Volatile Compounds of Mandevilla Guanabarica (Apocynoideae, Apocynaceae) from Three Restingas in Rio De Janeiro, Brazil

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Biochemical Systematics and Ecology 45 (2012) 102–107

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Biochemical Systematics and Ecology

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Volatile compounds of Mandevilla guanabarica (Apocynoideae, Apocynaceae) from three restingas in Rio de Janeiro, Brazil

Sandra Zorat Cordeiro a,*, Alice Sato b, Rosani do Carmo de Oliveira Arruda c, Naomi Kato Simas d

a Pós-graduação em Biotecnologia Vegetal, 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 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 22290-240, Brazil c Laboratório de Anatomia Vegetal, Departamento de Biologia, Universidade Federal do Mato Grosso do Sul (UFMS), Campus de Campo Grande, s/n, Cidade Universitária, CCBS, Campo Grande, MS 79070-900, Brazil d Laboratório de Fitoquímica, Departamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho, s/n, CCS, Bloco A, sala A2-16, Cidade Universitária, Rio de Janeiro, RJ 21941-590, Brazil

article info abstract

Article history: The chemical composition of volatile compounds in leaves of Mandevilla guanabarica from Received 19 April 2012 three restingas in Rio de Janeiro was characterized by GC/MS after SDE. The volatiles Accepted 7 July 2012 common were 3-hexen-1-ol, b-caryophyllene and germacrene D, which were majority Available online 10 August 2012 compounds for plants from Grumari, Jurubatiba and Maricá, respectively. M. guanabarica also contained other compounds: a-cubebene, a-copaene and cubebene (Grumari); Keywords: b-ocimene, a-caryophyllene and germacrene B (Jurubatiba); and b-elemene and alloar- Mandevilla omadendrene (Maricá). Although some compounds were common to all locations, the Apocynaceae ﬁ Volatile compounds qualitative and quantitative chemical pro les of volatile compounds of M. guanabarica Histochemistry from three different areas in the same season, with similar conditions of temperature and precipitation, were distinct. Leaf histochemical analysis revealed the presence of fatty acids in idioblasts, and rubber grains and droplets of lipophilic substances in the mesophyll, for all three samples. These results indicate the possible inﬂuence of physiological variations and/or environmental conditions on the composition of volatiles in M. guanabarica. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The genus Mandevilla Lindley (Apocynaceae, Apocynoideae) comprises at least 150 species in the Neotropical region. The material characteristics of the genus are the presence of colleteres at the base of the leaf, and a racemose inﬂorescence, with wide variety in the morphology of the corolla. Most species have large, showy and colorful ﬂowers, which confer high ornamental and landscaping value. Most species have a climbing habit, but others are shrubs, subshrubs, herbs or epiphytes (Sales et al., 2006). Some species have a hard subterranean structure capable of sprouting, termed a xylopodium, with proven pharmacological properties (Calixto et al., 1985; Appezzato-da-Glória and Estelita, 2000). About 70 Mandevilla species occur in Brazil, and although their major distribution centers are the Espinhaço Range in the states of Minas Gerais and Bahia, and

* Corresponding author. Tel.: þ55 21 2562 6330; fax: þ55 21 2562 2010. E-mail addresses: [email protected] (S.Z. Cordeiro), [email protected] (A. Sato), [email protected] (R.doC.deO. Arruda), naomisimas@ yahoo.com (N.K. Simas).

0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.07.019 S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 45 (2012) 102–107 103 the adjacent areas of Cerrado and rocky grasslands, Mandevilla is also represented in the east, especially in areas of restingas and inselbergs (Sales et al., 2006). Mandevilla guanabarica Casar ex. M.F.Salles, Kin.-Gouv. & A.O.Simões is endemic to shrubby restinga vegetation in the states of Rio de Janeiro and Espírito Santo, southeastern Brazil. It is a liana, with twining and latescent branches; the flowers have a yellow funnel-shaped corolla, and inflorescences with 3–6 flowers (Sales et al., 2006). Studies on the genus Mandevilla have treated aspects of the taxonomy (Sales et al., 2006), anatomy (Martins and Alves, 2008), pharmacological properties and ethnobotany (Calixto et al., 1985), and in vitro development (Handro et al., 1988). Several studies have described new compounds with biological activities, obtained from the subterranean system of Man- devilla, including steroids, cardenolide glycosides and terpenoids (Cabrera et al., 1993). Chemical studies of the aerial part are few, related to the composition of the latex emulsion (Lopes et al., 2009) or to the composition of the epicuticular waxes (Cordeiro et al., 2011). Histochemical studies with Mandevilla revealed rubber grains and fatty acids in the idioblasts, present in the palisade parenchyma and laticifer secretion with lipophilic substances rich in neutral lipids and resin oils (Alves and Oliveira, 1992; Lopes et al., 2009). Other studies revealed the presence of polyisoprene hydrocarbons (rubber), triterpenes, and phytosterols in the latex, which occurs abundantly in the leaves and stems of Mandevilla (Demarco et al., 2006; Konno, 2011; Van Die, 1955). Because the genus contains no aromatic species, no studies have yet examined volatile compounds. Analysis of Mandevilla leaves by a specific extraction method for plants with low volatiles content, followed by identification of compounds, would complement the limited information that is presently available. This study investigated the volatile chemical composition of leaves of M. guanabarica from three different restingas, comparing their components obtained by the simultaneous distillation-extraction (SDE) method. This study also evaluated the presence of lipophilic compounds via histochemical analyses of the leaves.

2. Materials and methods

2.1. Sampled areas and plant materials

Samples of M. guanabarica 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 the Environment and Natural Renewable Resources). All samples were collected in the same season (November 2009). Taxonomists Prof. Dr. Jorge Fontella Pereira, Marcelo Fraga Castilhiori, and Inaldo do Espírito Santo undertook the taxonomic identiﬁcations. Vouchers are deposited at the Herbarium Bradeanum (HB) in the State University of Rio de Janeiro (UERJ), Rio de Janeiro, Brazil, under accession numbers HB 93040 (M. guanabarica from Grumari), HB 93039 (M. guanabarica from Maricá), and HB 93038 (M. guanabarica from Jurubatiba). Table 1 shows the locations (latitude and longitude), mean annual precipitation, and mean annual temperature of the sampling areas.

2.2. Simultaneous distillation-extraction (SDE)

Fully expanded leaves (5 g) of M. guanabarica from the three areas were homogenized with 50 mL distilled water and submitted to SDE by SDE apparatus (Godefroot et al., 1981) for 2 h, using 2 mL of dichloromethane as organic collecting solvent. The sample and solvent ﬂasks were kept in a mineral-oil bath under agitation, respectively at 110–130 C and 55– 60 C. The vapors from the sample were condensed by cooling in the SDE apparatus, collected in the dichloromethane ﬂask, and placed in the GC/FID and GC/MS for analysis.

2.3. Volatile analysis

Samples were analyzed in a GC-2010-Shimadzu gas chromatograph ﬁtted with a DB-1MS fused silica capillary column (30 m 0.25 mm 0.2 mm) and equipped with a ﬂame ionization detector (FID). The oven temperature was programmed

Table 1 Collection areas of Mandevilla guanabarica, with location, latitude and longitude, mean annual precipitation, and mean annual temperature.

Area Location (Municipality) Latitude / longitude Mean annual Mean annual precipitation (mm) temperature (C) Grumari Environmental Rio de Janeiro 23030S 1173 23.7 Protection Area 43310W Maricá Environmental Maricá 22540S 1230 23.2 Protection Area 42490W Restinga de Jurubatiba Carapebus, Macaé, 22220S 1164 22.6 National Park and Quissamã 41470W 104 S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 45 (2012) 102–107

from 140 to 300 Cat10C min 1. The injector was maintained at 290 C and the detector at 300 C, injection of 1 mL, split ratio 1:50. Hydrogen was used as carrier gas at a flow rate of 1 mL min 1. Quantification was performed from GC/FID profiles, using the normalized peaks area of each compound. Mass spectra were obtained in a Shimadzu Model GC MS-QP 2010 Plus apparatus equipped with ZB-5MS fused silica capillary column (30 m 0.25 mm, film thickness 0.2 mm). The mass spectrometer was operated in electronic ionization (EI) at 70 eV. The oven and injector temperatures were the same as above. Injection of 1 mL, split ratio 1:50, and helium was used as the carrier gas at a flow rate at 1 mL min 1. Linear retention indices were measured by injecting a homologous series of n- alkanes (C9–C30) in the same column and conditions indicated above for GC analyses and of calculated by van den Dool and Kratz method (van den Dool and Kratz, 1963). The top 5% of the components with respect to relative abundance were identified by comparison of their mass spectra (MS) and Linear Retention Index (LRI) with those reported in literature (Adams, 2001) and also in National Institute of Standards and Technology (NIST) mass spectral database.

2.4. Histochemistry

The presence of lipids and rubber grains was investigated in fresh cross sections of leaves of M. guanabarica from the three different areas, using the following histochemical tests: Sudan III (Johansen, 1940), Sudan IV (Pearse, 1980), Sudan Red 7B (Brundrett et al., 1991), and Sudan Black (Pearse, 1980) for lipids; Nile Blue sulfate (Cain, 1947) for acid and neutral lipids; and Ò Oil Red O (Jayabalan and Shah, 1986) for rubber grains. The images were obtained by the software Image Pro-Plus through an Ò Ò Olympus digital camera coupled to an Olympus BX41 light microscope.

3. Results and discussion

In this study, the volatile compounds from M. guanabarica leaves from three different restingas were extracted using the simultaneous distillation method. The dichloromethane extracts from the M. guanabarica leaves, enriched in volatile compounds, were identified by GC/MS (Table 2). The volatile compounds were characterized and identified as sesquiterpenes, monoterpenes, and a hemiterpene. It was not possible to calculate the LRI of the hemiterpene, 3-hexen-1-ol. This compound was identified by comparison of the corresponding mass spectra fragmentation with library data of the GC/MS-QP2010 Plus Shimadzu mass detector, with 98% similarity, and with literature data (Adams, 2001). The major volatile compounds of M. guanabarica are predominantly sesquiterpenes, with the exceptions of the hemiterpene 3-hexen-1-ol, and b-ocimene, a monoterpene. The sesquiterpenes constitute a large group of structures, present in certain essences and resins. There are few known linear sesquiterpenes compared with the number of cyclic sesquiterpenes. These show antioxidant, cytotoxic, and antibacterial activities, observed especially for germacrenes (Niero and Malheiros, 2009). Only three compounds were common to this species from each area: 3-hexen-1-ol, b-caryophyllene, and germacrene D, and each of them was the major compound in one area. M. guanabarica from Grumari contained 3-hexen-1-ol (20.3%), germacrene D (20.2%), a-copaene (19.1%), and b-caryophyllene (17.5%) as the main compounds. M. guanabarica from Juru- batiba contained, in addition to b-caryophyllene (45.7%) and 3-hexen-1-ol (13.7%), b-ocimene (8.0%), the only monoterpene identified, and germacrene B (8.0%). In turn, M. guanabarica from Maricá showed germacrene D (34.0%) as the major compound, followed by b-caryophyllene (18.2%), b-elemene (11.8%), and 3-hexen-1-ol (9.0%). Excepting cubebene, alloaromadendrene and germacrene B, all the other compounds identified in M. guanabarica play a role as semiochemicals for many animals (El-Sayed, 2011). Semiochemicals are known like signaling chemicals that organisms can detect in its environment, which may induce changes in its behavior or its physiology (Dudareva et al., 2006). Recently, Koschnitzke (2011) investigated

Table 2 Percentages of relative abundance of major volatile compounds (>5%) obtained from Mandevilla guanabarica leaves collected in their respective locations, determined by GC/MS (n.d. ¼ not determined).

Compounds LRIcalculated Relative abundance (%) of foliar volatile compounds M. guanabarica Grumari M. guanabarica Jurubatiba M. guanabarica Maricá 3-hexen-1-ol n.d.a 20.3 13.7 9.00 b-ocimene 1043 – 8.0 – a-cubebene 1343 5.9 –– a-copaene 1371 19.1 –– Cubebene 1383 6.9 –– b-elemene 1385 –– 11.8 b-caryophyllene 1413 17.5 45.7 18.2 a-caryophyllene 1449 – 6.1 – alloaromadendrene 1453 –– 6.1 Germacrene D 1475 20.2 6.4 34.0 Germacrene B 1489 – 8.0 –

a Not determined: compound determined only by comparison of the corresponding mass spectra with library data. S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 45 (2012) 102–107 105 the floral biology of M. guanabarica and described the behavior of Trigona spinipes (Fabricius) (Hymenoptera, Apidae) in drilling the flowers of this species. There are no data about the chemical volatile composition of species of the genus Man- devilla in the literature neither about semiochemicals detected in this genus, which difficult the discussion of the results. Most studies on the volatiles in Apocynaceae are related to the composition of floral (Jürgens et al., 2008), fruit (Pélissier et al., 1996), or root extracts (Moudachirou et al., 1998). There are few studies on volatiles from the aerial parts of species of Apocynaceae (Brun et al., 2001; Cornélio et al., 2004, 2005; London et al., 2011; Mirza and Navaei, 2009; Zito et al., 2010, 2012) and this is the first report to characterize the volatile compounds of Mandevilla leaves. Cornélio et al. (2004) reported about essential oil from aerial parts of Aspidosperma polyneuron, which contained a- copaene (1.2%) and b-caryophyllene (1.7%). For Aspidosperma cylindrocarpon, the volatile extract of aerial parts was characterized by the presence of a major percentage of sesquiterpene hydrocarbons, with b-caryophyllene as the major compound (Cornélio et al., 2005). This was one of the compounds found in M. guanabarica, regardless of the sampling area. A high percentage of sesquiterpenes (38.5%) was reported in volatiles of Amsonia illustris, which contained a-humulene, b-caryophyllene, and guaiol (London et al., 2011). The volatile compounds from leaves of Catharanthus roseus, obtained by hydrodistillation, showed 85% fatty acids and fatty-acid esters, whereas the terpene component was only 6% (Brun et al., 2001). Similar results were obtained by hydrodistillation of stems of Caralluma europaea, which showed essential oils with

Fig. 1. Histochemistry of fresh leaves of Mandevilla guanabarica. Cross sections showing: orange lipid droplets stained with Sudan III (A), Sudan IV (B), and Sudan Red 7B (C); dark-blue lipid droplets stained with Sudan Black (D); blue idioblasts with acid lipids stained with Nile Blue sulfate (E); and orange rubber grains stained with Oil Red O (F). Legend: cut ¼ cuticle, dr ¼ druses, ep ¼ epidermis, id ¼ idioblasts, pm ¼ palisade mesophyll, sm ¼ spongy mesophyll, vb ¼ vascular bundle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 106 S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 45 (2012) 102–107 a high percentage (88.3%) of non-aromatic hydrocarbons and fatty acids (Zito et al., 2010) and leaves of Marsdenia erecta, which contained 80.6% anethol, an essential oil (Mirza and Navaei, 2009). Zito et al. (2012) reported about essential oils from different organs of Periploca laevigata subsp. angustifolia obtained by hydrodistillation. These authors identified 57 compounds in the branches and 6 in the leaves. Branches showed as major classes hydrocarbons (24.7%) and fatty acids (21.2%), with predominance of heneicosane (11.6%) and hexadecanoic acid (9.9%), respectively. In the leaves, the hydrocarbons heneicosane (38.2%) and tricosane (35.6%) and the alcohol eicosanol (12.9%) were the major compounds, beside sesquiterpenes (14.6%). The sesquiterpenoidal composition obtained for M. guanabarica indicate the prevalence of mevalonate pathway instead acetate or shikimate in the essential oil composition. Because these differing extracts were obtained from the same species, M. guanabarica, from three different areas, the influence of environmental conditions must be considered (Table 1). The mean annual temperature and precipitation of the three areas are very close, and the leaves were collected in the same month; this argues against any association between temperature, precipitation, or seasonal variation and the different volatile compositions shown by the plants. Although the soil composition was not analyzed in this study, other studies have reported on the influence on volatile compounds of edaphic factors such as organic-matter content, moisture, and aridity (Alañón et al., 2011; Boira and Blanquer, 1998). In addition to climate, seasonal variations, and soil, many factors influence the production and composition of volatiles and essential oils, including physiological variations such as organ development, pollinator activity cycle, and mechanical or chemical injuries; environmental conditions such as climate, pollution, diseases, and pests, not to mention geographical and genetic variations (Figueiredo et al., 2008). In another study, on epicuticular waxes of M. guanabarica collected in the same three areas, the chemical profiles analyzed were qualitatively and quantitatively similar, regardless of the origin of the plants (Cordeiro et al., 2011). Further studies are needed to investigate the cause of variation observed in the volatile compounds, and the lack of variation in epicuticular waxes. The histochemistry of M. guanabarica was investigated for each area sampled, but was very similar for all of them, and therefore only the results for samples from Maricá are presented (Fig. 1). The histochemistry results revealed droplets of lipophilic substances in cells of palisade and spongy mesophyll, detected by Sudan III, Sudan IV, and Sudan Red 7B; these droplets were stained orange, while Sudan Black stained the droplets dark blue. Sudans are organic dyes commonly used as stains for lipids (Brundrett et al., 1991), including terpenes, the main compounds found in the extracts of M. guanabarica by GC/MS. The cross sections clearly showed an absence of trichomes or secretory cavities, suggesting that the volatiles are stored in the palisade cells, as revealed by histochemistry. Acid lipids, also known as fatty acids, were detected in idioblasts, present in the parenchyma cells and stained dark blue by Nile Blue sulfate. Rubber grains, stained orange by Oil Red O, also were detected in the parenchyma cells. This work presents the first study on the volatile compound contents of M. guanabarica. The common volatile compounds of M. guanabarica from Grumari, Jurubatiba and Maricá were only 3-hexen-1-ol, b-caryophyllene, and germacrene D, with different percentages for each area sampled. These results indicate that, although some compounds were common to all localities, the qualitative and quantitative chemical profiles of volatile compounds of M. guanabarica are distinct in plants collected from different areas in the same season, with similar conditions of temperature and precipitation. These volatile compounds were stored in palisade cells, as revealed by histochemical evaluation.

Acknowledgments

The authors thank CAPES (Conselho de Administração de Pessoal de Ensino Superior) for a doctoral scholarship for the first author, PBV-UFRJ (Programa de Pós-graduação em Biotecnologia Vegetal, Universidade Federal do Rio de Janeiro) and FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro) for financial support, INEA (State Environmental Institute) and IBAMA (Brazilian Institute for Environment and Natural Renewable Resources) for authorization to collect in the sampling areas, taxonomists Jorge Fontella Pereira of the Museu Nacional (UFRJ) and Marcelo Fraga Castilhiori and Inaldo do Espírito Santo of the Herbarium Bradeanum (HB) for species identification, Ricardo Machado Kuster (UFRJ) for enabling the analysis of extracts, Janet W. Reid (JWR Associates, USA) for editing the English text, and the Universidade Federal do Estado do Rio de Janeiro (UNIRIO) for providing transport to the collection areas.

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