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Leaf volatiles and secretory cells ofAlpinia zerumbet (Pers.) Burtt et Smith(Zingiberaceae)C.P. Victório a b , R. do Carmo de O. Arruda c , C.A.S. Riehl d &C.L.S. Lage ea Instituto de Biofísica Carlos Chagas Filho , Universidade Federaldo Rio de Janeiro , Rio de Janeiro, Brazilb Colegiado de Ciências Biológicas e da Saúde , UniversidadeEstadual da Zona Oeste , Rio de Janeiro, Brazilc Departamento de Botânica , Universidade Federal do Estado doRio de Janeiro , Rio de Janeiro, Brazild Departamento de Química Orgânica , Instituto de Química,Universidade Federal do Rio de Janeiro , Rio de Janeiro, Brazile Instituto Nacional da Propriedade Industrial (INPI) , Rio deJaneiro, BrazilPublished online: 31 May 2011.

To cite this article: C.P. Victório , R. do Carmo de O. Arruda , C.A.S. Riehl & C.L.S.Lage (2011) Leaf volatiles and secretory cells of Alpinia zerumbet (Pers.) Burtt et Smith(Zingiberaceae), Natural Product Research: Formerly Natural Product Letters, 25:10, 939-948, DOI:10.1080/14786419.2010.514575

To link to this article: http://dx.doi.org/10.1080/14786419.2010.514575

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Natural Product ResearchVol. 25, No. 10, June 2011, 939–948

Leaf volatiles and secretory cells of Alpinia zerumbet (Pers.)Burtt et Smith (Zingiberaceae)

C.P. Victorioab*, R. do Carmo de O. Arrudac, C.A.S. Riehld and C.L.S. Lagee

aInstituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,Rio de Janeiro, Brazil; bColegiado de Ciencias Biologicas e da Saude, Universidade Estadualda Zona Oeste, Rio de Janeiro, Brazil; cDepartamento de Botanica, Universidade Federal doEstado do Rio de Janeiro, Rio de Janeiro, Brazil; dDepartamento de Quımica Organica,Instituto de Quımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;eInstituto Nacional da Propriedade Industrial (INPI), Rio de Janeiro, Brazil

(Received 6 March 2010; final version received 8 July 2010)

Plant leaves are commonly used in folk medicine and food industry. Theirvolatile composition is an important determinant in such applications.However, to properly assess the quality of volatiles, proper analytic toolsmust be utilised. Accordingly, the static headspace technique was used toevaluate the main volatiles emitted from in vitro-grown Alpinia zerumbetplants cultured with indole-3-acetic acid, thidiazuron, benzyladenine orkinetin, under standard physical conditions, as compared to those of field-grown donor plants. Although the leaf aroma of the donor plants wasfound to be a complex mixture, mainly consisting of sabinene, � and�-terpinene, 1,8-cineole and caryophyllene, volatile analyses from most ofthe in vitro samples only revealed the presence of sabinene andcaryophyllene. Many alkanes were found in the aromas after treatingplantlets with cytokinins. Histochemical analysis of leaf sections was alsocarried out. Secretory cells found in the epidermis and mesophyll showeda strong positive reaction to lipophilic compounds using Oil red and Nileblue reagents. These findings demonstrated how in vitro conditions mayalter the quality of volatiles in micropropagation systems, while leafanatomy analysis revealed a large quantity of oil cells in the mesophyll asa constant feature responsible for the production of volatile compoundsin both donor and in vitro-grown plants.

Keywords: volatile compounds; headspace; micropropagation; leafhistochemistry; plant growth regulators

1. Introduction

Plant leaves are commonly used in folk medicine, cosmetics and food industry.Particularly, Alpinia zerumbet (Pers.) Burtt et Smith. (Zingiberaceae), popularlyknown as ‘colonia’ and shell ginger, has remarkable worldwide importance based onthe essential oils found in all of its parts (Kress, Liu, Newman, & Li, 2005; Victorio,Leitao, & Lage, 2010). Both aroma and volatile composition are important

*Corresponding author. Emails: crispv@biof.ufrj.br; cris.pvictor@gmail.com

ISSN 1478–6419 print/ISSN 1478–6427 online

� 2011 Taylor & Francis

DOI: 10.1080/14786419.2010.514575

http://www.informaworld.com

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in commercial and medicinal applications and have significant economic andconsumer impact in several industries (Marsili, 2002; Rohloff, 2002). Specifically,volatile composition has applications in health and biosciences, the environment andthe food industry, as well as the identification of aromas (Marsili, 2002). More to thepurpose of this study, the development of an efficient micropropagation protocol ofaromatic and medicinal plants has considerable potential as an alternative for theoverproduction of secondary metabolites (Affonso, Bizzo, Lage, & Sato, 2009; Silvaet al., 2005; Victorio, Kuster, & Lage, 2009a). With regard to volatile terpenoids,several applications in tissue cultures have shown changes in their production andindicated a way to optimise them. Furthermore, in vitro plant propagation allows forthe standardisation of microenvironmental conditions and selected superior geno-types, since the production of secondary metabolites is widely influenced by heredity,plant ontogeny and external factors.

However, in order to evaluate the quality of volatiles against that of field-growndonor plants, it is necessary to develop tools capable of analysing and characterisingvolatile composition in a laboratory setting using in vitro culture media.To accomplish this, we employed headspace techniques which use a very simplifiedprocedure to isolate volatiles not otherwise obtained by distillation methods(Rohloff, 2002). Specifically, to analyse our plant material, samples were heated,and the resulting vapour was then injected into a gas chromatograph (Tholl et al.,2006). The advantage of headspace techniques lies in their ability to (1) analysesolvent-free volatiles, (2) reduce the quantity of plant material and (3) avoidalteration in the plant material that normally occurs in other extractive techniques,eliminating, at the same time, the distillation and fractionation steps (Huie, 2002;Stashenko, Jaramillo, & Martınez, 2004). It should also be noted that biosynthesis ofvolatile terpenoids in plants is compartmentalised and involves mevalonic acid andmethylerythritol phosphate pathways that occur in the cytosol and chloroplast ofsome plant cells and tissues. Consequently, we also employed light microscopy andhistochemical staining to observe the leaf tissues responsible for the production andaccumulation of terpenoids. This study carried out two mutually inclusive objectives:(1) evaluate the set of changes in aroma quality and leaf structure of A. zerumbetcultured in a microenvironment under the effect of growth regulators and (2) identifythe location of volatiles in leaf secretory cells by histochemical staining.

2. Results and discussion

Data of in vitro development under different growth regulators were compared withthose obtained for the control medium. The regenerative frequency was 100% for alltested media. The morphogenic aspects of plantlets can be seen in Figure S1 (onlineonly). Thidiazuron (TDZ) was the best medium to induce new shoots and leaves inplantlets, but harmful in elongating the shoots and roots. However, no significantdifference was observed between plantlets cultured on this media and the Murashigeand Skoog (1962; hereafter MS) control. The use of kinetin (KIN) induced a slightincrease in the number of leaves compared with MS control. The lowest fresh and dryleaf biomass of plantlets was cultured in TDZ and showed significant differences whencompared to control plants, although such differences were compensated by themaximum percentage of leaves and shoots produced by their plantlets. Plantlets

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cultured in control medium presented the highest fresh and dry leaf weight. Comparedto control medium, plantlets exposed to KIN-, BA- and IAA-supplemented (BA,benzyladenine; IAA, indole-3-acetic acid) media resulted in similarity of dry leaf massproduction; however, plantlets from MS control and IAA media accumulatedsignificantly more water in the in vitro development process.

Morpho-anatomical analysis using light microscopy made it possible to elucidatethe leaf anatomy of A. zerumbet grown in vitro and indicated the distribution ofvolatile terpenoids content in their tissues (Figure S2 – online only).

Leaf anatomy of plantlets from the in vitro environment bore striking differenceswhen compared with donor plants. The epidermis consists of long and large cells andthin walls, without silica bodies in the upper vascular bundles of epidermal cells. Thehypodermis was verified only in the midrib region, with cells similar to thosedescribed for donor leaves, but with thin walls. Below the epidermis, outside themidrib region, palisade cells are strongly photosynthetic and form one layer withshort and long cells. Spongy parenchyma consists of two or three cell layers, withpronounced reduction in leaf thickness. A reduction in vascular tissue developmentwas observed in plantlets of A. zerumbet. The presence of oil cells marked by denseand yellow content could already be observed in the mesophyll from leaves of2-month-old plantlets. Within 4 months of in vitro culture, oil cells presentedchloroplasts with dense cytoplasm, and evident nucleus was sometimes duplicated.In contrast to the oil cells of donor plants, it is interesting to note that oil cells inplantlets presented a higher number of chloroplasts. Moreover, a greater quantity ofsecretory cells was also observed. In donor plants, however, secretory cells weredispersed throughout the mesophyll. Oil cells from donor plants did not showchloroplasts, but numerous oil idioblasts containing yellow and brilliant lipophilicmaterial were observed (Figure S2 – online only).

Histochemical results indicate that the secretory material amassed in thesecretory cells of A. zerumbet is lipophilic. Among tabular cells of the epidermis,some trapezoidal cells with lipophilic compounds were revealed by Nile blue stainingthat reacted with carboxyl groups of acid lipids. The use of Oil red also showed apronounced content of lipophilic compounds located in secretory cells of themesophyll of 4-month-old plantlets. The use of formic acid to dilute Oil red reagentgave evidence of drop oils caused by this low polarity. Since Sudan IV reagentspecifically detects lipophilic compounds in the liquid phase and only accented dropoils in mesophyll, Nile blue and Oil red turned out to be the most efficient testsshowing lipophilic compounds in A. zerumbet cells (Figure S2 – online only).

Volatile contents in A. zerumbet are reported for the first time using in vitrocultures and the static headspace (SHS) technique based on the equilibrium betweenplant volatiles and the surrounding atmosphere. A total of 13 different compoundswere isolated by SHS from the leaves of donor plants. Specifically, leaf oil obtainedby hydrodistillation (HD) and analysed chromatographically using SHS showed thepresence of sabinene, �-terpinene, linalool and caryophyllene. The same leaf oil afterdirect injection in GC/MS and GC/FID (FID, flame ionisation detector) showeda high amount of sabinene, �-terpinene, 1,8-cineole and terpinen-4-ol (Victorio,Riehl, & Lage, 2009b). SHS also detected the predominance of monoterpenes indonor leaves, and leaf oil aroma presented terpinolene and �–bisabolene, which werenot verified through direct injection of leaf oil in GC. The aroma of donor leaves wascharacterised by the presence of sabinene and 1,8-cineole, with a predominance

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of �-terpinene. For most in vitro treatments, leaf aroma uniformly presentedsabinene and caryophyllene together with its oxide, except MS (control) and IAA 2.Plantlets from KIN and BA treatments revealed a high percentage of alkanes amongtheir constituents. Plantlets obtained from in vitro cultures produced a similarproportion between monoterpenes and sesquiterpenes, with the exception of IAA 2and TDZ 2 treatments in which about 40% and 22% of sesquiterpenes were isolatedagainst 20% and 11% of monoterpenes, respectively. By comparing chromato-graphic profiles, no significant changes in the composition of volatiles were achievedamong plantlets treated with the same growth regulator and collected from differentculture glasses, which guarantees the reproducibility of the protocol. Terpinen-4-ol,one of the main volatile compounds of A. zerumbet (Victorio et al., 2009b), was notdetected on the leaves of either donor plants or in vitro-grown plants by the SHStechnique. This result tells us that this technique may lack accuracy in detecting somemonoterpenes or that A. zerumbet plants under in vitro conditions may producea lower concentration of some terpenoids (Table 1). The absence of terpinen-4-ol wasalso verified in leaf oils of A. zerumbet, as analysed by HD when collected duringpast years in Rio de Janeiro (Victorio et al., 2009b).

As described above, the tissue culture technique resulted in the reproduction ofplants under controlled growth conditions. Specifically, the effects of auxins andcytokinins on the in vitro cultivation of A. zerumbet plants were evaluated for theirinfluence on aroma quality. Such evaluation of in vitro development makes itpossible to verify the capacity of the cultures to produce healthy plantlets withadequate biomass and, consequently, secondary metabolites. Increases in the numberof shoots and leaves from cytokinin effects are in agreement with properties of thisclass of growth regulators in stimulating adventitious shoot production. However,multiple shoots formed in shorter plantlets, as observed in media containing TDZ,makes the individual isolation of nodal segments too difficult, which is not desiredfor micropropagation. The lower dry weight (DW) of plantlets from the TDZtreatment compared with other growth regulator supplemented media indicates thata greater biomass requirement is necessary when TDZ is used.

Important insights into the efficacy of micropropagation may be gained from ananalysis of leaf anatomy. In this regard, the results of our study showed that theshort size of palisade cells of the cultured plantlets can be explained by the low lightlevels to which they are exposed in room cultures and the high dependence on culturemedium to obtain their nutrients. Consequently, the low light intensity caused adrastic reduction in leaf thickness, as also reported by Hazarika (2006). According toDickison (2000), the light level that leaves receive during their development is arelevant factor affecting mature leaf structure. In relation to leaf thickness, thereduction of hypodermis indicated morpho-anatomical changes of leaves adapting tothe high humidity found in the microenvironment of in vitro cultures. As notedpreviously, the hypodermis is a specialised water reservoir tissue commonly found inplants that adapt to xeromorphic environmental conditions. The presence ofhypodermis was first described in A. zerumbet by Tomlinson (1956). In addition, thereduced development of vascular tissues and mesophyll makes plantlets highlysusceptible to ex vitro acclimatisation. Although palisade thickness is reduced inplantlets, their palisade cells compensate for this by presenting a large number ofchloroplasts. It may be inferred from the absence of chloroplasts in oil cells of donorplants that they are lost in adult plants from developmental ontogeny stage.

942 C.P. Victorio et al.

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Table

1.Themain

leafaromacompoundsofdonorand4-m

onth-old

invitroplants

ofA.zerumbet

culturedunder

differentgrowth

regulators,

asobtained

bySHS–GC/M

S.

Donorplants

Invitroplants

RI calculated

Volatile

Odoura

Leafoil

Donorplants

MS

IAA

2(m

gL�1)

BA

2KIN

2TDZ2

809

Ethylacetate

––

DD

DD

DD

860

Hexenal

––

DD

DD

DD

Monoterpenes

(%)

81

69

29

20

18

16

11

979

Sabinene

Spicyblack

pepper

DD

DD

DD

D1015

�-Terpinene

Weaklemon

DD

––

––

–1023

�-C

ymene

Weakcitrus

–D

––

––

–1026

D-lim

onene

Weakly

lemon-like

–D

––

––

–1034

1,8

Cineole

Camphoraceous

DD

D–

DD

–1043

Trans-�-ocymene

––

D–

––

––

1063

�-Terpinene

Citrus-like,

herbal

DD

––

––

–1085

Terpinolene

Sweet-piney

DD

––

––

–1095

Linalool

Fresh,floral

DD

––

––

–

Sesquiterpenes

(%)

19

15

29

40

18

16

22

1399

Caryophyllene

Spicy-piney

DD

DD

DD

D1497

Pentadecaneb

––

––

DD

–1562

Caryophylleneoxide

Floral

DD

––

DD

D1598

Hexadecaneb

––

––

–D

DD

1697

Heptadecaneb

––

––

–D

D–

1798

Octadecaneb

––

––

–D

D–

1999

Eicosaneb

––

––

––

D–

Alkanes

(%)

––

––

36

42

11

Notes:D,detectedcompounds.

aOlfactoricdata

obtained

from

theliterature

ofGCtechniqueofidentified

volatilecompounds(C

hyauet

al.,2007;Seo

&Baek,2005;Wise,

Urbansky,Helms,Coates,&

Croteau,2002);

bAlkanes.

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Despite significant leaf plasticity, the greater number of oil cells found in plants

grown in vitro guarantee the production of volatile terpenoids. Positive reaction to

Nile blue and Oil red confirmed that oil cells are the main site for the production and

accumulation of monoterpenes and sesquiterpenes in A. zerumbet.Production of terpenoids partly occurs in chloroplasts and is strongly associated

with photosynthesis that depends on the efficiency of irradiance absorbed by

photosynthetic pigments (Chappell, 2002). Additionally, volatile composition results

from a set of factors that affect plant development, including genotype, microen-

vironment and culture protocol. As observed in this study, the status of plants

cultured in vitro commonly alters secondary metabolites as a result of microenvi-

ronment pressure. By using the SHS technique, it was possible to detect the genuine

odour perceived from the fresh plant of A. zerumbet and evaluate its aromatic

quality. Linalool, limonene, �-terpinene and terpinolene were the main compounds

found in leaf aroma from the donor plants of A. zerumbet, but they were not

observed in plantlet samples. To account for this, it is suggested that under in vitro

conditions, limonene production (1) and its conversion to �-terpinene (2) and/or

terpinolene (3) have been inhibited (Figure S3 – online only) (Wise, Savage,

Katahira, & Croteau, 1998). Phatak and Heble (2002) suggested a reduction in

enzyme activity of monoterpene biosynthesis depending on in vitro developmental

stages of Mentha arvensis, which may also provide an explanation for the absence of

some monoterpenes in A. zerumbet plantlets observed in this study. The failure of

plantlet aroma quality to improve when compared to donor plants, as well as the

absence of changes in the quality of volatile composition in A. zerumbet, may result

from low light intensity conditions where plantlets were developed, despite the great

quantity of chloroplasts observed by light microscopy. Evidence of the large number

of hydrocarbons in leaf volatiles when KIN and BA were applied indicates the

influence of cytokinins in the biosynthesis of hydrocarbons. Similar results related to

the production of alkanes were verified in the tissue cultures of Eucalyptus

camaldulensis that presented more than 50% of alkanes in oil (Giamakis, Kretsi,

Chinou, & Spyropoulos, 2001).Most studies report on the in vitro production of volatiles for eudicotyledons and

few studies on using monocotyledons. However, several species from the

Zingiberaceae present important economic potential because of their fragrance and

flavour. Attractive aspects of A. zerumbet use are associated with its aroma. Our

results demonstrated that the main volatiles of A. zerumbet aroma consisted of a

combination of spicy black pepper, in addition to camphoraceous and spicy-piney

odours also found in donor plants (Chyau et al., 2007; Seo & Baek, 2005; Wise,

Urbansky, Helms, Coates, & Croteau, 2002).In conclusion, the SHS technique coupled with GC/MS made it possible to

evaluate the main volatiles emitted from in vitro-grown plants cultured with growth

regulators under standard physical conditions. Analysis of volatile composition

revealed a qualitative reduction in the production and emission of terpenoids by

plantlets and a high percentage of alkanes resulting from culture in media containing

cytokinins. These findings demonstrate how in vitro conditions may alter the quality

of volatiles in micropropagation systems. At the same time, leaf anatomy analysis

revealed the large quantity of oil cells in the mesophyll as a constant feature

responsible for the production of terpenoid compounds in in vitro-grown plants.

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3. Experimental

3.1. Plant material

Leaves from donor plants of A. zerumbet were collected in February 2008, in theGarden of Nucleo de Pesquisas de Produtos Naturais, which is located in Rio deJaneiro, Brazil. A voucher specimen was deposited at the Herbarium of Rio deJaneiro Botanical Garden under accession no. RB 433485. These plants were used asthe source of plant material to initiate in vitro cultures.

3.2. Tissue cultures

Cultures were established according to Victorio (2008). Plantlets were subculturedinto glasses (16� 8 cm) containing Murashige and Skoog (1962; MS) culturemedium, added with 3% sucrose, vitamins and myo-inositol. Five treatments weretested: control (MS), MS plus 2mgL�1 IAA 2, MS plus 2mgL�1 TDZ 2, MS plus2mgL�1 BA 2 and MS plus 2mgL�1 (KIN 2). Cultures were maintained at 25� 2�Cwith a photoperiod of 16 h under fluorescent tubs (Duramax Universal, GeneralElectric�) at light intensity of 30Wm�2. Four-month-old plantlets were evaluated todetermine the percentage of shoot per explant, leaves per shoot, fresh weight (FW)and biomass DW of leaves. DW was determined after plantlets were dried at 40�C inan oven until constant weight was attained. Data obtained were subjected to analysesof variance (ANOVA), and averages were compared by the Tukey’s test at 5%significance, using the Statistica� software for Windows, version 5.0.

3.3. Leaf anatomy and histochemistry

Leaf samples from donor and in vitro plants of A. zerumbet which grew for 2 and4 months in MS were fixed in formalin, acetic acid, ethanol (FAA; 70%) for 48 h,dehydrated using a tertiary butanol series according to Johansen (1940) (70%, 85%,95%, 100%, 1 h 30min, respectively), then infiltrated in paraffin and sectionedtransversely in rotary microtome at a thickness of 15–18 mm. The sections werestained with 1% fucsin in 50% ethanol and 1% alcian blue and sealed with syntheticresin. The following histochemical tests were performed using fresh leaf bladessectioned transversally in a table microtome: Sudan black B (Pearse, 1980) Sudan IIIand IV (Johansen, 1940) for total lipophilic compounds; Nile blue test for acid andneutral lipophilic compounds (Cain, 1947); and Oil red O for lipophilic compounds(Clark, 1981). Sudan tests were carried out by placing leaf sections in 70% ethanolfor 1min, staining in 0.03% filtered solutions of each Sudan type in 70% ethanol for30min at 40�C in water bath and washing with 70% ethanol. Solutions of Nile bluewere dissolved in water, and leaf sections were treated for 30min. Controlprocedures for Sudan and Nile blue tests were carried out by placing the leafsection in a mixture of methanol, chloroform, water and chloridric acid (66 : 33 : 4 : 1)for 3 h at room temperature. Oil red was resuspended in 90% formic acid, andsections were immersed in solution and stained for 10min at room temperature.Control slides were prepared. Tests with hydrochloric acid were used to detectcalcium oxalate crystals (Jensen, 1962). Silica crystals were detected by the reactionof phenol in heated clove oil (1 : 1) (Johansen, 1940). All samples were rinsed with

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distilled water and mounted in glycerin on slides with coverslips. Observations werecarried out and captured using light microscopy (Olympus� BX-41).

3.4. Isolation procedures of volatiles

3.4.1. Hydrodistillation

Fresh leaves from donor plants ofA. zerumbet (250 g) were cut, subjected toHD for 3 husing a Clevenger-type apparatus, and analysed according to Victorio et al. (2009b).

3.4.2. SHS analysis

For each extraction, 0.3mL of leaf oil obtained by HD and 160mg of fresh leavesfrom donor and plantlets were placed in a 20mL vial, and then each vial was sealedand subjected to SHS. The vial was incubated for orbital agitation at 60�C, shakenfor 15min (equilibrium time) at 300 rpm with shaking cycle at 4 s/on and 3 s/off. Theprocedure was carried out on a Varian 1200L mass selective detector connected to agas chromatograph (Varian CP 3800). GC/MS (TIC) analyses were performed usinga Zebron ZB-5HT column (30m� 0.25mm, 0.10 mm) (Phenomenex�) under thefollowing conditions: split ratio 1:10; carrier gas, helium at 1mLmin�1; injectortemperature, 270�C; column temperature, 30–210�C at 3�Cmin�1; and mass spectra,70 eV. The injection consisted of 500mL of vapour phase taken by a heated syringe(60�C) from a vial and injected directly and automatically into the GC. All analyseswere conducted four times, either in duplicate per culture glass of each treatment orin duplicate to donor samples. Compounds were identified by comparison ofretention indices (RI) calculated for all volatile contents using a homologous series ofn-alkanes (C10–C20) recorded under the same operating conditions, comparing MSdata and their GC with those of standard samples found in a computer library searchof the National Institute of Standards and Technology (NIST), as well as theliterature (Adams, 2001).

Supplementary material

Figures S1–S3 relating to this paper are available online.

Acknowledgements

In memoriam of our dear laboratory technician Mr Valter Pereira Rodrigues. The authorsthank CAPES/PROEX for the PhD fellowship of the first author. We acknowledge thetechnical assistance of Mrs Priscila Maia Pereira (IQ/UFRJ), and Mr David Martin, whorevised the English text.

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