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Composition of Epicuticular Wax Layer of Two Species of Mandevilla (Apocynoideae, Apocynaceae) from Rio De Janeiro, Brazil

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Composition of Epicuticular Wax Layer of Two Species of Mandevilla (Apocynoideae, Apocynaceae) from Rio De Janeiro, Brazil Biochemical Systematics and Ecology 39 (2011) 198–202 Contents lists available at ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Composition of epicuticular wax layer of two species of Mandevilla (Apocynoideae, Apocynaceae) from Rio de Janeiro, Brazil Sandra Zorat Cordeiro a,*, Naomi Kato Simas b, Rosani do Carmo de Oliveira Arruda c, Alice Sato d a Universidade Federal do Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho, s/n – CCS, Bloco K, sala K2-032, Cidade Universitária, Rio de Janeiro – RJ 21941-902, Brazil b Laboratório de Fitoquímica, Curso de Farmácia, Universidade Federal do Rio de Janeiro (UFRJ), Campus Macaé, Av. Aluizio da Silva Gomes, 50 – Granja dos Cavaleiros - Macaé - RJ 27930-560, Brazil c Laboratório de Anatomia Vegetal, Departamento de Biologia, Universidade Federal do Mato, Grosso do Sul (UFMS), Campus de Campo Grande, s/n – Cidade Universidade, CCBS, Campo Grande - MS 79070-900, Brazil d Laboratório de Cultura de Tecidos Vegetais, Departamento de Botânica, Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Av. Pasteur 458 – CCBS, sala 414, Rio de Janeiro - RJ 22290-040, Brazil article info abstract Article history: The chemical composition of epicuticular waxes of Mandevilla guanabarica and Mandevilla Received 26 November 2010 moricandiana was comparatively analyzed by extraction in n-hexane and chloroform. The À Accepted 26 February 2011 mean wax content per unit of leaf area in the n-hexane extract was about 13–30 mgcm 2 Available online 22 March 2011 for M. guanabarica, containing 20–28% n-alkanes and 55–63% triterpenes; for M. mori- À candiana, the mean content was 19 mgcm 2, containing 73% n-alkanes and 14% triterpenes. À Keywords: In the chloroform extract, the wax yield was 40–80 mgcm 2 for M. guanabarica, with about Mandevilla 9–11% n-alkanes and 75–82% triterpenes; while for M. moricandiana, the wax yield was Apocynaceae m À2 fi Epicuticular waxes 110 gcm , with 52% n-alkanes and 14% triterpenes. The major compounds identi ed a b Chemotaxonomy were lupeol, pentacyclic triterpenes of the - and -amyrin class, and n-alkanes such as nonacosane, hentriacontane and tritriacontane. These results indicate that the quantitative chemical profiles of epicuticular waxes of M. guanabarica and M. moricandiana are distinct and could be used as an additional feature in taxonomic identification. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Mandevilla Lindley (Apocynaceae, Apocynoideae) consists of approximately 150 species distributed from Mexico to Argentina. Most of these plants show a climbing habit, although they may occur as shrubs, subshrubs, herbs, and epiphytes (Sales et al., 2006). The flowers are showy and colorful, which gives them ornamental and landscape potential (Metcalfe and Chalk, 1950). Mandevilla is well represented in Brazil. At least 50 species had been recorded, mainly in Atlantic Forest areas, in the sandy coastal environments known as “restingas”, rocky grasslands, and inselbergs (Sales et al., 2006). Mandevilla species show morphological and anatomical adaptations in response to dry conditions and intense sunlight, such as the presence of a xylo- podium, stolons and deciduous or xeromorfic leaves (Appezzato-da-Glória and Estelita, 2000; Martins and Alves, 2008). Despite these adaptations, characteristics related to the chemical composition of the epicuticular waxes have not received attention. * Corresponding author. Universidade Federal do Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho, s/n – CCS, Bloco K, sala K2-032, Cidade Universitária, Rio de Janeiro – RJ 21941-902, Brazil. Tel.: þ55 21 2224 5851; fax: þ55 21 2562 2010. E-mail addresses: [email protected] (S.Z. Cordeiro), [email protected] (N.K. Simas), [email protected] (R.C.O. Arruda), [email protected] (A. Sato). 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.02.009 S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 39 (2011) 198–202 199 Cuticular waxes are a complex mixture of aliphatic chain compounds such as n-alkanes, fatty acids, alcohols, aldehydes, ketones, or n-alkyl esters, and other compounds including flavonoids and pentacyclic triterpenoids (Barthlott et al., 1998; Dragota and Riederer, 2007). The physical and chemical properties of cuticular waxes allow the cuticle to perform certain ecophysiological functions, such as reducing water loss by cuticular transpiration and protecting against UV radiation, both with potential significance in plants that occur in environments with intense solar radiation. The cuticular waxes also promote interaction with other organisms and chemicals (Barthlott et al., 1998; Oliveira et al., 2003; Schreiber and Riederer, 1996). Epicuticular waxes are considered an important taxonomic marker that can be used as criteria to distinguish groups of species within families or genera, reflecting ecological and genetic relationships (Medina et al., 2006; Mimura et al., 1998; Motta et al., 2009). Mandevilla guanabarica Casar. ex M. F. Salles, Kin.-Gouv. & A. O. Simões is an endemic species of the restinga shrub formation, occurring in the states of Rio de Janeiro and Espírito Santo in southeastern Brazil. Mandevilla moricandiana (A. DC.) Woodson has been reported from northeastern and southeastern Brazil, where it is found mainly in regions of restinga and rocky grasslands. Both species have twining branches and a trailing habit; M. guanabarica has showy flowers with a yellow corolla; M. moricandiana has equally showy flowers with a pink corolla and the corolline tube may have a white or yellow interior (Sales et al., 2006; Woodson, 1933). The aim of this study was to analyze the chemical composition of the leaf epicuticular waxes obtained from n-hexane and chloroform extracts of M. guanabarica and M. moricandiana. In addition, this study investigated whether the chemical profile varied among plants of the same species obtained from three different regions, which could be used as a taxonomic tool. 2. Materials and methods 2.1. Sampled areas and plant materials Samples were collected in the Grumari Environmental Protection Area, the Maricá Environmental Protection Area and the Restinga de Jurubatiba National Park, all in the state of Rio de Janeiro. Authorization to collect was granted by the Brazilian government authorities (Municipality of Rio de Janeiro, INEA, State Institute of the Environment, and IBAMA, Brazilian Institute for Environment and Natural Renewable Resources). All samples were collected in the same season (November 2009) in order to avoid possible variations in the chemical composition of the waxes, as reported in other studies (Faini et al., 1999). Table 1 shows the locations (latitude and longitude), mean annual precipitation, and mean annual temperature of the sampling areas (Henriques et al., 1986; Nimer, 1989), the species collected, and the herbarium voucher number. All species were kindly identified by taxonomists Prof. Dr. Jorge Fontella Pereira, Marcelo Fraga Castilhiori, and Inaldo do Espírito Santo, from herbarium specimens deposited in the Bradeanum Herbarium (HB), in the State University of Rio de Janeiro (UERJ), Rio de Janeiro, Brazil. 2.2. Leaf area determination For each species and sampling location, ten fully expanded and undamaged leaves were removed from the branches collected. The leaves were photographed individually on a known area, for determination of leaf area using the software ImageJ 1.42q (Rasband, 2010) for subsequent calculation of the yield of extracts. The experiment was conducted in a randomized design with three replicates for each species and locality. The means of three replicates and their standard deviations were calculated. 2.3. Epicuticular waxes extraction and chemical analysis by gas chromatography (GC) coupled with mass spectrometry (MS) After leaf area determination, each leaf was placed in a separate pre-weighed flask containing 50 ml of n-hexane or chloroform, and maintained for 30 s under gentle agitation. This procedure extracts only surface n-hexane-soluble compounds or chloroform-soluble compounds, without disturbing the leaf interior. Table 1 Collection areas, location data, latitude and longitude, mean annual precipitation, mean annual temperature, number of species collected, and Bradeanum Herbarium voucher number. Area Location Latitude/longitude Mean annual Mean annual Species collected Voucher (HB) (municipality) precipitation (mm) temperature (C) Grumari Environmental Rio de Janeiro 23030S43310W 1173 23.7 M. guanabarica HB 93040 Protection Area Maricá Environmental Maricá 22540S42490W 1230 23.2 M. guanabarica HB 93039 Protection Area Restinga de Jurubatiba Carapebus, Macaé, 22220S41470W 1164 22.6 M. guanabarica HB 93038 National Park and Quissamã M. moricandiana HB 93033 200 S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 39 (2011) 198–202 Chloroform and hexane extracts were maintained at room temperature (25 Æ 1 C) for solvent evaporation to obtain the À solid residue. The amount of waxes was expressed per unit leaf area (mgcm2). The experiment was conducted in a randomized design with three replicates for each species, locality, and solvent. The means of three replicates and their standard deviations were calculated. Two mg of dried extract was dissolved in 200 mL chloroform. Subsequently, 1 mL was injected into a gas chromatograph (CG-2010-Shimadzu), flame ionization detector (FID), DB-1MS column (30 m  0.25 mm  0.2 mm), using He as carrier gas at À À 1 mL min
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