Volatile Organic Compounds of Marine Sponge Petrosia ficiformis from the Adriatic Sea

: Volatile organic compounds (VOCs) of Petrosia ficiformis were investigated for the first time. The VOCs from fresh and air -dried sponge were obtained by hydrodistillation and headspace-solid phase microextraction and they were analysed by coupled gas chromatography-mass spectrometry. Aliphatic compounds with oc tan- 3 - one (up to 24.02 %), oct- 1 -en- 3 - ol (up to 8.65 % ) and heptadecane (up to 39.37 %) were the most abundant in the fresh sponge along with benzaldehyde (up to 18.59 % ) and diisobutyl phthalate (up to 8.44 % ). Higher percentage of N , N -dimethylmethanamin e (up to 19.08 %) was found in dried sample headspace and the loss of octan- 3 -one and benzaldehyde and increase of benzyl alcohol (up to 18.90 % ) were noted. The great difference among the fresh and dried sponge VOCs obtained by hydrodistillation was notic ed for fatty acids and derivatives abundance and 1H - indole increased (up to 6.00 % ) in the dried sponge. Both methods enabled obtaining more complete VOCs profile and drying signific antly changed their composition . -ol, octan- 3 -one, benzyl alcohol, benzaldehyde, diisobutyl phthalate, N , N - dimethylmethanamine, dimethyl trisulphide, 1H -indole .


INTRODUCTION
etrosia is one of 4 genera of Petrosiidae family belonging to the order Haplosclerida, which is known among sponges as the most prolific source of secondary metabolites. [1] Petrosia genus includes 122 species belonging to two subgenera (Petrosia and Strongylophora) according to the Word Register of Marine Species (WoRMS). A recent review paper compared secondary metabolites isolated from the sponges of the genus Petrosia. [2] It was reported that associated microorganisms can account for up to 60 % of the fresh weight of marine sponges and it is believed that these sponge-associated microorganisms (such as bacteria, fungi, cyanobacteria, and unicellular algae) may be involved in the biosynthesis of natural products. [3] Petrosia ficiformis (Poiret, 1789; Haplosclerida, Petrosiidae) is a marine sponge found across the Mediterranean and in the Eastern Atlantic. [4] The mixtures of high-molecular weight polyacetylenes with 46-55 carbons were isolated from P. ficiformis. [5] Five additional polyacetylenes up to 52 carbons, isolated from P. ficiformis from the Mediterranean Sea, were also reported. [6] Polyacetylenes with two terminal 1-yn-3-ol-4-ene moieties and 46 total carbons isolated from Mediterranean P. ficiformis were identified and evaluated for their biological activity. It was found that they inhibit sea urchin egg development and act as a potent toxin against Artemia salina. [7,8] Petroformynes, isopetroformynes, and various oxidized or isomerized analogues were reported in P. ficiformis and most of these analogues exhibited lethality against brine shrimp. [7][8][9][10] Sterols have rarely been isolated from Petrosia sponges in temperate regions, but P. ficiformis contains sterol compounds with cyclopropane in the branch at C-17. [2] Petrosterol (26,27-cycloaplysterol), a steroid with a cyclopropane ring at C-25 and C-26, was isolated from P. ficiformis collected in the Bay of Naples and later ficisterol (23-ethyl-24-methyl-27-norcholesta-5,25dien-3β-ol) was found as the minor component. [11][12][13] The relative abundance of phospholipids classes varies with sponge species, but amino phospholipids, especially phosphatidylethanolamine and phosphatidylcholine were found in P. ficiformis. [14] Its phospholipids also included numerous branched fatty acids (e.g. (Z,Z)-25-methylhexa-

Headspace Solid-Phase Microextraction (HS-SPME)
HS-SPME was performed with PAL Auto Sampler System (PAL, RSI 85, CTC Analytics AG, Switzerland) using SPME fibres with PDMS/DVB (polydimethylsiloxane/divinylbenzene) and DVB/CAR/PDMS (divinylbenzene/carboxene/ polydimethylsiloxane) coatings. Both fibres were obtained from Agilent Technologies (Santa Clara, CA, USA). Prior to the use, the fibres were conditioned according to the manufacturer's instructions. 1 g of fresh or dry sponge sample was placed into 20 mL glass vials and hermetically sealed with a screw cap containing polytetrafluoroethylene (PTFE)/silicone septum. The samples were equilibrated for 15 min at 60 °C and then extracted for 45 min. The injector temperature was set to 250 °C and thermal desorption was carried out for 6 min directly to the GC column. HS-SPME was performed in triplicate.

Hydrodistillation (HD)
Modified Clevenger apparatus with pentane (Kemika, Zagreb, Croatia) and diethyl ether (Kemika, Zagreb, Croatia) solvent trap (v/v ratio 1:2) was used for distillation. 100 g of fresh sample and 30 g of air-dried sample were used separately. HD was carried out for 2 hours. After HD, the solvent trap layer was carefully separated using a glass pipette, concentrated by the slow flow of nitrogen up to 0.05 mL and later used for GC-MS analysis.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of VOCs
GC-MS analysis was carried out on Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 8890A equipped with mass selective detector model 5977E. Separation of the compounds was achieved on an HP-5MS column (Agilent Technologies, Santa Clara, CA, USA) 30 m x 0.25 mm containing a non-polar stationary phase (5 % diphenyl / 95 % dimethylpolysiloxane) and 0.25 μm film thickness. The following operating conditions for the gas chromatograph were used: 250 °C injector temperature; 300 °C detector temperature; column temperature program: 2 min isothermally at 70 ° C, then temperature gradient from 70 °C to 200 °C at 3 °C/min and further retention for 15 min.

No.
Compound RI Area (%) ± SD* Pyridine 753 Phenol Octanal  Figure 2). A higher percentage of heptadecane in the dry sample can be attributed to the presence of cyanobacteria living symbiotically with sponges. It has been discovered that the biosynthetic pathway from cyanobacteria consists of the acyl-acyl carrier protein reductase and the aldehyde decarbonylase, which together convert the intermediates of fatty acid metabolism into alkanes and alkenes. [17] Several other saturated hydrocarbons such as hexadecane and pentadecane were identified in smaller proportions in almost all samples ( Table 1).
The second most dominant group of compounds were benzene derivatives (Figure 1). In HS-FrPF higher presence of benzaldehyde was found compared to HS-DrPF (6.9 times, f1; 2.0 times, f2) ( Figure 2). As already explained in the previously published research [18][19][20] the loss of benzaldehyde during air drying could be due to its high volatility. On the other hand, benzyl alcohol percentage increased in the dry samples (9.2 (f1) -18.9 (f2) times). Phenylpropane derivatives are derived from phenylalanine with the side-chain shortened by two carbon atoms which can take place β-oxidatively or non-oxidatively. [21] The compounds containing nitrogen showed similar abundance when analysed with f1 (8.30 %, HS-FrPF; 7.94 %, HS-DrPF), but when analysed with f2 they were 15.3 times higher in HS-DrPF (1.50 %, HS-FrPF; 22.90 %, HS-DrPF) (Figure 1). The volatile amine, N,N-dimethylmethanamine (trimethylamine) has been frequently used as a freshness parameter of marine organisms. Trimethylamine is formed from trimethylamine oxide (TMAO) as the result of the action of bacteria that cause spoilage, [22] which may explain the higher percentage of this amine in dry samples (2.1 (f1) -19.1 (f2) times). The aromatic heterocyclic compound pyridine was found in all samples and its percentage decreased 1.0 (f2) -1.7 (f2) times after air-drying. Pyridine derivatives are often part of biomolecules in marine sponges such as alkaloids. [23] Among terpenes, p-cymene, eucalyptol, fenchone, linalool, β-thujone and β-citral were detected in a low percentage. Other compounds such as norisoprenoids, organohalogen compounds and fatty acid derivatives were detected with minor abundance in the total headspace composition.

Composition of the VOCs Obtained by Hydrodistillation
Analysis of the VOCs in the hydrodistillate resulted in 88.53 % in FrPF (HD-FrPF) and 77.02 % in DrPF (HD-DrPF) of the total identified compounds ( Table 2).
In both samples, the group of aliphatic compounds predominated with 44.99 % in HD-FrPF and 36.67 % in HD-DrPF (Figure 3). Oct-1-en-3-ol, oxylipin known as global metabolome that induces the defence of marine invertebrates and algae, [15] had the greatest abundance in HD-FrPF. It decreased 30.1 times after drying probably because of its high volatility (Figure 4). Nine more oxylipins were detected and identified ( Table 2). Heptadecane, as the second most abundant aliphatic compound in HD-FrPF, was present with lower abundance than in the headspace of the samples extracted by HS-SPME (Figure 4).
In HD-FrPF 15.06 % of the total identified compounds belonged to the group of fatty acids and derivatives ( Figure 3). In HD-DrPF, the portion of this group decreased 41.1 times. Two hexadecanoic acid esters, methyl (Z)-hexadec-7enoate (3.77 %) and ethyl (E)-hexadec-9-enoate (3.66 %), were identified in HD-FrPF with higher abundance, but were not detected in HD-DrPF (Figure 4). The reason for fatty acid esters decrement after the drying may be oxidation reactions and lipid decomposition that can occurred during the drying.