n-alkanes from Paepalanthus Mart. species (Eriocaulaceae)

n-alcanos de espécies de Paepalanthus Mart. (Eriocaulaceae)

n-alkanes from Paepalanthus Mart. species (Eriocaulaceae)

n-alcanos de espécies de Paepalanthus Mart. (Eriocaulaceae)

Lourdes Campaner dos Santos^I,1; Anne L. Dokkedal^II; Miriam Sannomiya^I; Maria Carla Piza Soares^I; Wagner Vilegas^I

^IUniversidade Estadual Paulista, Departamento de Química Orgânica, Instituto de Química, C. Postal 355, CEP 14800-900, Araraquara, SP, Brasil (FAPESP, CNPq)

^IIUniversidade Estadual Paulista, Faculdade de Ciências, Departamento de Ciências Biológicas, C. Postal 473, CEP 17033-360, Bauru, SP, Brasil (FUNDUNESP)

ABSTRACT

This work presents the study of nonpolar compounds from plants belonging to the genus Paepalanthus Mart. (Eriocaulaceae). Long-chain linear aliphatic hydrocarbons were identified by GC-FID and GC-MS. The results indicate that Paepalanthus subg. Platycaulon species present a very homogenous profile, with carbon chains of n-alkanes ranging from C₂₅ to C₃₁, most samples presenting higher frequencies of C₂₇ and C₂₉ homologues. Paepalanthus subg. Paepalocephalus species may be distinguished from one another by the distribution of main n-alkanes. P. macrocephalus, subsect. Aphorocaulon species, presents alkanes with odd-carbon numbers and P. denudatus and P. polyanthus, Actinocephalus species, present alkanes with quite distinctive profiles, with many shorter chains and a high frequency of even-carbon number, especially P. polyanthus. The results obtained indicate that the distribution of alkanes can be a useful taxonomic character, as do polar compounds like flavonoid glycosides.

Key words:Paepalanthus, n-alkanes, GC-FID, GC-MS, chemotaxonomy

RESUMO

Este trabalho apresenta o estudo de substâncias apolares obtidas a partir de plantas pertencentes ao gênero Paepalanthus Mart. (Eriocaulaceae). Hidrocarbonetos alifáticos de cadeias longas lineares foram identificados por CG-DIC e CG-EM. Os resultados indicam que as espécies de Paepalanthus subg. Platycaulon apresentam perfil homogêneo, com cadeias carbônicas de n-alcanos variando de C₂₅ a C_31, com a maioria das amostras apresentando freqüências maiores dos homólogos C₂₇ e C_29. As espécies do subgênero Paepalocephalus podem ser diferenciadas pela distribuição dos n-alcanos principais. P. macrocephalus, uma espécie da subseção Aphorocaulon, apresenta perfil com alcanos de cadeia ímpar, enquanto P. denudatus e P. polyanthus, espécies da seção Actinocephalus, apresentam perfil bem distinto, com grande número de cadeias mais curtas e alta freqüência de cadeias com número par de carbonos, especialmente P. polyanthus. Os resultados obtidos indicam que a distribuição de nalcanos pode ser útil como caráter taxonômico, assim como as substâncias mais polares, como os flavonóides glicosilados.

Palavras-chave:Paepalanthus, n-alcanos, GC-FID, GC-MS, quimiotaxonomia

Introduction

Eriocaulaceae comprises around 1,200 species in 10 genera (Giulietti et al. 2000). It is a natural group of herbaceous monocotyledons, characterized by small flowers densely arranged in capitula. Despite the low number of genera when compared to other plant families, the Eriocaulaceae encompasses many infrageneric levels. Paepalanthus is the largest genus of this family, comprising about 500 species (Giulietti & Hensold 1990) and it is subdivided in many subgenera, sections and subsections (Sano 2004). Although the huge amount of botanical work on this group, it is still difficult to clearly define these levels (Giulietti et al. 1995; Sano 2004). On the other hand, little is known about the chemical composition of the plants from this family. Previous reports (Andrade et al. 1999; Vilegas et al. 1999a; Vilegas et al. 1999b; Dokkedal et al. 2004) have shown that polar compounds like flavonoids glycosides can be usefull as a taxonomic character.

Among plant secondary metabolites, alkanes from epicuticular waxes have acquired a very consolidated condition as indicators of taxonomic relations between plant groups. Considerable interest has been shown in the systematic distribution of such compounds throughout the plant kingdom. Attention has been directed towards the possibility of using their distribution as a means of establishing a taxonomic system based on chemical characteristics (Eglinton et al. 1962a; Eglinton et al. 1962b; Eglinton & Hamilton 1963; Herbin & Robins 1968; Manners & Davis 1984; Gneco et al. 1988; Salatino et al. 1988; Salatino et al. 1989). However, inconsistencies of the alkanes of plant waxes as taxonomic markers have been pointed out; some authors have observed that the alkane distribution could be strongly affected by several factors, among them the age of the plant organ (Wilkinson & Kasperbauer 1972; Stocker & Wanner 1975; Nordby & Nagy 1977). Others (e.g. Smith & Martin-Smith 1978) concluded that no chemotaxonomic relationship could be derived from the composition of n-alkanes as the intraspecific variation was greater than the interspecific variations. Gradually, it seems that alkanes have regained the chemotaxonomists confidence (Sorensen et al. 1978; Faboya et al. 1980; Cowlishaw et al. 1983; Broschat & Bogan 1986). Salatino et al. (1991) have analysed a variable number of individuals of 12 species of Velloziaceae. Three degrees of plasticity of alkane profiles were recognized, depending on the species considered. They found that for most taxa, alkanes may be taxonomically useful at the species level if some precautions are taken. Skorupa et al. (1998) found that foliar epicuticular hydrocarbon patterns of 11 species of Pilocarpus represent useful evidences for its taxonomy at the interspecific, specific e infraspecific hierarchic levels. Merino et al. (1999) observed that alkane patterns are taxonomically valuable to explain Lupinus species relationships.

In the present study we have undertaken the investigation of nonpolar extracts by GC-FID and GC-MS from capitula of 11 additional species of Paepalanthus, distributed in two subgenera as follows: P. subg Platycaulon (P. bromelioides, P. latipes, P. planifolius, P. speciosus, P. vellozioides) and P. subg. Paepalocephalus (P. macrocephalus, P. denudatus, P. hilairei, P. polyanthus, P. ramosus and P. robustus).

Material and methods

The plants were collected in February 1995 in Serra do Cipó, Minas Gerais state, Brazil and were identified by Prof. Dr. Paulo Takeo Sano and by Prof. Dr. Ana Maria Giulietti, of the Instituto de Biociências - USP, where the exsicatas were kept: P. denudatus Koern. (CFSC 13853); P. hilairei Koern. (CFSC 13843); P. polyanthus (Bong.) Kunth (CFSC 13849); P. ramosus (Wilkstr.) Kunth (3210 HUEFS, SPF); P. robustus Silv. (CFSC 13840) belonging to P. sect. Actinocephalus and P. macrocephalus Ruhl. (CFSC 13847) of P. subsect. Aphorocaulon (all species of P. subg. Paepalocephalus); P. bromelioides Silv (CFSC 13856); P. latipes Silv. (CFSC 13846); P. planifolius (Bong.) Koern. (CFSC 13848); P. speciosus Gardner (CFSC 13851); P. vellozioides Ruhl. (CFSC 13842), of P. subg. Platycaulon.

Capitula of each plant (1g) were extracted with 10 mL of hexane by maceration for one week. The extracts were concentrated at 40 ºC in a rotary evaporator and the final solutions were evaporated under a gentle nitrogen flow until almost dry, and diluted to 200 µL with hexane. The extracts were transferred onto a silica gel Sep-Pak cartridge (690 mg 8 µm), which was previously conditioned with 5 mL of hexane and sequentially eluted with 1.5 mL of hexane. Fractions from each plant were evaporated under a stream of nitrogen until almost dry. The residues were dissolved in 100 µL of hexane and then analyzed by GC-FID with standards of n-alkanes (C₂₀, C₂₆ and C₃₂ - Aldrich Chemie) and GC-MS. Gas chromatography (GC) analyses were performed using a Varian 3380 gas chromatograph equipped with a fused silica CBP-5 capillary column (25m×0.33 mm i.d.; film thickness 0.5 m) and a flame ionization detector (FID). Hydrogen was used as the carrier gas (60 Kpa), and the injection split ratio was 1:30. The injection temperature was 250 ºC; the column temperature was held at 100 ºC for 2 min, then increased to 280 ºC at 10 ºC/min, and this temperature was held for 15 min; the detector temperature was 280 ºC. Samples of 1 µL were injected using a 10 µL Hamilton syringe. High resolution (HR) chromatography-mass spectrometry (GC-MS) analyses were performed using a Hewlett Packard (HP) 5970 MSD, with electron impact ionization (70eV), coupled to an HP 5890 GC. The column was a 25 m× 0.25 mm i.d. HP-1 (cross-linked methyl silicon; 0.3 µm film thickness). Samples of 1 µL were injected using the split mode (split ratio 1:30), with the injector and the interface both maintained at 280ºC. The temperature program used was the same as described above. Hydrogen was used as carrier gas (100 Kpa). The MS scan range was 50-500 a.m.u. Data were processed on an HP 7946/HP 9000-300 CPU.

The calibration curve was constructed injecting standard hydrocarbons C₂₀, C₂₆, C₃₂. The calibration curve graph was constructed using log Retention Time vs Carbon atom number. The first fractions of each hexane extract from each Paepalanthus species were then analyzed by HRGC-FID under the same condition as that of the hydrocarbons standard aforementioned to obtain the chromatograms.

The first fractions of each hydrocarbon standard were injected into the GC-MS equipment using the same conditions as described above. The identification of the compounds was based on interpretation of the fragmentograms and retention index calculations.

Results and discussion

The chromatograms of the fractions analyzed by GC-FID and GC-MS show a typical profile of well-resolved peaks separated by 14 a.m.u (CH₂) corresponding to long-chain aliphatic hydrocarbons. The molecular ion is weak in every case. Major peaks were identified as being C₂₅H₅₂, C₂₇H₅₆, C₂₉H₆₀ and C₃₁H₆₄, respectively. Figures 1 and 2 show the nalkanes profile of each plant based on the intensity of the peaks obtained from GC-FID analyses.

The results indicate that Paepalanthus subg. Platycaulon (Fig. 1) species present a very homogenous profile, with carbon chains of the nalkanes ranging from C₂₅ to C₃₁, most samples presenting higher frequencies of C₂₇ and C₂₉ homologues. In all species C₂₇ is the main alkane, excluding P. latipes, where C₂₉ is the main one. These results agree with data of previous reports using polar compounds like flavonoid glycosides (Vilegas et al. 1999a; Vilegas et al. 1999b) and reinforce the homogeneity of these taxa, since subg. Platycaulon is characterized by naphthopyranone derivatives and 7-methoxy flavonol derivatives. On the other hand, in the subg. Paepalocephalus species (Fig. 2), although C₂₇-C₂₉ homologues present higher frequencies in all samples, they may be distinguished from one another by the distribution of n-alkanes, taking into account the main alkanes quoting the main one outside and the second one inside parentheses: P. macrocephalus - C₂₁, C₂₇ and C₂₉ (no real predominance of either alkane); P. hilairei - C₂₇ (C₂₉); P. ramosus - C₂₉ (C₂₇); P. robustus - C₂₉ (C₂₇); P. denudatus - C₂₇, C₂₈; P. polyanthus - C₂₇ (C₂₅, C₂₉). We also can see that P. macrocephalus (subsect. Aphorocaulon species) presents just alkanes with odd-carbon numbers and P. denudatus and P. polyanthus (Actinocephalus species) present a quite distinctive profile, with many shorter chains and a high frequency of alkanes with even-carbon numbers, especially P. polyanthus. These results are different from those described by Salatino et al. (1988), who detected C₂₇ as the main homologue in all species of P. subg. Paepalocephalus. These data are in agreement with the cladistic analysis (Giulietti et al. 2000) where P. subg. Platycaulon form a clade, which is sister to P. subsect. Aphorocaulon and P. sect. Actinocephalus (both belonging to P. subg. Paepalocephalus). The results obtained indicate that the distribution of alkanes can be useful as a taxonomic character, although a more detailed inventory of alkanes profiles of species of Paepalanthus are needed, based on wide sampling for each species.

Aknowledgments

We thank the financial support of FAPESP, CNPq and FUNDUNESP.

Received: February 09, 2004. Accepted: March 04, 2005

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Publication Dates

• Publication in this collection
  15 Mar 2006
• Date of issue
  Dec 2005

History

• Received
  09 Feb 2004
• Accepted
  04 Mar 2005