Phytochemical study of the headspace volatile organic compounds of fresh algae and seagrass from the Adriatic Sea (single point collection)

Performed phytochemical study contributes to the knowledge of volatile organic compounds (VOCs) of Halopteris filicina (Grateloup) Kützing, Dictyota dichotoma (Hudson) J. V. Lamouroux, Posidonia oceanica (L.) Delile and Flabellia petiolata (Turra) Nizamuddin from the Adriatic Sea (single point collection). VOCs were investigated by headspace solid-phase microextraction (HS-SPME) and analysed by gas chromatography and mass spectrometry (GC-MS/FID). H. filicina headspace contained dimethyl sulfide (DMS; 12.8%), C8-compounds (e.g. fucoserratene (I; 9.5%)), benzaldehyde (II; 8.7%), alkane C17, dictyopterene D and C (III, IV), tribromomethane (V), 1-iodopentane, others. F. petiolata headspace was characterized by DMS (22.2%), 6-methylhept-5-en-2-one (9.5%), C17 (9.1%), II (6.5%), compounds I-V. DMS (59.3%), C15 (14.5%), C17 (7.2%) and C19 (6.3%) dominated in P. oceanica headspace. Sesquiterpenes were found in D. dichotoma, predominantly germacrene D (28.3%) followed by other cadinenyl (abundant), muurolenyl and amorphenyl structures. Determined VOCs may be significant for chemosystematics and chemical communications in marine ecosystem.


Introduction
Volatile organic compounds (VOCs) are low-molecular compounds with low to moderate hydrophilicity that can not only be dissolved in water, but also dissipate into the gas phase at air-water interfaces. Prior to 1966, only one volatile substance was identified from wet, undecomposed seaweed and described in the literature as dimethyl sulfide (DMS). Afterwards, the number of identified marine plant VOCs has been constantly growing. In 1976, VOCs from a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and Flavours: Chemical Fingerprinting and Unlocking the Potential) and by the Croatian Ministry of Science and Education (MZO grant no. KK.01.1.1.01.0002 -The Scientific Center of Excellence for Marine Bioprospecting-BioProCro). We confirm that the field study did not involve endangered or protected species.
The samples of 2 brown algae (Halopteris filicina (Grateloup) Kützing, Dictyota dichotoma (Hudson) J. V. Lamouroux) and 1 green alga (Flabellia petiolata (Turra) Nizamuddin) as well as 1 seagrass (Posidonia oceanica (L.) Delile) were collected in the middle part of the Adriatic Sea, close to Mala Smokvica island, in July 2017 at the same geographic location (43 o 31'04.4"N, 15 o 56'32.6"E). Single point sample collection provided representative robust samples for first time chemical profiling. The samples were collected at 11 a.m. under sunny conditions-constantly illuminated, and area receive direct sunlight for >80% of the time from sunrise to sunset. During the samples collection there was no wind and the sea temperature was 22˚C. The algae were collected from depth of 0.5-3 m and the sea was collected from the same depth (directly into the plastic bag where collected algae were placed). The samples were separately collected and separately placed in air tight plastic bags containing seawater and were immediately transported to the laboratory. The samples were kept in dark at 4˚C until the extractions that were performed within 24 h after the collection. Before HS-SPME, each sample was taken out of the bag, cut into small pieces, and the excess of seawater was removed by placing it between filter paper layers for 2 min (the seawater was not removed completely).

Headspace solid-phase microextraction (HS-SPME)
The headspace extraction was performed with a manual SPME holder using polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre obtained from Supelco Co. (Bellefonte, PA, USA). The fibre was conditioned prior to the extraction according to the instructions by Supelco Co. For HS-SPME, previously 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 o C during equilibration (15 min) and HS-SPME (45 min). After sampling, the SPME fibre was withdrawn into the needle, removed from the vial, and inserted into the injector (250 o C) of the GC-FID and GC-MS for 6 min where the extracted volatiles were thermally desorbed directly to the GC column. HS-SPME was done in triplicate for each sample.

Gas chromatography and mass spectrometry (GC-FID and GC-MS) analyses
The GC-FID analyses were carried out with 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 similar to those described previously [21]. In brief, the oven temp. was programmed isothermal at 70 o C for 2 min, increasing from 70-200 o C at 3 o C min -1 , and held isothermally at 200 o C for 15 min; carrier gas was helium (He 1.0 mL min -1 ). The GC-MS analyses were performed using an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7820A equipped with a mass selective detector (MSD) model 5977E (Agilent Technologies) and a 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 [22] and their mass spectra with the spectra listed in 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 Table 1 were calculated from GC-FID and GC-MS analyses (triplicates).

Results and discussion
Headspace solid-phase microextraction (HS-SPME) with the fibre polydimethylsiloxane/ divinylbenzene (PDMS/DVB) was successfully applied first time to investigate VOCs from four fresh marine plants (Halopteris filicina (Grateloup) Kützing, Dictyota dichotoma (Hudson) J. V. Lamouroux, Posidonia oceanica (L.) Delile and Flabellia petiolata (Turra) Nizamuddin) that were collected from the Adriatic Sea from the same location. Single point sample collection provided representative robust samples for first time chemical profiling. Representative chemical profiles of their headspace composition were obtained without the application of heat that can lead to the artefacts (as in other methods for VOCs isolation e.g. hydrodistillation). The first insight into the results (Table 1) reveals striking differences among the obtained chemical profiles. As was expected, significant contrast with common VOCs released by terrestrial plants can also be immediately noticed. The results are further discussed in separate subtitles for each seaweed or seagrass and for possible biosynthetic origin of identified VOCs.

Halopteris filicina (Grateloup) Kützing headspace VOCs
Halopteris filicina (Grateloup) Kützing is a greenish-brown, fern-like seaweed that belongs to the family Stypocaulaceae and it has been usually found in the Mediterranean and warm seas. Preliminary phytochemical screening of its crude extracts revealed the presence of alkaloids, saponins, flavonoids and terpenes [23]. Free amino acids, amino sulfonic acids, sugars and sterols have been examined and quantitatively determined [24], but there is no report on its volatile constituents. Its methanolic extract showed inhibition against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus spp., Salmonella typhi, and Escherichia coli [23].

Flabellia petiolata (Turra) Nizamuddin headspace VOCs
Flabellia petiolata (Turra) Nizamuddin is a green alga that belongs to Udoteaceae family (Chlorophyta, Bryopsidales) commonly found in the Mediterranean basin [36]. Compared to many other green algae, F. petiolata appears to be a particularly interesting species, since antibacterial, antiviral, antimitotic, antifungal and cytotoxic activities of its raw extract have been observed [37]. Selected constituents from F. petiolata from Turkey were determined [38] such as: the average cellulose content (18.86±0.69%), the average crude protein content (22.45 ±0.62%) and the crude fat content (1.08±0.24%). There were no available data on its VOCs composition.
The main compound of P. oceanica headspace was DMS (59.3%; Table 1) that was expected since its precursor DMSP was previously identified in the leaves of this seagrass [43]. DMSP has been proposed to exhibit physiological roles (e.g. as an intracellular osmolyte and antioxidant), and also serves as a chemoattractant [26,44]. DMS (mediated by both bacterial and algal DMSP lyases) has been generated in oceans at remarkably high amounts (>107 tons per year) and is a key component of the ocean sulfur cycle and has a global role in atmosphere-ocean feedback processes [45,46]. Chlorophyceae, especially Ulva, Enteromorpha, and Codium, and red alga, Polysiphonia, are capable of producing large amounts of DMS in contrast to Phaeophyceae (brown algae) which produce little [47,48] that is in agreement with present research (Table 1). Namely, DMS percentage was the highest in P. oceanica, than in green alga (F. petiolata) and the lowest in brown algae (Cystoseira sp. and D. linearis). Besides higher alkanes pentadecane (14.5%) and heptadecane (7.2%) that were abundant in F. petiolata and Cystoseira spp. (Table 1), nonadecane was also found (6.3%).

Possible biosynthetic origin of identified VOCs
The formation of DMS [1] results from an enzymatic decomposition of dimethyl-β-propiothetin, a metabolite of methionine that is fairly widespread in marine plants. Formed dimethylsulfoniopropionate (DMSP), a tertiary sulfonium compound involved in osmoregulation in algae, is the precursor of DMS. Recently, the algal enzyme responsible for formation of DMS from DMSP has been identified and characterized in algae Emiliania huxleyi [26].
Marine macroalgae exhibit a high ability to fix halide ions and form a variety of halogenated secondary metabolites [1]. In present research only tribromomethane and 1-iodopentane were found. A halogenating enzyme, haloperoxidase [29] is considered to participate in their synthesis in the presence of halides and hydrogen peroxide. Among them bromoperoxidases were detected in seaweeds (e.g. from Corallina pilulifera or Ascophyllum nodosum). The enzyme produces CHBr 3 by its reaction with ketoacids, halide ions and hydrogen peroxide [51].
Both in terrestrial and aquatic ecosystems, VOCs of primary producers are usually dominated by lipid degradation products, and the overall mechanism for their enzymatic release is identical as in terrestrial plants [11] and algae [13]. The enzyme cascade is initiated by activated phospholipase, followed by lipoxygenase and hydroperoxide lyase, which leads to VOCs liberation. However, the particular enzymes are highly species-and sometimes even strainspecific [13] that can explain large biodiversity of volatile lipid degradation products (e.g. carbonyl compounds, alcohols, hydrocarbons). Marine algae contain C 20 , C 22 and C 18 unsaturated fatty acids, and they can produce both plant (C 18 ) and animal type (C 20 and C 22 ) fatty acid hydroperoxides. Short-chain aldehydes (e.g. C 6 , C 9 ) and middle-chain aldehydes (e.g. C 10 ) that were particularly present in H. filicina and F. petiolata are mainly formed from fatty acids (C 20 ) in marine algae (via hydroperoxides), whereas they are formed from C 18 fatty acids in higher plants [52,53]. For example, the formation of hexanal is proposed via linoleic acid cascade and arachidonic acid cascade through their hydroperoxides as intermediates by the lipoxygenase/fatty acid hydroperoxide lyase pathway. It could also be provided by oxidation from other polyunsaturated fatty acids, as well as heptanal [54]. Lower aliphatic alcohols may be formed by decomposition of secondary hydroperoxides of fatty acids by the reduction of the corresponding aldehydes [55]. Octanal and nonanal could originate from ω9 mono-unsaturated fatty acids (MUFAs) and also from ω6 PUFAs such as linoleic acid [55]. Following this general concept of lipid peroxidation, and subsequent oxidative cleavage of the carbon skeleton, the biosynthesis of C 11 -and C 8 -hydrocarbons could start from a single precursor (e.g. eicosapentaenoic acid). The polyunsaturated fatty acid substrate could be activated [56] either by 9-lipoxygenase or by 12-lipoxygenase, and resulting 9-or 12-hydroperoxides that cleave oxidatively to produce characteristic C 11 -and C 8 -hydrocarbons (e.g. fucoserratene). However, the origin of 3-methylbutanal is well known. It is obtained from amino acids, and more particularly from leucine, during Maillard reactions by Strecker degradation [16].
Benzenoid and phenylpropanoid volatile compounds, primarily derived from phenylalanine require shortening of the carbon skeleton side chain by a C 2 -unit, which can potentially occur via either the β-oxidative pathway or non-oxidatively [60].

Conclusions
Considering limited data available on the chemical composition of marine plants from the Adriatic Sea, the present research is contribution toward their better chemical characterization. Significant differences were found among the headspace VOCs from 3 seaweeds and 1 seagrass. High abundance of DMS was found in P. oceanica followed by F. petiolata and H. filicina indicating those plants as source of sulfur compounds in marine ecosystem. Their headspace contained individually variety of C 8 -compounds (e.g. fucoserratene), benzaldehyde, alkanes C 15 , C 17 and C 19 , dictyopterene D and C, others. Sesquiterpenes were found in D. dichotoma, predominantly germacrene D indicating similarity to terrestrial aromatic plants. Identified VOCs contain different types of organic compounds that may be significant for chemosystematics and ecology (chemical communications in marine ecosystems).