Biochemical Systematics and Ecology 39 (2011) 198–202
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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
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
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 November 2010
Accepted 26 February 2011
Available online 22 March 2011
The chemical composition of epicuticular waxes of Mandevilla guanabarica and Mandevilla
moricandiana was comparatively analyzed by extraction in n-hexane and chloroform. The
mean wax content per unit of leaf area in the n-hexane extract was about 13–30 mg cm2
for M. guanabarica, containing 20–28% n-alkanes and 55–63% triterpenes; for M. moricandiana, the mean content was 19 mg cm2, containing 73% n-alkanes and 14% triterpenes.
In the chloroform extract, the wax yield was 40–80 mg cm2 for M. guanabarica, with about
9–11% n-alkanes and 75–82% triterpenes; while for M. moricandiana, the wax yield was
110 mg cm2, with 52% n-alkanes and 14% triterpenes. The major compounds identified
were lupeol, pentacyclic triterpenes of the a- and b-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.
Keywords:
Mandevilla
Apocynaceae
Epicuticular waxes
Chemotaxonomy
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 xylopodium, 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
collected. The leaves were photographed individually on a known area, for determination of leaf area
ImageJ 1.42q (Rasband, 2010) for subsequent calculation of the yield of extracts. The experiment
a randomized design with three replicates for each species and locality. The means of three replicates
deviations were calculated.
from the branches
using the software
was conducted in
and their standard
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
(municipality)
Latitude/longitude
Mean annual
precipitation (mm)
Mean annual
temperature ( C)
Species collected
Voucher (HB)
Grumari Environmental
Protection Area
Maricá Environmental
Protection Area
Restinga de Jurubatiba
National Park
Rio de Janeiro
23 030 S 43 310 W
1173
23.7
M. guanabarica
HB 93040
Maricá
22 540 S 42 490 W
1230
23.2
M. guanabarica
HB 93039
1164
22.6
M. guanabarica
M. moricandiana
HB 93038
HB 93033
Carapebus, Macaé,
and Quissamã
0
0
22 22 S 41 47 W
200
S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 39 (2011) 198–202
Chloroform and hexane extracts were maintained at room temperature (25 1 C) for solvent evaporation to obtain the
solid residue. The amount of waxes was expressed per unit leaf area (mg cm2). 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
1 mL min1 and a split ratio (50:1). Temperature was increased by 10 C min1 from 140 to 300 C, and then maintained at
300 C for 15 min. The injector was maintained at 290 C and the detector at 300 C. Quantification was performed from GC/
FID profiles, using relative area (%). The top 5% of the components with respect to relative abundance were identified.
Identification of the n-alkanes was based on injection of commercial standards (Sigma Fluka Alkane standard solution
C21-C40) and analysis of a subsample in a GC/MS-QP2010 Plus Shimadzu mass detector, using the same operating conditions as
above (except the column; ZB-5MS column 30 m 0.25 mm 0.2 mm) and the MS scanned for 50–650 da at 2 s decade1
with an electron impact ionization potential of 70 eV. The triterpenoid compounds were identified by comparison of the
corresponding mass spectra with library data (Spectrum Libraries: NIST 05.LIB), complemented with proton nuclear magnetic
resonance (1H NMR) spectrometry (Bruker DRX 400 MHz) using deuterated chloroform as solvent. The NMR spectra of the
mixture were evaluated by comparison with literature data.
3. Results and discussion
The solvents used to extract epicuticular waxes must be chosen carefully, because depending on the solvent used, one type
of component will be extracted more easily than others (Bakker et al., 1998). Chloroform is most often used for the extraction
and study of epicuticular waxes (Dragota and Riederer, 2007; Oliveira et al., 2003). However, some studies have used other
solvents such as dichloromethane, n-hexane, and toluene (Bakker et al., 1998; Medina et al., 2006).
For all the species and areas studied, the n-hexane extractions gave lower yields than the chloroform extractions. For
n-hexane extractions, the highest yield was obtained from M. guanabarica collected in Jurubatiba (30.6 mg cm2) and Grumari
(25.8 mg cm2), followed by M. moricandiana (18.9 mg cm2), although the differences were not significant (Fig. 1). The lowest
yield was obtained for M. guanabarica collected in Maricá (13.2 mg cm2). Yields of 2–30 mg cm2 for n-hexane extraction in
Clusia leaves were reported by Medina et al. (2006).
For the chloroform extractions, M. moricandiana had the highest yield, up to about 110 mg cm2. For M. guanabarica
collected from different areas, the wax yields were significantly lower, especially for plants from Maricá (43.7 mg cm2),
followed by Grumari (66.9 mg cm2) and Jurubatiba (85.3 mg cm2) (Fig. 1). Yields between 60 and 75 mg cm2 (Oliveira and
Fig. 1. Mean yields of waxes extracted with n-hexane and chloroform, for both species in their respective localities. Different small letters next to the bars
indicate statistical differences in the efficiency of extraction with n-hexane, by the Tukey–Kramer test (P < 0.05). Different capital letters next to the bars indicate
statistical differences in the yield of extractions with chloroform, by the Tukey–Kramer test (P < 0.05). The mean yields of all extractions carried out with nhexane and chloroform for each species and location showed statistically significant differences by the Tukey–Kramer test (P < 0.001).
S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 39 (2011) 198–202
201
Salatino, 2000) and between 14 and 700 mg cm2 (Amaral et al., 1985) to chloroform extractions were reported for leaves of
trees in the Brazilian Cerrado. There are no data about the extraction yield of epicuticular waxes of Mandevilla in the literature
that can be compared with the data obtained.
The hexane-extract samples from M. guanabarica and M. moricandiana showed n-alkanes and triterpenes as the main
constituents. The n-alkanes were composed of nonacosane (C29), hentriacontane (C31), and tritriancontane (C33). The triterpenes were composed of three pentacyclic triterpenes of the a- and b-amyrin class, and the mixture of lupeol and its
acetate derivative (Table 2). Methylation products with diazomethane of extracts resulted in the same retention time as
chromatograms obtained from the original samples. This feature confirmed the absence of acid compounds and highlighted
the presence of these n-alkanes and neutral triterpenes.
Lupeol and its acetate derivative were efficiently extracted using chloroform as solvent. The gas chromatogram of the
chloroform extracts showed the main large signal at 22.0 min, except for M. moricandiana where this was a small signal. The
mass spectrum showed a molecular ion at 426 da followed by m/z 411. These fragments are initiated by C14–C27 cleavage and
consequent methyl elimination. They can easily decompose into smaller fragments such as m/z 218, m/z 207, and m/z 189,
which is characteristic for the fragmentation of triterpenes with a lupane skeleton. The latter two fragments are characteristic
for the fragmentation of triterpenes with a lupane skeleton bearing a hydroxyl group in position C3. In addition to these
fragments, the presence of m/z 383 was evidenced, indicating an acetate moiety (Carvalho et al., 2010; Shiojima et al., 1992).
The lupeol mixture was identified on the basis of NMR data. The characteristic signals of lupeol were shown by a signal at
1.68 ppm corresponding to methyl group (C30) at the sp2 C20 position, and two olefinic large singlets at 4.56 and 4.68 ppm,
characteristic of olefinic protons at C29. The presence of a carbinol methine proton at C3 was confirmed by multiplet at
3.2 ppm. Also, the acetyl methyl moiety was confirmed by the singlet at 2.0 ppm (Aragão et al., 1990; Barbosa et al., 2010;
Sobrinho et al., 1991). The mass fragmentation data complemented by the resonance spectrum data confirmed the
mixture of lupeol and lupeol acetate in the chloroform extracts.
The additional constituents of the chloroform extract at retention times of 21.4, 22.9, and 23.6 min in the chromatogram
revealed the common fragments to be m/z 218 and m/z 203. These are the typical retro-Diels-Alder fragmentation, employed
as a characteristic diagnostic tool for the presence of a 12-13 double bond in pentacyclic triterpenes, as in a- and b-amyrin
(Budzikiewicz et al., 1963; Zanon et al., 2008). The constituent at 21.4 min showed the molecular ion m/z 426, and at 23.6 min
showed m/z 468, suggesting an additional acetate moiety in the structure. The constituent at 22.9 showed incomplete
fragmentation, and a molecular ion was absent. The NMR data confirmed the pentacyclic triterpenes of the a- and b-amyrin
class by the large triplets at 5.13 and 5.18 ppm characteristic for olefinic proton in C12 of this class, respectively (Aragão et al.,
1990; Furukawa et al., 2002). The presence of carbinolic methine and acetyl moiety were found at the same value as described
for the lupeol characterization. These data emphasize the constitution of a- and b-amyrin, acetylated and not acetylated, in
the chloroform extract obtained from waxes of Mandevilla leaves.
The hexane extracts from the M. guanabarica samples showed a total relative abundance of n-alkanes of 20–28%; for
M. moricandiana, the relative abundance of n-alkanes reached 73%. For the triterpenes, this proportion was reversed: the
relative abundance of these compounds reached total values of 55–63% for M. guanabarica, whereas for M. moricandiana, the
relative abundance reached only 14%. The total relative abundance between n-alkanes and triterpenoids in the chloroform
extract showed a content of 10% n-alkanes for M. guanabarica samples, and for M. moricandiana this abundance reached 52%.
The total relative abundance of triterpenes was 75–82% for the M. guanabarica samples, and only 13% for M. moricandiana.
In studies with plant species of the Brazilian cerrado and caatinga, Oliveira and Salatino (2000) and Oliveira et al. (2003)
found that species with high proportions of n-alkanes C27 to C33 (above 70%) in the epicuticular waxes, had lower water
permeability, whereas leaves containing waxes with higher proportions of triterpenes were more permeable to water. This
information suggests that the leaves of M. moricandiana are less permeable to water than those of M. guanabarica, in plants
from all three sites.
Another difference in chemical characteristics between these species is the lupeol content. The concentration of this
triterpene was over 50% in all chloroform extracts from the M. guanabarica samples, and only 9% in the M. moricandiana
Table 2
Retention time and percentage of relative abundance of major compounds (>5%) obtained from the hexane (HEX) and chloroform (CHL) extracts of the
species collected in their respective locations, determined by CG-MS.
Retention
time (minutes)
16.6
18.3
20.6
21.4
22.0
22.9
23.6
Compound
Nonacosane (C29)
Hentriacontane (C31)
Tritriacontane (C33)
a- and b-amyrin
Lupeol and lupeol acetate
Pentacyclic triterpene
a- and b-amyrin acetate
Relative abundance (%) of compounds from foliar waxes of Mandevilla in hexane (HEX)
and chloroform (CHL) extract
M. guanabarica
Grumari
M. guanabarica
Jurubatiba
M. guanabarica
Maricá
M. moricandiana
Jurubatiba
HEX
CHL
HEX
CHL
HEX
CHL
HEX
CHL
6.5
15.6
6.0
–
6.4
13.8
28.8
2.0
4.8
2.0
14.4
61.4
2.3
4.3
6.2
11.0
3.0
–
6.0
14.0
42.1
3.2
5.3
1.0
9.7
54.8
4.4
12.5
6.6
13.5
5.4
–
5.1
10.6
47.8
2.0
6.2
2.5
8.8
50.5
3.1
12.1
14.1
30.6
29.9
–
10.0
–
4.2
11.8
18.8
22.0
0.6
9.1
–
4.0
202
S.Z. Cordeiro et al. / Biochemical Systematics and Ecology 39 (2011) 198–202
sample (Table 2). The presence of triterpenoids in plant waxes has been described, with quantities ranging from a trace to
>50% of the total wax mixture (van Maarseveen and Jetter, 2009).
The chemical profiles of M. guanabarica were qualitatively and quantitatively constant, regardless of the origin of the
plants. The chemical profiles of the waxes of M. guanabarica and M. moricandiana, showed qualitative similarities, but differed
in their quantitative constitution. The waxes of these species were chemically distinguishable regardless of possible climate
differences, nutritional conditions, and water availability, suggesting that this feature can be used as a taxonomic marker.
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; taxonomists Prof. Dr. Jorge Fontella
Pereira of the Museu Nacional (UFRJ) and Marcelo Fraga Castilhiori and Inaldo do Espírito Santo of the Herbarium Bradeanum
for species identification; Prof. Dr. Ricardo Machado Kuster from the Núcleo de Pesquisas de Produtos Naturais (UFRJ) for
reading the manuscript; and the Universidade Federal do Estado do Rio de Janeiro (UNIRIO) for providing transport to the
collection areas.
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