Chemical Diversity of Headspace and Volatile Oil Composition of Two Brown Algae (Taonia atomaria and Padina pavonica) from the Adriatic Sea

Two selected brown algae (Taonia atomaria and Padina pavonica from the family Dictyotaceae, order Dictyotales) growing in the same area (island Vis, central Adriatic Sea) were collected at the same time. Their phytochemical composition of the headspace volatile organic compounds (HS-VOCs; first time report) was determined by headspace solid-phase microextraction (HS-SPME). Hydrodistillation was applied for the isolation of their volatile oils (first report on T. atomaria volatile oil). The isolates were analyzed by gas chromatography (GC-FID) and mass spectrometry (GC-MS). The headspace and oil composition of T. atomaria were quite similar (containing germacrene D, epi-bicyclosesquiphellandrene, β-cubebene and gleenol as the major compounds). However, P. pavonica headspace and oil composition differed significantly (dimethyl sulfide, octan-1-ol and octanal dominated in the headspace, while the oil contained mainly higher aliphatic alcohols, trans-phytol and pachydictol A). Performed research contributes to the knowledge of the algae chemical biodiversity and reports an array of different compounds (mainly sesquiterpenes, diterpenes and aliphatic compounds); many of them were identified in both algae for the first time. Identified VOCs with distinctive chemical structures could be useful for taxonomic studies of related algae.


Introduction
Marine secondary metabolites possess outstanding structural and functional diversity related to their different metabolic pathways [1]. While the volatile organic compounds (VOCs) of terrestrial plants have attracted attention since antiquity, the VOCs of marine algae have been much less investigated. Therefore, the target of the present research are VOCs of two brown seaweeds from family Dictyotaceae, order Dictyotales: Taonia atomaria (Woodward) J. Agardh, 1848 and Padina pavonica (Linnaeus) Thivy, 1960.
T. atomaria (family Dictyotaceae, order Dictyotales, class Phaeophyceae) is a brown seaweed widespread in the Mediterranean Sea. Taondiol and atomaric acid, cyclised meroditerpenoids, were isolated from this alga collected in Canary Islands [2][3][4]. The chemical investigation of T. atomaria from composition of their volatile oils isolated by hydrodistillation (first report on T. atomaria volatile oil); (c) compare the results of the corresponding headspace and volatile oil chemical composition; (d) discuss the obtained results in comparison to the literature data, particularly regarding the extracts composition or the algal volatile oils from different geographic areas; and (e) indicate possible biosynthetic formation pathways of the major identified compounds according to the literature data.

Results and Discussion
In order to investigate the chemical diversity of the headspace and volatile oil composition from two brown algae of the family Dictyotaceae, order Dictyotales collected from the Adriatic Sea from the same area (island Vis, Croatia), two complementary methods were used: headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) followed by gas chromatography and mass spectrometry (GC-FID; GC-MS) analysis.
In order to avoid the changes that could occur in VOCs from the native samples after a long time of collection or drying, both algae were investigated as fresh samples. Two fibres of different polarity were used for HS-SPME (Polydimethylsiloxane/Divinylbenzene (PDMS/DVB) and Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS)) to obtain more complete chemical profiles. The results pointed out striking differences between the chemical profiles for the headspace and volatile oil of the same algae (depending on the compounds molecular mass and volatility). It should be taken into consideration that the headspace composition does not reflect the composition percentage of the sample (only of the headspace) and hydrodistillation is not an adequate method for the isolation of water-soluble, high-molecular compounds and less volatile compounds such as diterpenes.
The major VOCs found in the headspace and oil are known to exhibit different biological activities. Germacrene D is involved in plant-insect interaction, acting as a pheromone on receptor neurones [25] and is also an important deterrent and insecticidal agent against different parasites such as mosquitos, aphids and ticks [26,27]. Germacrene D could be considered responsible for the cytotoxic activity of Kundmannia sicula (L.) D.C. essential oil being the main compound present in the oil (81.2%) [28]. This is in agreement with the results reported by Setzer et al. [29] where germacrene D resulted active against human breast adenocarcinoma (MDA-MB 231 and MCF-7), human ductal carcinoma (Hs 578T) and human hepatocellular carcinoma (Hep G2). Epi-bicyclosesquiphellandrene could be connected with antidermatophytic activity [30]. Gleenol exhibited the following biological activities: termiticidal, antihelminitic and growth regulation effects on plant seeds [31].

Possible Biosynthetic Origin of the Major Identified VOCs
Sesquiterpenes, dominant in the T. atomaria headspace and volatile oil, are C 15 -compounds containing the assembly of three isoprenoid units. The large number of sesquiterpenoid carbon skeletons arises from the common precursor, farnesyl pyrophosphate (FPP), by various modes of cyclizations usually followed by skeletal rearrangement (Figure 1).
Selinane and cadinane are two main types of sesquiterpenes reported in brown algae [36]. According to the currently accepted hypotheses, in addition to farnesyl pyrophosphate (FPP), neryl pyrophosphate (NPP) may be a precursor in sesquiterpenes formation (Figure 1). It can be assumed [37] that in the cyclisation process (after pyrophosphate (PP) loss) germacrenyl cations A and B are formed, and after hydride migration, cations C and D are formed (Figure 1). For the biosynthesis of cadinene-type sesquiterpenes, two alternative pathways were suggested [38]. The primarily formed cation A may be transformed to cation C and after deprotonated to germacrene D (1b). The subsequent change from cisstructure to trans-1b is crucial for the formation of cadinane type sesquiterpenes (with Z double bond in the ring) followed by protonation and rearrangement to cation D. As an alternative, NPP may serve as a substrate for cadinene-type sesquiterpene biosynthesis. In this case, the cyclization to the cadinanes would proceed via cations B and D. Moreover, it can be assumed that germacrene D and germacrenyl cation D are the biosynthetic precursors of the major isolated sesquiterpenes from T. atomaria. Major sesquiterpenes of this alga with cadinane skeleton that could be derived from germacrene D were (Figure 1): cadina-1(6),4-diene, δ-cadinene, cadina-3,5-diene, 4-epi-bicyclosesquiphellandrene; ylangene; cubebol, β-cubebene, α-cubebene, zonarene, and gleenol. Bicyclogermacrene is probably derived from germacrenyl cation C.
Dimethyl sulfide (DMS), a major compound of the P. padina headspace, results from an enzymatic decomposition of dimethyl-β-propiothetin [33], a metabolite of methionine that is fairly widespread in marine plants. Formed dimethylsulfoniopropionate (DMSP), a tertiary sulfonium compound, is the precursor of DMS. Recently, the algal enzyme responsible for the formation of DMS from DMSP was found and characterised in alga Emiliania huxleyi [39].
In general, the overall mechanism of enzymatic lipid degradation is identical in terrestrial plants [40] and algae [41]. The enzyme cascade is initiated by activated phospholipase, followed by lipoxygenase and hydroperoxide lyase. However, the particular enzymes are highly species-and sometimes even strain-specific [41], and this can explain the large biodiversity of lipid degradation VOCs (e.g., carbonyl compounds, alcohols, and hydrocarbons). Marine algae contain unsaturated fatty acids, and they can produce C 18 , C 20 and C 22 fatty acid hydroperoxides. Following the general concept of lipid peroxidation, and subsequent oxidative cleavage of the carbon skeleton, the biosynthesis of C 8 -hydrocarbons from P. pavonica could start from the polyunsaturated fatty acid substrate that could be activated [42] either by 9-lipoxygenase or by 12-lipoxygenase producing 9-or 12-hydroperoxides that further cleave oxidatively to produce C 8 -compounds. Octanal could originate from ω9 mono-unsaturated fatty acids (MUFAs) and also from ω6 poly-unsaturated fatty acids (PUFAs) such as linoleic acid [43].

Materials and Methods
The samples of two brown algae Taonia

Materials and Methods
The samples of two brown algae Taonia  The samples were separately collected and placed in air-tight plastic bags containing surrounding seawater and were immediately transported to the laboratory. The samples were kept in the dark at 4 • C, and the extractions were performed within 48 h of the collection. Before headspace solid-phase microextraction (HS-SPME), each sample was separately cut into small pieces and the excess seawater was removed by placing it between the filter paper layers for 2 min (the seawater was not removed completely) as was done in previous research [32].

Headspace Solid-Phase Microextraction (HS-SPME)
Headspace solid-phase microextraction (HS-SPME) was performed with a manual SPME holder using two fibres covered with PDMS/DVB (Polydimethylsiloxane/Divinylbenzene) or DVB/CAR/PDMS (Divinylbenzene/Carboxen/Polydimethylsiloxane) obtained from Supelco Co. (Bellefonte, PA, USA). The fibres were conditioned prior to the extraction according to the instructions by Supelco Co. For HS-SPME, prepared samples (1 g) were placed separately in 5 mL glass vials and hermetically sealed with PTFE/silicone septa. The vials were maintained in a water bath at 60 • C during equilibration (15 min) and HS-SPME (45 min). After the sampling, the SPME fibre was withdrawn into the needle, removed from the vial, and inserted into the injector (250 • C) of GC-FID and GC-MS for 6 min where the extracted volatiles were thermally desorbed directly to the GC column. The procedure was similar as in previous paper [32]. HS-SPME was done in triplicate for each alga.

Hydrodistillation (HD)
Hydrodistillation was performed in a modified Clevenger apparatus for 2 h with the use of 1 mL of solvent trap (pentane:diethyl ether 1:2 v/v). The prepared samples (10 g; cut into small pieces) were used separately for the hydrodistillation. The volatile oil dissolved in the solvent trap was removed with a pipette, passed through the layer of MgSO 4 in a small glass funnel and carefully concentrated by the slow flow of nitrogen until the volume of 0.2 mL. The hydrodistillation for each sample was performed in triplicate. One microlitre was used for GC-FID and GC-MS analyses.

Gas Chromatography and Mass Spectrometry (GC-MS) Analyses
Gas chromatography and mass spectrometry (GC-MS) analyses were done on an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7890A equipped with a flame ionization detector (FID) and a HP-5MS capillary column (5% phenyl-methylpolysiloxane, Agilent J and W). The GC conditions were the same as described previously [32]. In brief, the oven temperature was set up isothermally at 70 • C for 2 min, then increased from 70-200 • C at 3 • C·min −1 , and held isothermally at 200 • C for 15 min; the carrier gas was helium (He 1.0 mL·min −1 ). The GC-MS analyses were done on an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7820A equipped with a mass selective detector (MSD) model 5977E (Agilent Technologies) and HP-5MS capillary column, under the same conditions as for the GC-FID analysis. The MSD (EI mode) was operated at 70 eV, and the mass range was 30-300 amu.
The identification of VOCs was based on the comparison of their retention indices (RI), determined relative to the retention times of n-alkanes (C 9 -C 25 ), with those reported in the literature (National Institute of Standards and Technology [44]) and their mass spectra with the spectra from Wiley 9 (Wiley, New York, NY, USA) and NIST 14 (D-Gaithersburg) mass spectral libraries. The percentage composition of the samples was computed from the GC peak areas using the normalization method (without correction factors). The average component percentages in Tables 1 and 2 were calculated from GC-FID and GC-MS analyses.

Conclusions
HS-SPME and hydrodistillation were adequate and complementary methods for the research of headspace and volatile oil composition of T. atomaria and P. pavonica. Although these two seaweed species belong to the same botanical family and order, and were collected from the same area at the same time, significant diversity in their VOCs composition was found. The headspace and oil composition of T. atomaria were quite similar (containing germacrene D, epi-bicyclosesquiphellandrene, β-cubebene and gleenol as the major compounds). However, the headspace and oil composition of P. pavonica differed significantly (dimethyl sulfide, octan-1-ol and octanal dominated in the headspace, while the oil contained mainly higher aliphatic alcohols, trans-phytol and pachdityol A). The current research contributed to the knowledge of algae chemical biodiversity since the obtained chemical profiles reveal an array of different compounds (mainly sesquiterpenes, diterpenes and aliphatic compounds); many of them were identified in both algae for the first time. Identified VOCs with distinctive chemical structures (among them biologically active compounds can be found) could be useful for algae taxonomic studies.