Sesquiterpenes from the essential oil of Laurencia dendroidea (Ceramiales, Rhodophyta): isolation, biological activities and distribution among seaweeds

Sesquiterpenes from the essential oil of Laurencia dendroidea (Ceramiales, Rhodophyta): isolation, biological activities and distribution among seaweeds

Vanessa Gressler^*,I; Érika M. Stein^II; Fabiane Dörr^I; Mutue T. Fujii^III; Pio Colepicolo^I; Ernani Pinto^I

^IDepartamento de Análises Clínicas e Toxicológicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580, Bloco 17, 05508-900 São Paulo-SP, Brazil

^IIDepartamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, 05422-970 São Paulo-SP, Brazil

^IIINúcleo de Pesquisas em Ficologia, Instituto de Botânica, Av. Miguel Estéfano, 3687, 04301-902 São Paulo-SP, Brazil

ABSTRACT

Two known sesquiterpenes (1R*,2S*,3R*,5S*,8S*,9R*)-2,3,5,9-tetramethyltricyclo[6.3.0.0^1,5]undecan-2-ol and (1S*,2S*,3S*,5S*,8S*,9S*)-2,3,5,9-tetramethyltricyclo-[6.3.0.0^1,5]undecan-2-ol were isolated for the first time from the essential oil of the red seaweed Laurencia dendroidea collected in the Brazilian coast. These compounds were not active against eight bacteria strains and the yeast Candida albicans, but showed some antioxidant activity. Both compounds were also found in other seaweed species showing that they are not exclusive taxonomic markers to the genus Laurencia.

Keywords: Seaweeds, Laurencia dendroidea, sesquiterpenes, essential oil, antioxidant activity, antimicrobial activity

Introduction

Species of algae from the genus Laurencia J.V. Lamour. (Ceramiales, Rhodomelaceae) have been a subject of intensive research since an earlier study of marine natural products (Faulkner, 2000). The genus Laurencia is a rich source of secondary metabolites of diverse structures with biological activity (Rashid et al., 1995), which can vary significantly with species and location.

The volatile organic compounds which comprise the essential oil are molecules with low molecular weight, low to moderate hydrophilicity and high vapor pressure. Since these compounds can cross the membrane and be released in the atmosphere, they play an important role as chemical communications for algae in marine ecosystems (Fink, 2007; Gressler et al., 2009; Gressler et al., 2011).

A wide range of volatile secondary metabolites such as halogenated and sulfur compounds (Kladi et al., 2004; Karabay-Yavasoglu et al., 2007), hydrocarbons, alcohols, phenols, aldehydes, ketones, acids, esters, terpenes and other compounds (Gressler et al., 2009), are distributed among the seaweeds. However, few works reported the essential oil composition of seaweeds.

This study describe, for the first time, the isolation of two triquinane alcohols (1R*,2S*,3R*,5S*,8S*,9R*)-2,3,5,9-tetramethyltricyclo[6.3.0.0^1,5]undecan-2-ol (SPF-1), also known as 7-epi-silphiperfolan-6β-ol (1), and (1S*,2S*,3S*,5S*,8S*,9S*)-2,3,5,9-tetramethyl-tricyclo[6.3.0.0^1,5]undecan-2-ol (SPF-2), also known as silphiperfolan-7β-ol (2), from the essential oil of Laurencia dendroidea.

In addition, this paper describes a screening of these two compounds in a range of 21 species of seaweeds collected in different places and dates, and their antimicrobial and antioxidant properties.

Materials and Methods

Seaweed samples

The red macroalga Laurencia dendroidea was collected at Parati beach in Anchieta, Espírito Santo State, Brazil, in October, 2006 and identified by Dr. Mutue Toyota Fujii. An exsiccate (SP 399.947) is deposited on Herbário do Estado (SP) "Maria Eneyda P. K. Fidalgo" at Instituto de Botânica de São Paulo. Other 21 algae species distributed in 41 samples were used for analyses. Their description is shown in Table 1.

Distillation of the volatile components

Four hours of steam distillation of 200 g of fresh material was done in a Clevenger-type apparatus. The obtained distillate was removed from the apparatus with ethyl ether and the water residue was removed with sodium sulfate. The samples were concentrated by rota-evaporation and then diluted 100 times with ethyl acetate prior to analysis by GC/MS.

Isolation and identification of the compounds

The two major compounds of the essential oil were separated by preparative TLC (Silica Gel 60 F₂₅₄ 0.5 mm Merck) eluted twice with hexane:ethyl acetate (95:5). The compounds SPF-1 and SPF-2 were analyzed by GC/MS and uni- and bidimensional NMR.

Gas chromatography/mass spectrometry (GC/MS) analysis

The essential oil and the isolated compounds were analyzed in a gas chromatograph coupled to a mass spectrometer (GCMS-QP2010 Plus, Shimadzu, Japan). The chromatographic separation was achieved with a Rtx-5MS capillary column (30 m x 0.25 mm x 0.1 µm) and helium was used as carrier gas (with a flow rate of 1 mL.min^-1). Oven temperature was increased at a rate of 3 °C.min^-1 from 60 to 260 °C, and hold for 40 min. Injection and transfer line temperatures were 220 and 240 °C. The detection was performed in the full scan mode, with a mass range of 50-650 m/z. Electron impact ionization was employed with collision energy of 70 eV, and the mass spectrometer ion source was maintained at 240 °C. The target compounds were identified by GC retention times and from their mass spectra. The relative concentration (%) was determined by the integration of all peaks obtained in the total ion chromatogram (TIC) mode. The proportion between the compounds SPF-1 and SPF-2 in different marine seaweed species was obtained using the extraction of specific fragments, characteristic of each compound (m/z 98 for SPF-1 and m/z 86 for SPF-2).

NMR spectra

¹H and ¹³C uni- and bi-dimensional NMR spectra were recorded using a DPX 500 (Bruker, Germany) spectrometer with CDCl₃ as solvent and TMS as internal standard.

HS-SPME extraction and analysis

The lyophilized algae material (50 mg) were kept in a 10 mL vial. After the equilibration time (15 min at 60 °C) the SPME fiber (PDMS, 100 µm) was exposed during 15 min. The volatile organic compounds composition of the species were analyzed by GC/MS at the same conditions described for the essential oil and isolated compounds.

Test microorganisms

In vitro antimicrobial studies were carried out against eight bacterial strains (Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Klebsiella pneumoniae ATCC 13883, Salmonella typhi ATCC 19430, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853), and one yeast strain (Candida albicans ATCC 10231).

Antimicrobial testing

The Minimal Inhibitory Concentration (MIC) was determined in 96-well culture plates by a micro-dilution method using a microorganism suspension with a density of 10⁵ CFU.mL^-1 in Mueller Hinton Broth (MHB) for bacteria, and Sabouraud Broth (SB) for yeast, as recommended by NCCLS (NCCLS M100-S15 and M38-A). The bacterial suspension was incubated for 24 h at 35±1 °C and the yeast suspension for 72 h at 35±1 °C. Proper blanks were assayed simultaneously with samples tested in triplicate. The concentrations tested were 500, 250, 125, 62.5, 31.25 and 16.125 µg.mL^-1.

Antioxidant testing

DPPH radical scavenging assay

Radical scavenging activity of L. dendoidea essential oil and isolated compounds against stable DPPH (2,2-diphenyl-2-picrylhydrazyl, Sigma-Aldrich Chemie, Steinheim, Germany) was determined spectrophotometrically according to Yen & Chan (1995) with some modifications. The absorbance decrease was measured at 517 nm on a UV-visible light spectrophotometer (Spectrum Power Wave_x 340, Bio-Tec Instruments, INC). Sample solutions were prepared by dissolving the essential oil or the isolated compounds in methanol. The solution of DPPH in methanol (1 mM) was prepared daily, before UV measurements. Ten µL of this solution were mixed with 100 µL sample solution of final concentrations of 500, 250, 100, 50 and 5 µg.mL^-1 in a 96-well plate. The samples were kept in the dark for 20 min at room temperature and then the decrease in absorption was measured. Absorption of blank sample containing the same amount of methanol and DPPH solution was prepared and measured daily. Vitamin E was used as a positive control. The experiment was carried out in triplicate. Radical scavenging activity was calculated by the following formula:

where Abs_DPPH is the absorption of blank sample (MeOH+DPPH) and Abs_sample is the absorption of tested solution+DPPH.

Chemiluminescence determination in the presence of HRP

This method is based on light emission and, for antioxidant determination, solutions of 1 mg.mL^-1 of the test sample in phosphate buffer 5% DMSO were mixed with H₂O₂, yielding a final concentration of 5 x 10^-5 mol.L^-1. Then luminol (5-amino-2,3-dihydrophthalazine-1,4-dione, in DMSO) was added to a final concentration of 1.13 x 10^-4 mol.L^-1. The 96-well plate was incubated for 3 min at 30 °C and then HRP at a final concentration of 0.2 IU.mL^-1 was added to initiate the chemiluminescence reaction (Krol et al., 1994). Phosphate buffer/5% DMSO was used as a blank for the maximum luminescence and N-acetyl-L-cysteine was used as the positive control. Chemiluminescence was measured for 15 min at 25 °C with a microplate reader (Tecan Infinite^®M200). Final concentrations of 500, 250, 100, 50 and 5 mg.mL^-1 of the solutions were analyzed and the experiment was carried out in triplicate. The antioxidant activity was calculated by the following formula:

where A_control is the area under the curve of the blank (phosphate buffer/5% DMSO+H₂O₂+luminol+HRP) during 15 min and A_sample is the area under the curve of the sample (sample+H₂O₂+luminol+HRP) during 15 min.

Results and Discussion

The extraction of the essential oil of L. dendroidea yielded 0.066% of fresh weight. The chromatogram of the essential oil obtained showed one major peak at 29.5 min (61.67%). Since this peak was not identified by the available libraries (NIST 08 and NIST 08S), the essential oil was submitted to TLC analysis and it was possible to see two strong spots, which were further isolated by preparative TLC. The two isolated compounds were analyzed by GC/MS and NMR techniques and both showed the same retention time (29.5 min) and a 1529 Kovats index.

The ¹H-NMR spectrum of the compound SPF-1 showed that all proton resonances are between δ 0.87 and 1.86 ppm. Only four signals were useful to structure determination (doublets at δ 0.87 and 0.97, singlets at δ 1.07 and 1.10) with integration equivalent for three hydrogen atoms. Similarly as SPF-1, in the ¹H-NMR spectrum of the compound SPF-2 all proton resonances were between δ 0.89 and 1.69 ppm. Four methyl resonances at δ 0.89 (d), 0.92 (d), 1.11 (s) and 1.17 (s) with integration equivalent for three hydrogen atoms were also seen.

Table 2 shows the ¹H and the ¹³C NMR data of both non-halogenated sesquiterpenes. A comparison between the values obtained in this study and literature data showed that the compounds are SPF-1 and SPF-2, previously isolated from Laurencia majuscula Saito & Womersley and L. dendroidea J. agardh (as L. scoparia) (Coll & Wright, 1989; Wright et al., 1990; Davyt et al., 2006). However, the ¹³C-NMR data published by Wright et al. (1990) have one incorrect signal-carbon number attribution. The authors indicated that the δ 27.8 ¹³C-NMR signal is the ethylene group bonded to C3 (δ 81.1 ppm) to SPF-1, but our HMBC-NMR correlation indicates that δ 27.8 ppm signal is bounded to C5. The ¹H-¹³C long range correlations are also given in Table 2.

Both triquinane alcohols smell camphoraceous and woody and have oil aspect.

To verify if the isolated compounds are molecules that could represent taxonomic markers, the presence of the mixture of these compounds was verified in 21 seaweed species which grow in Brazilian coast by HS-SPME-GC/MS analysis.

As previously informed, both sesquiterpenes have the same retention time (probably because of the similarity in their structure), but different mass spectra (Figure 1). To analyze the proportion of each compound in the peak of a crude sample, m/z 98 and m/z 86 were chosen as characteristic fragments to identify SPF-1 and SPF-2, respectively. The Table 3 shows the species tested and the relative amount of the mixture.

As can be seen in Table 3, both compounds SPF-1 and SPF-2 were observed in different seaweed species, in amounts ranging from 1.12±0.37 to 53.25± 0.21%.

Regarding the proportion of the fragments analyzed, most of the species that have the sesquiterpenes SPF-1 and SPF-2 in his composition showed higher percentage of the m/z 86 (in a proportion of 2:1), indicating that the compound SPF-2 is twofold higher. On the other hand, the proportion found between m/z 86 and m/z 98 was 1.5:1 for Palisada perforata (Bory) K.W. Nam (PP3).

Even though both fragments were seen in the Pterosiphonia pennata (C. Agardh) Sauvageau (PT) samples, it was not possible to obtain the total amount of SPF-1 and SPF-2 because there was another compound (with a different mass spectra) in the same retention time.

The sesquiterpenes evaluation also showed large occurrence in the Ceramiales group, which is known to have high potential of secondary metabolites production (Fenical & Norris 1975). In the Laurencia dendroidea, samples collected in different places at different dates showed that both compounds represented around 50% of composition (53.25±3.21 and 48.67±0.32% for LD3 and LD4, respectively). Among the Gigartinales, only O. secundiramea, a known producer of secondary metabolites with various activities (Paul et al., 1988), is capable to produce the referred compound. Sesquiterpenes were not found in Sargassum sp., Ulva sp. species (Heteroconthophyta and Chlorophyta group representatives, respectively). Green seaweeds produce mainly acyclic sesqui- and diterpenes while brown seaweeds are known by polyphenols, terpenoids, volatile hydrocarbons and sulfur compounds production (Pereira, 2002). The non occurrence of SPF-1 and SPF-2 might be an indication that these compounds are only observed in red algae.

For some species, like Gracilaria domingensis (Kützing) Sonder ex Dickie and Gracilaria birdiae Plastino & Oliveira, the presence of the sesquiterpenes was not expected, since no previous evidence of this metabolite class was found in the literature. The very small amount found in GB and GD2 might be explained by the sampling method used. HS-SPME integrates extraction and concentration of volatile compounds into a single step, leading to a high sensitivity (Gressler et al., 2009), not achieved with other extraction methods (like hydrodistillation and solvent extract). In Palisada perforata (Bory) K.W. Nam, previous called as Laurencia papillosa, the SPF-1 and SPF-2 production was seen in our study, which is consistent with other terpenes already indentified by Kamenarska et al. (2006).

The essential oil and the two isolated compounds (SPF-1 and SPF-2) were tested to verify their antimicrobial and antioxidant activities. No significant antimicrobial inhibition was observed to the microorganism in the concentration tested. The essential oil of L. dendoidea J. Agardh did not show good antioxidant activity (8.10% at 500 µg.mL^-1), however, the isolated compounds SPF-1 and SPF-2 showed higher potencies 27.5 and 30.3% at 500 µg.mL^-1, respectively when analyzed by the DPPH method, Figure 3(a). The antioxidant activity observed by the chemiluminescence method was 14.9, 34.6 and 20.8% at 500 µg.mL^-1 for the essential oil, SPF-1 and SPF-2, respectively, Figure 3(b).

Overall, DPPH assay was fairly consistent with the chemiluminescence assay. The small difference between the results could be consequence of the differential sample solubility in the reactions media (organic medium for DPPH assay and aqueous medium for chemiluminescence assay), difference in sensitivity among both methods (according to Hirayama et al., 1997, chemiluminescence method is more sensitive because of the high light emission produced by the H₂O₂/luminol/HRP system) and/or different mechanisms of radical scavenging.

Conclusions

Both tricyclic sesquiterpenes (SPF-1 and SPF-2) have never been reported in Brazilian seaweed species. Although they were observed in variable quantities in different seaweed samples, they showed to be very common between distinct species. However, more systematic studies (considering environmental physicochemical parameters) should be done to allow a better comprehension of SPF-1 and SPF-2 algae synthesis and seasonal distribution.

Based on the results obtained, it can be inferred that these compounds do not act as taxonomic markers to the genus Laurencia.

It was also seen that the isolated compounds were not active against the bacteria and yeast strains tested up to 500 µg.mL^-1. On the other hand, these compounds showed some antioxidant properties.

Acknowledgements

This work was supported by the Brazilian research funding agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Amparo a Pesquisa do Estado de São Paulo and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Ministério da Saúde, Ministério de Ciência e Tecnologia and CNPq-INCT-Redoxoma.

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Received 23 Dec 2010

Accepted 15 Jan 2011

Publication Dates

• Publication in this collection
  15 Apr 2011
• Date of issue
  Apr 2011

History

• Accepted
  15 Jan 2011
• Received
  23 Dec 2010