontinuous sclerenchymatic sheath. Collenchyma occurring near both epidermis and also near to the adaxial xylem. Calcium oxalate cluster crystals near the phloem are readily visible in LM (arrowhead). Scale bars: a and b 5 100 mm. allowed the identification of terpenoids containing carbonyl group on the collenchyma (Fig. 6d) and vacuoles of the mesophyll cells (Fig. 6f). Alkaloids were detected by Dittmar’s reagent chromatic reaction, showing brownish-content cells near the midribs and parenchymatous cells (Fig. 6h). Total lipids were identified on epidermal cells and cuticle, showing the typical red coloration of Sudan III staining (Fig. 6j). Blue/violet stained amyloplasts are found in the mesophyll, which were evidenced by Lugol’s reaction. Figures 6a, 6c, 6e, 6g, and 6i show negative the controls for the staining tests, respectively. DISCUSSION The botanical identification is the first official and mandatory step for the authentication of herbal medicines, and the identity of the herbal medicine is further confirmed by the analysis of the respective chemical profile. Even though actually molecular biology techniques are applied for the authentication of plant Microscopy Research and Technique 1005 materials, these techniques are not yet recognized to the identification of herbal medicines in Pharmacopoeias. The leaf macroscopic morphology features of G. arenicola follow the description by Jordaan and van Wyk (1999), which are distinctive from Gymnosporia senegalensis (Lam.) Loes. These distinctive features are important because both medicinal species are apparently similar, co-exist in some geographical regions and can be used for some common ethnopharmaceutical purposes (Serrano et al., 2008). The co-existence of both species in traditional markets may cause the presence of herbal adulterants/contaminants and mislead the correct species identification among the population. Our results revealed that the most distinctive microscopic features in G. arenicola were the presence of a dorsiventral mesophyll, multicellular covering trichomes and papillae, which are not previously detected in G. senegalensis (Serrano et al., 2009). Microscopic analysis of the leaf of G. arenicola revealed the presence of a striated and papillose cuticle. The presence of papillae is useful for diagnostic purposes. The observation of a conspicuous thick cuticle coating the epidermis is a character already reported for the Celastraceae family and for the Gymnosporia genus (Metcalfe and Chalk, 1950). This morphological feature was particularly emphasized on South American species of the Celastraceae family, e.g. Maytenus boaria (Metcalfe and Chalk, 1981), M. ilicifolia (Bernardi and Wasicky, 1959), M. oleoides (Hlwatika et al., 1998), and M. rigida (da Rocha et al., 2004). According to the above-mentioned authors, the cuticle helps to prevent water loss efficiently against uncontrolled evaporation, and avoids collapsing during dehydration processes, which has been considered of taxonomic value and can be used for diagnostic purposes. The presence of papillae is useful for diagnostic purposes and these results are not consistent with the findings of Jordaan and van Wyk (2006) for the Gymnosporia sect. Buxifoliae Jordaan. In the Buxifoliae section, the presence of papillae is not expected, with some exceptions (G. arenicola is not mentioned as an exception). Angular collenchyma is present below the upper and above the lower epidermis, as reported in other species of this botanical genus, e.g., G. rothiana (Jain et al., 2009). As previously mentioned, a clearly differentiated layer of thick-walled hypodermal cells occur below the upper epidermis, in agreement with the observations of Jordaan and van Wyk (2006). Crystalliferous idioblasts containing calcium oxalate cluster crystals often occur on the hypodermal cells, and chlorenchyma. The distribution of these idioblasts follows the observations of Jordaan and van Wyk (2006). According to Metcalfe and other authors, these crystals are common in the Celastraceae family, being mentioned in Elaeodendron, Catha, and Maytenus (Metcalfe and Chalk, 1950; Jain et al., 2009). The presence of calcium oxalate crystals in the Celastraceae family has been registered by numerous authors. Plant crystalliferous idioblasts have been recognized to assume different functions as storing the calcium ion, avoiding the oxalate toxic accumulation, contributing to the mechanical support and protection against herbivory (Franceschi and Horner Jr, 1980). 1006 G. DA SILVA ET AL. Fig. 4. SEM (a), (c), and (f) and LM (b), (d), and (e) surface analysis of G. arenicola leaf. a: Surface view of the upper epidermis revealing absence of stomata and illustrates the morphology of the epidermal cells and cuticle. b: Paradermal section showing epidermal cells with straight walls, which are distinctly pitted and a few cells show larger shinning bodies. c: Surface view of the lower epidermis showing random distribution of the stomata. d: Paradermal section of the lower epidermis showing anomocytic stomata, with a ring of five subsidiary cells. e: Paradermal section of the lower epidermis also showing paracytic stomata (arrowhead). f: Surface view of the lower epidermis, showing a slightly sinuous cuticle. Scale bars: a–c and e–f 5 50 mm; d 5 100 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Anomocytic and paracytic stomata are present in this species as described for other species of the Maytenus genus (da Rocha et al., 2004). Anomocytic stomata also occur in the Gymnosporia genus (Jain et al., 2009). The observation of multicellular uniseriate nonglandular trichomes in this species is not according to Jordaan and van Wyk (2006) findings for the Buxifoliae section. In the Buxifoliae section, the presence of Microscopy Research and Technique LEAF IDENTIFICATION OF GYMNOSPORIA ARENICOLA 1007 Fig. 5. LM examination of the powdered leaf of G. arenicola. Details of fragment tissues with calcium oxalate cluster crystals on different locations: on hypodermis cell, on spongy parenchyma and over the veins and insular areas (a, b, and c, respectively); (d) Anom- ocytic and paracytic stomata in an epidermal fragment and some shining bodies (arrow) in the cells of the stomatal region. e: Pappilate cell occurring next to stomata. f: Fragment of vascular sclerenchyma fibers. Scale bars: a, b, and e 5 50 mm; c, d, and f 5 100 mm. nonglandular trichomes is not expected, with some exceptions (G. arenicola is not mentioned as an exception). The occurrence of nonglandular trichomes is not frequent in the Celastraceae family (Jain et al., 2009). The mesophyll reveals dorsiventral organization and the midrib has an amphicrival collateral vas- cular bundle. Metcalfe and Chalk (1950) have indicated both dorsiventral and isobilateral mesophyll in Gymnosporia sp. The presence of a dorsiventral mesophyll is consistent with Jordan and van Wyk (2006) findings for the Buxifoliae section. Microscopy Research and Technique 1008 G. DA SILVA ET AL. Fig. 6. The histochemical results show the staining in LM transverse sections of G. arenicola leaf. a: Negative control for ferric chloride; (b) Ferric trichloride; (c and e) Negative control for 2,4dinitrophenylhydrazine; (d and f) 2,4-dinitrophenylhydrazine; (g) Negative control for Dittmar’s reagent; (h) Dittmar’s reagent; (i) Negative control for Sudan III; (j) Sudan III. Scale bars: a–f 5 50 mm; g– j 5 100 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] The histochemical results suggest the presence of odihydroxyphenols, terpenoids with carbonyl group, alkaloids, total lipids, and starch. The detection of these constituents in situ can be partially correlated with previous phytochemical studies (Orabi et al., 2001). Histochemical analysis allowed the identification of total lipids on the surface of the epidermal cells and cuticle, terpenoids on the collenchyma cells near the midribs and alkaloids, o-dihydroxyphenols, and starch grains in the mesophyll. The detection of phenols confirms previous chemical studies on this species (da Silva G, 2009) and highlights the presence of tanniniferous cells in the Buxifoliae section (Jordaan and van Wyk, 2006). Concerning the phenolic-storing cells, Metcalfe and Chalk (1950) have mentioned them in the mesophyll of species of Cassine, Euonymus, Gymnosporia, Maytenus, Microtropis, Myginda, Pachystima, Siphonodon, Wimmeria, and Zinowiewia. From Beckman’s point of view, these specialized cells, distributed within most tissues, synthesize phenolics and store them in their vacuoles during the normal process of differentiation (Beckman, 2000). In addition, based on the Scalbert’s review, phenolic compounds, especially the tannins, exhibit antimicrobial properties involving the inhibition of extracellular microbial enzymes or oxidative phosphorylation, or deprivation of the substrates required for microbial growth (Scalbert, 1991). According to Metcalfe and Chalk (1983), the histochemical analysis is very interesting for systematic anatomy because these studies often add a distinctive/ restrictive cell distribution pattern and providing relevant phylogenetic relationships for the members of a certain group. As mentioned above G. arenicola is characterized by certain specific anatomical features of the leaf. The stomata type and the calcium oxalate crystals forms were also found to be useful anatomical characters for the authentication of this herbal medicine, which confirms the relevance of these botanical markers in the taxonomy of the Celastraceae family (Gomes and Lombardi, 2010). The epidermis, hypodermis, mesophyll organization, and the presence of trichomes/papillae revealed to be very useful characters for the identification of this plant, as suggested by Jordaan and van Wyk (2006). Our results confirm previous epidermal features described for some Celastraceae, namely the presence of papillae and uniserriate multicellular covering trichomes (Pant and Kidway, 1965). The present findings demonstrate that the G. arenicola leaf has both nonglandular trichomes and hypoderm, which was not previously described in Gymnosporia sect. Buxifoliae Jordaan (Jordaan and van Wyk, 2006). In addition to the conventional pharmacopoeial methods, SEM and histochemical tests provided additional useful information for diagnostic proposes. The use of SEM allowed the achievement of detailed images of certain structures previously observed in LM. This methodology also allowed the obtention of three-dimensional images and the investigation of the surface topology of plant materials, with higher resolution images. The histochemical results confirmed the presence of previously identified classes of compounds and allowed the in situ localization of these compounds. These results can be considered as a preliminary chemical identification criteria performed before the consequent Chemical Identification Test mandatory in all occidental official pharmacopoeias. The present study provides morpho-anatomical markers, based on macro- and microscopic features, for the botanical diagnosis of G. arenicola leaf that must be considered in an official quality monograph of this herbal medicine. ACKNOWLEDGMENTS The authors gratefully acknowledge Maria Adelia Diniz from the Institute for Tropical Scientific Research (Lisbon) for the provision of literature and identification of the plant material and Mariana Borges for her kind assistance in the graphical composition of Figures 1–5. We also thank Telmo Nunes from the Microscopy and Image Analysis Laboratory of the Centre for Environmental Biology (Faculty of Sciences, Microscopy Research and Technique LEAF IDENTIFICATION OF GYMNOSPORIA ARENICOLA University of Lisbon) for the technical assistance on the SEM analysis. REFERENCES Beckman CH. 2000. Phenolic-storing cells: keys to programmed cell death and periderm formation in wilt disease resistance and in general defense responses in plants? Physiol Mol Plant Pathol 57: 101–110. Bernardi HH, Wasicky M. 1959. Algumas pesquisas sobre a “Espinheira-santa” ou “Cancerosa” Maytenus ilicifolia, Martius, usada como rem edio popular no Rio Grande do Sul. 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