Perácio Rafael Bueno Ferreira et al. / Journal of Pharmacy Research 2011,4(4),942-944 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Morphoanatomy, Histochemistry and Phytochemistry of guineense Sw. () Leaves Perácio Rafael Bueno Ferreira*1, Cíntia Sorandra Oliveira Mendes1, Sarah Barbosa Reis,1, Cínthia Gracielly Rodrigues1, Dario Alves de Oliveira1, Maria Olívia Mercadante-Simões1 1Departamento de Biologia Geral, Centro de Ciências Biológicas e da Saúde, Universidade Estadual de Montes Claros, Avenida Ruy Braga, s/n, Campus Prof. Darcy Ribeiro, 39401-089 Montes Claros, MG, Brasil. Received on: 04-01-2011; Revised on: 17-02-2011; Accepted on:16-03-2011 ABSTRACT Psidium guineense, commonly known as “araçá”, is a traditional medicinal used to treat diarrhea. The present study examined the morphoanatomy, histochemistry and phytochemistry of P. guineense leaves to evaluate the presence and localization of pharmacologically active compounds. The anatomical study employed traditional light microscope techniques. Histochemical and phytochemical tests and thin-layer chromatography (TLC) were used to detect flavonoids, and essential oils. The leaves of P. guineense show characteristics common to other species of Myrtaceae, such a marginal vein, a brochidodromous enervation pattern, simple tector trichomes, paracitic stomata, and secretory cavities. Histochemical tests indicated the occurrence of flavonoids and tannins in the mesophyll and vascular system, and flavonoids and essential oils in secretory cavities. Phytochemical tests confirmed the presence of these compounds. The TLC demonstrated the diversity of the chemical substances revealed by many Rfs for the different groups of compounds evaluated. The knowledge of the morpho-anatomical characteristics of P. guineense can aid botanical diagnoses of this species, which has secondary compounds of therapeutic application.

Key words: Araçá, flavonoids, leaf anatomy, tannins, terpenoids, TLC.

INTRODUCTION Psidium guineense is a native of the Brazilian savanna (González et al., 2005) used to pulverized in a Willey mill; the resulting material was stored in paper sacks at -20 ºC. treat diarrhea and for tanning leather (Rodrigues & Carvalho, 2001). Many classes of bio-active Phytochemical prospecting for flavonoids, tannins and essential oils was performed using the substances have been isolated from species of Myrtaceae that have therapeutic or deterrent qualitative protocols described by Mouco et al (2003) utilizing extracts prepared from 1 g of properties such used to treat diabetes (Gutiérrez et al., 2008) and P. plant material dissolved in 10 mL of solvent. Flavonoids were extracted using 80% methanol widgrenianum that shows anti-microbial activity (Donato & Morretes, 2005). In order (with 5% hydrochloric acid) warmed to 80 °C for 2 hours; tannins were extracted in methanol to insure the correct identification of medicinal , unequivocal botanical characterizations for 10 days, and essential oils were obtained by hydro-distillation in an adapted Clevenger of these species are required (Farias, 2007). Then, the present work examined the morpho- apparatus (Jacobs, 1958). anatomical, histochemical, and phytochemical characters of the leaves of P. guineense. Thin-layer chromatography (TLC) was performed using commercially produced silica gel MATERIALS AND METHODS chromatographic plates (2.5x10x10 cm) (60 G F254 - 0.25 mm). The mobile phases utilized were butanol/water/acetic acid (30:20:5) for flavonoids, acetone/toluene/formic acid (3:3:1) for Botanical material tannins, and hexane for terpenoids. The flavonoids were identified by comparison with the Leaves of Psidium guineense were collected during March and April/2009 from specimens retention time of quercetin; the tannins were stained with 1% ferric chloride, and the essential occurring in the municipality of Glaucilandia, Minas Gerais State, (-16º51’00'’ and - oils stained with sublimated iodine. The stains were compared and the retention factors (Rf) of 43º41’49'’) in an area of cerrado (open savanna vegetation, with small that are exposed the bands were calculated using the formula Rf = distance traveled by the sample / distance to high solar radiation levels). The species was identified by Marcos Sobral (University of traveled by the mobile phase. Minas Gerais) and fertile branches were deposited in the Montes Claros’ Herbarium (HMC 456) at the University of Montes Claros as reference specimens. RESULTS Morphology The leaves of P. guineense are simple, oblong, pilose, with slightly undulated margins, with Morphological characterizations of the leaves were performed by measuring the lengths and obtuse base and apex (Figures 1 and 2). The leaf blades are from 10 to 16 cm long and from 4 widths of 10 fully-expanded blades from 10 different individuals. To evaluate the venation to 9 cm wide. The petioles are about 1 cm long and 0.5 cm in diameter. The enervation is patterns, the leaves were cleared (Shobe & Lersten, 1967) and classified based on the types pinnate, with parallel lateral veins uniformly distributed over the blade and arched towards the defined by Hickey (1979). apex, and the marginal nerves form arcs (Figure 2). The secondary enervation pattern is brochidodromous, and the tertiary enervation is of the admedial ramified type (Figure 3). Anatomy Leaf sections removed from the midrib and median regions of the blade and petiole were fixed The adaxial face of the epidermis in the median region of the leaf blade is multiseriate, being composed of three cell layers (Figure 4), with a thick cuticle and cells with periclinal axis in FAA50, for 48 hours, dehydrated in an ethanol series, stored in 70% ethanol, and embedded in paraffin (Johansen, 1940). Serial, 8 to 10 µm thick sections were obtained on a rotatory larger than the anticlinal one. The leaves are hypostomatic, with paracitic stomata (Figure 5); microtome, stained with Astra blue and Safranin (Bukatsh, 1972), and mounted in Entelan as unicellular tector trichomes are present on both faces of the leaf; the trichomes have pointed tips permanent slides. Fragments of the leaf blade were dissociated and stained with crystal violet and cell walls thick and lignified (Figure 6). The mesophyll is dorsiventral; the palisade (Johansen, 1940) to characterize the stomata. parenchyma is composed of two cell layers; and the spongy parenchyma has five to six layers Histochemistry (Figure 4). Numerous cavities (Figures 3, 4 and 7) are seen having a secretory epithelium with Histochemical tests were performed on transversal sections of fresh, totally expanded leaves a cell located in the midst of the epidermal cells that have thin external periclinal cell wall from the central nerve region and the blade. Hand-cut sections were exposed to: DMACA to (Figure 4) and that is in contact with the exterior surface of the leaf. Lamellar collenchyma can stain flavonoids (Feucht et al., 1986); acidified vanillin to stain tannins (Mace & Howell, be seen on the midrib (Figure 7) surrounding the vascular system, with secretory cavities and 1974); and Nadi reagent to visualize essential oils (David & Carde, 1964). The material was idioblasts containing druses (Figure 8). The vascular system is composed of bi-collateral photographed using a Canon A 620 digital camera coupled to a Nikon optical microscope bundles that form a pronounced open arch having secondary structure (Figure 7). The leaf (model Eclipse E-200). mesophyll of the leaf margin has a homogeneous and compact parenchyma composed of isodiametric cells and is lined by epidermal cells that have thicker cuticles than the others Phytochemistry epidermal cells. The abaxial face of the epidermis is uniseriate and is composed of cells with Totally expanded leaves were dried at room temperature for 72 hours and subsequently smaller volumes than the cells on the adaxial face. The epidermis of the petiole has tector trichomes, a cortex with secretory cavities, and a vascular system in the shape of an open arch *Corresponding author. (Figure 9). Perácio Rafael Bueno Ferreira The histochemical and phytochemical analyses indicated the presence of flavonoids, tannins, 1Departamento de Biologia Geral,Centro de and essential oils distributed throughout the leaf tissue (Tables 1 and 2). The TLC of the Ciências Biológicas e da Saúde, Universidade flavonoids demonstrated an Rf 0.41 band similar to the pattern of quercetin (Figure 10). The Estadual de Montes Claros, Avenida Ruy Braga, TLC of the tannins revealed seven bands, with the Rf 0.08 band indicating the presence of s/n, Campus Prof. Darcy Ribeiro, 39401-089 hydrolysable tannins identified by blue color (Figure 11); the bands with Rfs of 0.16, 0.32, Montes Claros, MG, Brasil 0.44, 0.52, 0.6 and 0.76 indicated the presence of condensed tannins, identified by green color. Journal of Pharmacy Research Vol.4.Issue 4. April 2011 942-944 Perácio Rafael Bueno Ferreira et al. / Journal of Pharmacy Research 2011,4(4),942-944

Figures 1 to 9: (ab) abaxial face of the epidermis; (ad) adaxial face of the epidermis; (co) lamellar collenchyma; (dr) druse; (ep) epidermis; (md) midrib; (me) multiseriate epidermis; (mv) marginal vein; (ph) phloem; (pp) palisade parenchyma; (pw) thin external periclinal wall; (sc) secretory cavity; (se) secretory epithelium; (sp) spongy parenchyma; (st) stomata; (sv) secondary vein; (tr) trichome; (tv): tertiary vein; (xi) xilem.

Table 1. Histochemical tests results for flavonoids, tannins, and essential oils for Psidum guineense Sw (Myrtaceae) leaves.

Chemical Reagent Color Epidermis Parenchyma Secretory Vascular Group cavities System

Flavonoids DMACA Blue + ++ + - Tannins Acidified vanillin Red ++ ++ + + Essential oils NADI Pink - - ++ - “+” = presence of the chemical group; “-“ = absence of the chemical group Table 2. Phytochemical tests results for flavonoids, tannins, and essential oils for Psidum guineense Sw (Myrtaceae) leaves. Chemical Group Reagent Coloration Result

Flavonoids Shinoda test Pink ++ sodium hydroxide Yellow + Figures 10 to 12. TLC. (10) Quer- ferric chloride Bluish ++ cetin. (11) Hydrolyzable and con- Tannins 1% ferric chloride Black ++ densed tannins. (12) Essential oils. Lead acetate White ++ (E: end of the chromatographic Copper acetate Brown ++ Essential oils Salkowski Brown ++ run; S: start of the chromato- graphic run; Rf: retention factor). “ +” = presence of the chemical group Journal of Pharmacy Research Vol.4.Issue 4. April 2011 942-944 Perácio Rafael Bueno Ferreira et al. / Journal of Pharmacy Research 2011,4(4),942-944 Three well-defined purple color bands were observed in the TLC of essential oils, with Rfs of (FAPEMIG) and the Financiadora de Estudos e Projetos (FINEP) for their financial support; 0.59, 0.68 and 0.85 (Figure 12). FAPEMIG for BIPDT and PROBIC grants; the University of Montes Claros for their logistical support; and Suelaine Barbosa for her technical assistance. 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Rodrigues VEG, Carvalho DA. Plantas Medicinais no domínio dos Cerrados, 1ªed, Editora UFLA, characters of Myrtaceae species, such the predominance of paracitic stomata than other types Lavras, 2001, 180p. 34. Santos SC, Mello JCP. Taninos. In Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR and the presence of a marginal vein and secretory cavities; the histochemical and phytochemi- (org.) Farmacognosia: da planta ao medicamento 6. ed. Porto Alegre: UFRGS, Florianópolis: cal tests undertaken here confirmed the presence of pharmaceutically active secondary metabo- UFSC. 2007: 615-656. lites (principally flavonoids, tannins and terpenoids) that make Psidium guineense an impor- 35. Shobe WR, Lersten NR. A technique of clearing and staining gymnosperm leaves. Bot Gaz. 1967: tant medicinal plant. 128; 150-152. 36. Simões CMO, Sptizer V. Óleos voláteis. In Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR (org.) Farmacognosia: da planta ao medicamento 6. ed. Porto Alegre: UFRGS, Florianópolis: ACKNOWLEDGMENTS UFSC. 2007: 467-495. The authors would like to thank the Fundação de Amparo à Pesquisa de Minas Gerais 37. Zuanazzi JAS, Montanha JA. Flavonóides. In Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR (org.) Farmacognosia: da planta ao medicamento 6. ed. Porto Alegre: UFRGS, Florianópolis: UFSC. 2007: 577-614.

Source of support: The Foundation for Research Support of Minas Gerais (FAPEMIG) and Foundation Financier of Studies and Projects (FINEP), Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 942-944