Detailed Chemical Prospecting of Volatile Organic Compounds Variations from Adriatic Macroalga Halopteris scoparia

The present study aimed to isolate volatile organic compounds (VOCs) from fresh (FrHSc) and air-dried (DrHSc) Halopteris scoparia (from the Adriatic Sea) by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) and to analyse them by gas chromatography and mass spectrometry (GC–MS). The impact of the season of growth (May–September) and air-drying on VOC composition was studied for the first time, and the obtained data were elaborated by principal component analysis (PCA). The most abundant headspace compounds were benzaldehyde, pentadecane (a chemical marker of brown macroalgae), and pentadec-1-ene. Benzaldehyde abundance decreased after air-drying while an increment of benzyl alcohol after drying was noticed. The percentage of pentadecane and heptadecane increased after drying, while pentadec-1-ene abundance decreased. Octan-1-ol decreased from May to September. In HD-FrHSc, terpenes were the most abundant in June, July, and August, while, in May and September, unsaturated aliphatic compounds were dominant. In HD-DrHSc terpenes, unsaturated and saturated aliphatic compounds dominated. (E)-Phytol was the most abundant compound in HD-FrHSc through all months except September. Its abundance increased from May to August. Two more diterpene alcohols (isopachydictyol A and cembra-4,7,11,15-tetraen-3-ol) and sesquiterpene alcohol gleenol were also detected in high abundance. Among aliphatic compounds, the dominant was pentadec-1-ene with its peak in September, while pentadecane was present with lower abundance. PCA (based on the dominant compound analyses) showed distinct separation of the fresh and dried samples. No correlation was found between compound abundance and temperature change. The results indicate great seasonal variability of isolated VOCs, as well among fresh and dried samples, which is important for further chemical biodiversity studies.

Nunes et al. [15] identified minor and major constituents in Halopteris scoparia harvested off the northern coast of the Island of Gran Canaria, Spain. The authors found 5.20 g/100 g dw (dry weight) of moisture, 57.20 g/100 g dw of total minerals, 5.54 g/100 g dw of proteins, 3.64 g/100 g dw lipids, and 29.86 g/100 g dw of total carbohydrates. As volatilome was more comparable. From 91.50% (HS-FrHSc, June) to 99.31% (HS-DrHSc, July) of VOCs were identified with f1 and from 89.97% (HS-DrHSc, May) to 100% (HS-DrHSc, September) of VOCs were identified with f2. Identified VOCs could be classified into six different groups: saturated aliphatic compounds, unsaturated aliphatic compounds, benzene derivatives, terpenes, C 13 -norisoprenoids (carotenoids degradation products), and others (Figures 1 and 2). The dominant VOCs were aliphatic compounds followed by benzene derivatives, except for HS-FrHSc in June and August extracted with f2, where benzene derivatives represented the most prevalent group of compounds (Figures 3 and 4).
Among terpenes, sesquiterpene germacrene D and diterpene phytane were dominant. Germacrene D was detected only in HS-FrHSc with the peak in May (5.44%, f1; 1.87%, f2). In our previous research, germacrene D was found in D. dichotoma headspace as one of the most abundant terpenes at the blooming inception in May [20]. Phytane was detected only in July with the greatest portion in HS-DrHSc (4.46%, f1; 4,19%, f2). The increment in its abundance after drying implies possible chlorophyll degradation. Total ion chromatograms (TICs) of fresh samples from May and September isolated by HS-SPME are presented in Figure 2 for fibre f1 and Figure 4 for fibre f2.

Statistical Analysis of the H. scoparia Headspace VOCs
The principal component analyses (PCA) were used to describe the variations among the dominant volatiles (>2%) of HS-FrHSc and HS-DrHSc in relation to the material preparation (fresh or dry), seasonal changes, and fibre. The results are shown in Figures 5 and 6.
The PCA analysis of the data obtained by f1 fibre is shown in Figure 2a,b. The first two PCs described 67.4% of the initial data variability. A correlation between certain groups of the compounds was observed (Figure 5a). Pentadecane, nonanal, β-ionone, and tridecanal showed the highest variable contribution to PC1 and factor-variable correlation, while hexan-1-ol and benzyl alcohol contributed to PC2.
The correlation plot and score plot of the dominant components from data obtained by f2 fibre are shown in Figure 5c,d. The first two PCs described 62.38% of the initial data variability. The abundance of benzaldehyde, (E,E)-octa-3,5-dien-2-one, pentadecane, and tridecanal contributed to PC1, while hexanal and hexanoic acid had the highest contribution to PC2. May samples (fibre f2) were separated in the bottom left part of the plot as a function of the highest octan-1-ol and 3-hydroxybutan-2-one content.
A clear separation between the fresh and dried samples was obtained for both fibres (Figure 5b,d). For f1, fresh samples were positioned on the left part of the multivariate space, while the scores of the dried samples were vertically distributed on the right side of the score plot. Interestingly, the sampling month showed no effect on the fresh samples, while, in dry samples, there were similarities between May and June, and between August and September. Similarly, a clear separation of the dry and fresh samples was observed for f2 fibre. As it can be seen in Figure 5d, the fresh sample harvested in May showed great variation compared to other months. The main reason was the high portion of aliphatic saturated compounds (53.29%), as well as terpenes (6.09%) and others (3.23%).
When the data for f1 and f2 were analysed together, the first two PCs described 53.3% of the initial data variability, but the score plot showed a clear separation between fresh and dried samples ( Figure 6). As a function of the higher content of benzaldehyde and octan-1-ol, as well as low content of pentadec-1-ene and pentadecane in the dry samples, the samples from May (both fibres) were segregated in the bottom part of the plot and September samples in the top part. Some separation between the sampling months was seen, but no correlation with temperature change over months was observed.
Diterpene alcohol (E)-phytol was the most abundant compound in HD-FrHSc through all months except September. Its abundance increased from May (11.35%) to August (47.03%) when it was in its highest bloom. Recent studies have shown its antioxidant potential [27], antimicrobial [28], anti-inflammatory [29], antitumour [30], and antidiabetic activities [27]. Its derivative, phytane, was detected in July (6.11%, HD-FrHSc; 2.43%, HD-DrHSc). Two more diterpene alcohols, isopachydictyol A and cembra-4,7,11,15-tetraen-3-ol, and one sesquiterpene alcohol, gleenol, were detected in high abundance. Both diterpene alcohols were the most abundant in May (9.70%, isopachydictyol A; 6.88%, cembra-4,7,11,15tetraen-3-ol) in the sample after air-drying and were detected in the lowest content in the fresh sample in September. They were detected as the major compounds in HD of D. dichotoma [20] and isolated from several species of Dictyota genus [31,32]. Both diterpenes have various biological activity [33,34]. Gleenol was detected in the highest portion in May in HD-FrHSc (6.26%) and its portion decreased each month (Table 3).   Among aliphatic compounds, the dominant was unsaturated pentadec-1-ene with its peak in September in both HD-FrHSc (26.46%) and HD-DrHSc (18.98%). Higher aliphatic unsaturated alcohol (Z,Z,Z)-octadeca-9,12,15-trien-1-ol was prevalent in HD-FrHSc in May (8.09%) and decreased each month. Higher aliphatic unsaturated aldehyde (Z)-octadec-9-enal was the most abundant in HD-FrHSc in August (8.51%). Both the alcohol and the aldehyde were less abundant in air-dried samples throughout all the months since lipid oxidative degradation probably occurred, which means they were oxidised and further degraded. Six carboxylic acids were detected and identified with hexanoic acid as the most abundant one particularly in HD-DrHSc in May (4.80%). Three C 13 -norisoprenoids were detected, among which β-ionone was the most abundant. It was higher in air-dried samples, which was the result of carotenoid degradation during the process of air-drying.
Total ion chromatogram (TIC) of fresh samples from May and September obtained by HD are presented in Figure 8.

Statistical Analysis of the H. scoparia VOCs Obtained by Hydrodistillation
The PCA results for VOCs of fresh and air-dried H. scoparia obtained by hydrodistillation are shown in Figures 9 and 10.
In fresh samples, the first two PCs described 71.13% of the initial data variability. The correlation loadings of the first two PCs (Figure 9a) showed high correlations between germacrene D and (Z)-pentadec-6-en-1-ol and gleenol, between heptadec-1-ene and pentadec-1-ene, and between hexadecan-1-ol and hexadecanoic acid. Germacrene D, gleenol, (Z,Z,Z)-octadeca-9,12,15-trien-1-ol, and isopachydictyol A were the variables with the highest variable contributions, according to the correlations. The PC2 was associated with β-ionone, pentadec-1-ene, heptadec-1-ene, and (E)-phytol abundance in the samples. The score plot (Figure 9b) showed the position samples in the multivariate space of the first two PCs. There was a clear separation between the months. The dominant group in the fresh samples from May and September was aliphatic unsaturated compounds, while, in June, July, and August, it was the group of terpenes.
The impact of HS-SPME and HD on the obtained results was obvious from the composition of the headspace (Tables 1 and 2) and hydrodistillate (Table 3) of the fresh samples. The most abundant headspace compounds were benzaldehyde, pentadecane, and pentadec-1-ene. (E)-Phytol was the most abundant compound in HD-FrHSc (except September) followed by diterpene alcohols (isopachydictyol A and cembra-4,7,11,15-tetraen-3-ol) and sesquiterpene alcohol gleenol.
The used statistical test did not correlate compound abundance with the temperature change as the temperature varied.

Sample Collection
The seaweed H. scoparia (Linnaeus) Sauvageau 1904 samples were harvested in 2021 from May to September. The sampling location was in the Adriatic Sea on the southern side of the islandČiovo (43.493373 • N, 16.272519 • E). Seaweed samples were harvested at a depth ranging from 20 to 120 cm. A YSI Pro2030 probe (Yellow Springs, OH, USA) was used to measure the sea temperature ( Figure 11). The identification was performed by a marine botanist according to seaweed morphological characteristics. The samples were air-dried in the shade at room temperature for 10 days following a similar pattern, which is very common for terrestrial plants and similar to our other studies [20,23,24].

Headspace Solid-Phase Microextraction (HS-SPME)
Two SPME fibres (Agilent Technologies, Palo Alto, Santa Clara, CA, USA) covered with PDMS/DVB (polydimethylsiloxane/divinylbenzene) or DVB/CAR/PDMS (divinylbenzene/carboxene/polydimethylsiloxane) and placed on the PAL Auto Sampler System (PAL RSI 85, CTC Analytics AG, Schlieren, Switzerland) were used for HS-SPME. Prior to the extraction, both fibres were conditioned following the manufacturer instructions. The seaweed samples (1 g) were placed into 20 mL glass vials sealed with a stainless-steel cap with polytetrafluorethylene (PTFE)/silicon septa. The equilibration of the sample was conducted at 60 • C for 15 min. After equilibration, the sample was extracted for 45 min. Thermal desorption directly to the GC column was carried out at an injector temperature of 250 • C set for 6 min.

Hydrodistillation (HD)
Approximately 50 g of fresh and 20 g of air-dried H. scoparia samples were hydrodistilled. For HD, a v/v ratio 1:2 (3 mL) of pentane and diethyl ether was used as the solvent trap in a modified Clevenger apparatus. HD was performed for 2 h, and the solvent trap with dissolved VOCs was isolated. Concentration of the solvent trap was performed under a slow flow of nitrogen until the final volume was 0.2 mL. A volume of 2 µL was used for GC-MS analyses.

Gas Chromatography-Mass Spectrometry Analysis (GC-MS)
VOCs isolated from H. scoparia were analysed using a gas chromatograph (model 8890 Agilent Technologies, Palo Alto, Santa Clara, CA, USA) equipped with a mass spectrometer detector (model 5977E MSD, Agilent Technologies). For the separation of VOCs, HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness, Agilent Technologies, Palo Alto, Santa Clara, CA, USA) was used. The conditions for the GC-MS analysis and the process for the identification of the compounds were previously described in detail by Radman et al. [20]. The analyses were carried out in duplicate and shown as the average percentage of peak area.

Statistical Analyses
The relations between the dominant volatiles (>2%) of fresh and dried alga samples, analysed by HS-SPME and HD were determined using principal component analysis (PCA) [35]. The average percentage peak areas of the dominant volatiles from fresh and dried samples obtained by HS-SPME (two fibres) and HD were used for the analysis. For the PCA analyses, STATISTICA ® (version 13, StatSoft Inc, Tulsa, OK, USA) was used, and the data were log-transformed prior to analyses.

Conclusions
The present study reported complementary isolation of VOCs from FrHSc and DrHSc by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) and their analysis by gas chromatography and mass spectrometry (GC-MS). The use of both methods was justified for detailed chemical prospection of the headspace, volatile, and less volatile organic compounds from H. scoparia. The impact of the season of growth (May-September) and air-drying on VOC composition was also studied for the first time, and the obtained data were elaborated by principal component analysis (PCA).
The most abundant headspace compounds of H. scoparia were benzaldehyde, pentadecane (a chemical marker of brown macroalgae), and pentadec-1-ene. Benzaldehyde abundance decreased after air-drying, while an increment in benzyl alcohol after the drying was noticed. The percentage of pentadecane and heptadecane increased after air-drying while pentadec-1-ene abundance decreased. Octan-1-ol decreased from May to September. In HD-FrHSc, terpenes were the most abundant group in June, July, and August, while, in May and September, the dominant group was unsaturated aliphatic compounds. In HS-FrHSc, great variability was found within the 5 months by two fibres, while the HS-DrHSc volatilome was more comparable.
In HD-DrHSc terpenes, unsaturated and saturated aliphatic compounds dominated. (E)-phytol was the most abundant compound in HD-FrHSc through all months except September. Its abundance increased from May to August. Since phytol is a bioactive compound, the obtained hydrodistillates could be attractive for further bioactivity studies. Two more diterpene alcohols, isopachydictyol A and cembra-4,7,11,15-tetraen-3-ol, and one sesquiterpene alcohol, gleenol, were also detected in high abundance. However, due to lower volatility, phytol and other diterpene alcohols could not be isolated completely by HD. Among aliphatic compounds, the dominant was pentadec-1-ene with its peak in September, while pentadecane was present with lower abundance.
PCA (based on the dominant compound analyses) showed a distinct separation of the fresh and dried samples. However, the used statistical test did not correlate compound abundance with the temperature change as the temperature varied. The results indicate great seasonal variability of isolated VOCs, as well among fresh and dried samples, which is important for further chemical biodiversity studies.