Seasonal Monitoring of Volatiles and Antioxidant Activity of Brown Alga Cladostephus spongiosus

Cladostephus spongiosus was harvested once a month during its growing season (from May to August) from the Adriatic Sea. Algal volatile organic compounds (VOCs) were obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) and analysed by gas chromatography and mass spectrometry (GC-MS). The effects of air drying and growing season on VOCs were determined. Two different extraction methods (ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE)) were used to obtain ethanolic extracts of C. spongiosus. In addition, the seasonal antioxidant potential of the extracts was determined, and non-volatile compounds were identified from the most potent antioxidant extract. Aliphatic compounds (e.g., pentadecane) were predominantly found by HS-SPME/GC-MS. Hydrocarbons were more than twice as abundant in the dry samples (except in May). Aliphatic alcohols (e.g., hexan-1-ol, octan-1-ol, and oct-1-en-3-ol) were present in high percentages and were more abundant in the fresh samples. Hexanal, heptanal, nonanal, and tridecanal were also found. Aliphatic ketones (octan-3-one, 6-methylhept-5-en-2-one, and (E,Z)-octa-3,5-dien-2-one) were more abundant in the fresh samples. Benzene derivatives (e.g., benzyl alcohol and benzaldehyde) were dominant in the fresh samples from May and August. (E)-Verbenol and p-cymen-8-ol were the most abundant in dry samples in May. HD revealed aliphatic compounds (e.g., heptadecane, pentadecanal, (E)-heptadec-8-ene, (Z)-heptadec-3-ene), sesquiterpenes (germacrene D, epi-bicyclosesquiphellandrene, gleenol), diterpenes (phytol, pachydictyol A, (E)-geranyl geraniol, cembra-4,7,11,15-tetraen-3-ol), and others. Among them, terpenes were the most abundant (except for July). Seasonal variations in the antioxidant activity of the ethanolic extracts were evaluated via different assays. MAE extracts showed higher peroxyl radical inhibition activity from 55.1 to 74.2 µM TE (Trolox equivalents). The highest reducing activity (293.8 µM TE) was observed for the May sample. Therefore, the May MAE extract was analysed via high-performance liquid chromatography with high-resolution mass spectrometry and electrospray ionisation (UHPLC-ESI-HRMS). In total, 17 fatty acid derivatives, 9 pigments and derivatives, and 2 steroid derivatives were found. The highest content of pheophorbide a and fucoxanthin, as well as the presence of other pigment derivatives, could be related to the observed antioxidant activity.


Introduction
The genus Cladostephus currently consists of five taxonomically recognised species. These species are cosmopolitan brown algae that grow on rocks in the intertidal zone and at depths of up to six metres, mainly in temperate seas [1]. Cladostephus spongiosus occurs in the Adriatic Sea [2]. Its thalli are usually between 3 and 20 cm long, with the longest thallus present in summer, during its growing season, when the sea temperature is the highest.   Aliphatic alcohols were present with high percentages in all months studied, especially when extracted with the fibre f2 ( Figure 2), and they were more abundant in the fresh samples. The short-chain alcohols ethanol, hexan-1-ol, octan-1-ol, and oct-1-en-3-ol were the most abundant (Tables 1 and 2). Previous studies showed that the oxylipin oct-1-en-3-ol is a defence compound in marine algae [18][19][20].
Hexanal, heptanal, nonanal, and tridecanal constituted the majority of aldehydes (Tables 1 and 2). The algal aldehydes are formed through degradation of fatty acids, which can occur either via oxidation or via the enzymatic action of lipoxygenases [21]. Hexanal and heptanal are mainly derived from linoleic acid [22,23]. Nonanal could originate from ω9 monounsaturated fatty acids (MUFAs) and ω6 polyunsaturated fatty acids (PUFAs), such as linoleic acid [24]. The fatty aldehyde tridecanal could be derived from long-chain fatty acids. Aldehydes obtained with the polar fibre f2 were more abundant in the air-dried samples in all months. Table 1. The volatile organic compounds (VOCs) of Cladostephus spongiosus obtained via headspace solid-phase microextraction (HS-SPME) with divinylbenzene/carboxen/polydimethylsiloxane fibre (f1) and analysed via gas chromatography-mass spectrometry (GC-MS): fresh (Fr) samples, dry (Dr) samples.    No-order of the compounds elution on HP-5MS column; RI-retention index; SAC-Saturated aliphatic compounds; UnSAC-Unsaturated aliphatic compounds.
Benzene derivatives group dominated the fresh samples from May (33.86%, f1; 21.42%, f2), July (16.72%, f1; 27.87%, f2), and August (21.35%, f1; 35.34%, f2), as can be seen in Figures 1 and 2. Benzyl alcohol and benzaldehyde formed the majority of benzene derivatives. Benzaldehyde was the predominant compound in all fresh samples obtained by f2 (Table 2) and in all fresh samples (except June) obtained by f1 (Table 1). In contrast, benzyl alcohol was more abundant in the dry samples. The volatile benzene derivatives can be formed from phenyalanine when the side chain of a carbon skeleton shortens by C2-unit. This reaction most commonly occurs via the β-oxidative pathway [25]. The loss of benzaldehyde during air-drying could be due to its high volatility [26][27][28].
In the group of other compounds carboxylic acids, dictyopterenes, and C 13 -norisoprenoides were abundant. Carboxylic acids such as hexanoic and heptanoic acids contributed strongly to the increase in other compounds abundance, especially in the dry May sample.   Figure 4a,b shows the PCA of the data for f1 and f2 analysed together. The initial data variability of the first two PCs described 52%; however, the fresh and dried samples were clearly separated based on the higher content of (E,Z)-octa-3,5-dien-2-one and benzyl alcohol, as well as low content of pentadecane in the fresh samples. The samples from both fibres were segregated according to the sampling month, with clear vertical distribution from May to August.  Figure 4a,b shows the PCA of the data for f1 and f2 analysed together. The initial data variability of the first two PCs described 52%; however, the fresh and dried samples were clearly separated based on the higher content of (E,Z)-octa-3,5-dien-2-one and benzyl alcohol, as well as low content of pentadecane in the fresh samples. The samples from both fibres were segregated according to the sampling month, with clear vertical distribution from May to August.  The correlation plot and score plot of the dominant components from data obtained by f1 and f2 fibres are shown in Figure 3a-d, respectively. The first two PCs of the data obtained by f1 fibre described 53.4% of the initial data variability. The highest variable contribution to PC1 was observed for tridecanal, hexanal, pentadec-1-ene, and ß-ionone, while ethanol, hexan-1-ol, octan-1-ol, and phenol contributed to PC2.

Statistical Analysis of the Headspace VOCs
The first two PCs in the data obtained by f2 fibre described 62.2% of the initial data variability. The abundance of two aldehydes (nonanal and tridecanal) contributed to PC1, while alcohols (ethanol and hexan-1-ol) and phenol had the highest contribution to the PC2. The distribution of the samples in the multivariate space showed no significant difference between the fibres. However, a clear separation between the fresh and dried samples was obtained (Figure 3b,d). For both f1 and f2, the dry samples were positioned on the left part of the multivariate space and the fresh samples, taken in May, were separated in the bottom right part of the score plot. Interestingly, the sampling month showed no effect on the dry samples, while in fresh samples there are similarities between June, July, and August, while May was clearly separated in the upper right. Figure 4a,b shows the PCA of the data for f1 and f2 analysed together. The initial data variability of the first two PCs described 52%; however, the fresh and dried samples were clearly separated based on the higher content of (E,Z)-octa-3,5-dien-2-one and benzyl alcohol, as well as low content of pentadecane in the fresh samples. The samples from both fibres were segregated according to the sampling month, with clear vertical distribution from May to August.

Hydrodistillation Obtained Volatilome Variations of C. spongiosus
In the hydrodistillate, the percentages of the identified compounds ranged from 81.21% (Fr_July) to 89.11% (Dr_August) of the total compounds detected. These compounds were classified into four different groups: aliphatic compounds, sesquiterpenes, diterpenes, and others ( Figure 5). Terpenes, especially sesquiterpenes and diterpenes, were the most abundant in all samples (36.09%, Fr_August-52.08%, Fr_May), except in the fresh sample in July (33.13%). Sesquiterpenes were more abundant only in May's fresh sample (30.47%), mainly because of the high proportion of germacrene D (9.63%) and epi-bicyclosesquiphellandrene (4.49%) and gleenol (11.58%) ( Table 3). All three detected sesquiterpenes were the predominant sesquiterpenes in the hydrodistillate of brown alga T. atomaria which belongs to the same subclass Dictyotophycidae [29]. When observing the seasonal changes in H. scoparia, which belongs to the same order of Sphacelariales, the highest proportion of germacrene D and gleenol in the hydrodistillate of the fresh sample was also found in May and later decreased each month until September [30].
Fatty acid ethyl ester (FAEE) ethyl icosanoate, assigned to the group of others, was the dominant compound in all air-dried samples (12.85%, Dr_May-30.74%, Dr_July) and in the fresh samples in June (30.84%) and July (34.51%). Naturally occurring FAEEs as well as fatty acid methyl esters (FAMEs) could potentially be used as biofuels [36].
The saturated hydrocarbon heptadecane and aldehyde pentadecanal, as well as the unsaturated hydrocarbons (E)-heptadec-8-ene and (Z)-heptadec-3-ene, were the major aliphatic compounds with the highest percentage in August, contributing to the representation of the aliphatic compounds ( Figure 5). All were most abundant in the fresh sample in August except heptadecane which was not detected but was most abundant in the dry sample in the same month (Table 3).

Statistical Analysis of the VOCs Obtained via Hydrodistillation
The PCA results for VOCs of fresh and air-dried C. spongiosus obtained via hydrodistillation are shown in Figure 6a,b. The first two PCs described 76.3% of the initial data variability. The correlation loadings of the first two PCs (Figure 6a) showed correlations between germacrene D and gleenol, and between aliphatic compounds ((E)-heptadec-8ene, (Z)-heptadec-3-ene and pentadecanal). Epi-bicyclosesquiphellandrene and cembra-4,7,11,15-tetraen-3-ol were the variables with the highest variable contributions, based on the correlations. The PC2 was associated with germacrene D, phytol, and pachydictyol A abundance in the samples. The score plot (Figure 6b) showed the position samples in the multivariate space of the first two PCs. There was a clear separation between June and July samples in the centre right part of the plot, and segregation between May and August samples.

Statistical Analysis of the VOCs Obtained via Hydrodistillation
The PCA results for VOCs of fresh and air-dried C. spongiosus obtained via hydrodistillation are shown in Figure 6 a-d. The first two PCs described 76.3% of the initial data variability. The correlation loadings of the first two PCs (Figure 6a) showed correlations between germacrene D and gleenol, and between aliphatic compounds ((E)-heptadec-8ene, (Z)-heptadec-3-ene and pentadecanal). Epi-bicyclosesquiphellandrene and cembra-4,7,11,15-tetraen-3-ol were the variables with the highest variable contributions, based on the correlations. The PC2 was associated with germacrene D, phytol, and pachydictyol A abundance in the samples. The score plot (Figure 6b) showed the position samples in the multivariate space of the first two PCs. There was a clear separation between June and July samples in the centre right part of the plot, and segregation between May and August samples.
June and July samples, characterized with higher content of cembra-4,7,11,15-tetraen-3-ol and ethyl icosanoate, and lower percentage of germacrene D and gleenol were positioned in the right part of the plot. The vertical distribution between May and August samples was related to differences in germacrene D, gleenol, pachydictyol A, and phytol abundance. There were more differences in seasonal variation between fresh samples. The distribution was along the PC1 axis and cannot be related to one group of compounds but was in the relation to the abundance of epi-bicyclosesquiphellandrene, cembra-4,7,11,15tetraen-3-ol, ethyl icosanoate and pentadecanal, while the distribution along the PC2 axis was related to terpenes (germacrene D, phytol and pachydictyol A) abundance in the samples. No correlation was found between the compounds' content and temperature change.

Antioxidant Activity of Ethanol Extracts In Vitro
Seasonal variations in antioxidant activity of C. spongiosus extracts produced by MAE and UAE were evaluated by ORAC, FRAP, and DPPH assays (Figure 7). All extracts from MAE showed higher activity. The ORAC results ranged from 54.9 ± 1.5 to 69.8 ± 1.9 µM TE for UAE extracts and from 55.1 ± 1.1 to 74.2 ± 1.0 µM TE for MAE extracts. The highest peroxyl radical inhibition activity was observed for the samples harvested in May. The FRAP results ranged from 157.4 ± 3.9 to 238.5 ± 21.4 µM TE for UAE extracts, and from 176.4 ± 4.7 to 293.8 ± 9.2 µM TE for MAE extracts. The highest reducing activity was also observed for the samples harvested in May. The scavenging ability of DPPH radical ranged from 18.2 ± 0.7 to 27.3 ± 0.7% for UAE extracts, and from 24.4 ± 1.8 to 32.1 ± 0.5% for MAE extracts. The highest inhibition was recorded for August samples. Chiboub et al. [4] reported DPPH inhibition of C. spongiosus macerated with hexane, ethyl acetate, and methanol for 48 h at 15.6 ± 0.5%, 52.2 ± 0.0%, and 41.5 ± 0.1%, respectively, which is comparable to our results. Moreover, Yalç n et al. [37] extracted C. spongiosus f. verticillatum June and July samples, characterized with higher content of cembra-4,7,11,15-tetraen-3ol and ethyl icosanoate, and lower percentage of germacrene D and gleenol were positioned in the right part of the plot. The vertical distribution between May and August samples was related to differences in germacrene D, gleenol, pachydictyol A, and phytol abundance. There were more differences in seasonal variation between fresh samples. The distribution was along the PC1 axis and cannot be related to one group of compounds but was in the relation to the abundance of epi-bicyclosesquiphellandrene, cembra-4,7,11,15-tetraen-3-ol, ethyl icosanoate and pentadecanal, while the distribution along the PC2 axis was related to terpenes (germacrene D, phytol and pachydictyol A) abundance in the samples. No correlation was found between the compounds' content and temperature change.

Antioxidant Activity of Ethanol Extracts In Vitro
Seasonal variations in antioxidant activity of C. spongiosus extracts produced by MAE and UAE were evaluated by ORAC, FRAP, and DPPH assays (Figure 7). All extracts from MAE showed higher activity. The ORAC results ranged from 54.9 ± 1.5 to 69.8 ± 1.9 µM TE for UAE extracts and from 55.1 ± 1.1 to 74.2 ± 1.0 µM TE for MAE extracts. The highest peroxyl radical inhibition activity was observed for the samples harvested in May. The FRAP results ranged from 157.4 ± 3.9 to 238.5 ± 21.4 µM TE for UAE extracts, and from 176.4 ± 4.7 to 293.8 ± 9.2 µM TE for MAE extracts. The highest reducing activity was also observed for the samples harvested in May. The scavenging ability of DPPH radical ranged from 18.2 ± 0.7 to 27.3 ± 0.7% for UAE extracts, and from 24.4 ± 1.8 to 32.1 ± 0.5% for MAE extracts. The highest inhibition was recorded for August samples. Chiboub et al. [4] reported DPPH inhibition of C. spongiosus macerated with hexane, ethyl acetate, and methanol for 48 h at 15.6 ± 0.5%, 52.2 ± 0.0%, and 41.5 ± 0.1%, respectively, which is comparable to our results. Moreover, Yalçın et al. [37] extracted C. spongiosus f. verticillatum with methanol and ethanol solutions (100%, 80%, and 70%) for 120 min using UAE. The extracts showed antioxidant potential through 2,2-azino-bis(3-ethylbenzothiazoline-6sulfonate) (ABTS) radical scavenging and cupric ion reduction. Pinteus et al. [6] showed that C. spongiosus harvested from the Peniche coast, and extracted with dichloromethane, has the ability to scavenge peroxyl and DPPH radicals. The antioxidant activity results mentioned above cannot be compared with our results because the authors used a different methodology or expressed their results in different units [38]. with methanol and ethanol solutions (100%, 80%, and 70%) for 120 min using UAE. The extracts showed antioxidant potential through 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging and cupric ion reduction. Pinteus et al. [6] showed that C. spongiosus harvested from the Peniche coast, and extracted with dichloromethane, has the ability to scavenge peroxyl and DPPH radicals. The antioxidant activity results mentioned above cannot be compared with our results because the authors used a different methodology or expressed their results in different units [38].

Non-Target Screening of Non-Volatile Compounds in Ethanol Extract
The ethanolic extract of the freeze-dried algal sample with the best antioxidant potential according to ORAC and FRAP assay (May, MAE) was analysed using UHPLC-ESI-HRMS. The major compounds were selected according to signal intensity (peak area in

Non-Target Screening of Non-Volatile Compounds in Ethanol Extract
The ethanolic extract of the freeze-dried algal sample with the best antioxidant potential according to ORAC and FRAP assay (May, MAE) was analysed using UHPLC-ESI-HRMS. The major compounds were selected according to signal intensity (peak area in counts). From the extracted ion chromatograms (XICs) in positive ion mode, these compounds were identified based on the given elemental composition and MS/MS spectra with confidence level 2 (probable structure) and 3 (possible structure) [39]. Seventeen fatty acid derivatives, nine pigments and derivatives, and two steroid derivatives were found (Table 4).    No-order of the compounds elution on Acquity UPLC BEH Phenyl-Hexyl analytical column.
Three C18 FAAs, derivatives of linoleic acid (linoleamide, no. 9), oleic acid (oleamide, no. 13), and stearic acid (stearamide, no. 17), two C16 FAAs, derivatives of palmitic acid (palmitamide, no. 10) and palmitoleic acid (palmitoleamide, no. 8), and one C14, derivative of myristic acid (myristamide, no. 7), C20 derivative of (11Z)-icos-11-enoic acid (gondamide, no. 19), and C22 FAA derivative of erucic acid (erucamide, no. 24) were detected. In our previous studies, we detected oleamide, erucamide, and palmitamide in the green alga Dasycladus vermicularis [40] and two others, palmitoleamide and linoleamide, in the brown algae Ericaria crinita and Ericaria amentacea [28]. The FAAs detected and identified in C. spongiosus belong to the group of primary fatty acid amides (PFAMs) with the structure R-CO-NH 2 , where R represents a long-chain FA [41], and are bioactive signalling molecules. In general, PFFAs have a broad therapeutic spectrum including anticancer, antibiotic, anthelmintic and antidiabetic [42]. They play an important role in the nervous system of the mammals because they have the potential to bind to the receptors of many drugs, indicating effects on locomotion, angiogenesis, and sleep [43]. It has been reported that PFAMs have a great anticancer activity as they effect cell proliferation [44]. Oleamide is the most studied PFFA and has great potential against Alzheimer disease [45]. Certain plant essential oils are rich in PFFAs because they act as self-defence agents in plants [42], but data on the occurrence of PFAAs in marine algae are lacking.
In the group of pigments, one chlorophyll a derivative (pheophorbide a, compound 21), one demetallized chlorophyll b (pheophytin b, compound 27), three pheophorbide derivatives (compounds 22, 23, 25), one xanthophyll and two derivatives (compounds 15, 16, 28), and one monoterpene lactone (loliolide, compound 1) were detected ( Table 4). The pigments are known antioxidants [51,52]. Pheophorbide a, a derivative of chlorophyll a, has been extracted from marine algae and has shown great antitumor activity [53]. Among the pigments, it was the most abundant in the extract (Table 4), which contributes to the antioxidant activity of the extract. Cho et al. [54] studied the green alga Ulva prolifera (formerly Enteromorpha prolifera) and its ethanolic extract, which showed high antioxidant activities. The later subfraction with chloroform showed the strongest antioxidant activity of all subfractions, and further spectroscopic analysis revealed the pheophorbide a as the major compound.
Fucoxanthin, the major xanthophyll in brown algae [55,56], and xantophyll derivatives, halocynthiaxanthin acetate and loroxanthin decenoate, were detected. El Hattab et al. [57] reported fucoxanthin in C. spongiosus f. verticillatus. Halocynthiaxanthin acetate and loroxanthin decenoate are more abundant in green algae [58]. Xanthophylls exhibit diverse bioactive properties, such as antioxidant, antitumor, and anti-inflammatory activities [55,56,59]. The antioxidant activity of the extract might be related to the high amounts of fucoxanthin (Table 4). Ibrahim et al. [60] evaluated the potent antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi and the strong antioxidant activity of fucoxanthin extracted from Dictyota fasciola (formerly Dilophys fasciola). Fucoxanthin is one of the most studied xanthophylls against cancer cells because of its antiproliferative effect [59]. Halocynthiaxanthin is a metabolite of fucoxanthin and has shown even stronger cytotoxic results than fucoxanthin when tested on human neuroblastoma cells [61]. Sansone et al. [62] studied the biological activity of the marine green alga Tetraselmis suecica, whose ethanolic extract contained a high proportion of xhantophylls, among which loraxanthin esters were present. The extract showed potent antioxidant activity and antitumor activity against human lung cancer line (A549).

Macroalga Samples
Between May and August 2021, the samples of the brown alga C. spongiosus (Hudson) C. Agardh, 1817 were collected. The sampling took place in the Adriatic Sea, off the coast of the island ofČiovo (43.493373 • N, 16.272519 • E). Each sample was collected at a depth of 20 to 120 cm from the same lagoon. The sea temperature was measured during each sampling with a YSI Pro2030 probe (Yellow Springs, OH, USA) and increased from 20.1 • C in May to 28.1 • C in August. Alga species was determined according to its morphological attributes by marine botanist. For the determination of VOCs, the samples were dried in the shade at room temperature for 10 days, while samples for the determination of non-volatile compounds were freeze-dried (FreeZone 2.5, Labconco, Kansas City, MO, USA) prior the extraction. Algal samples were extracted in 50% ethanol using MAE in the advanced microwave extraction system (ETHOS X, Milestone Srl, Sorisole, Italy) and UAE based on the prior research [9]. The samples were pulverised and mixed with 50% ethanol at a 1:10 (w/v) algae to solvent ratio and extracted for: (1) 15 min at 200 W and 60 • C for MAE, and (2) 60 min at 40 kHz frequency and 60 • C in an ultrasonic bath for UAE.
to process the mass spectrometer data. Based on the mass spectra and the given elemental compositions of the compounds, and in conjunction with the results of the search in the ChEBI, Lipid Maps, and MassBank databases, the identification of the compounds was proposed.

Antioxidant Activity of Extracts
Three different methods were used to assess in vitro antioxidant activity of crude algal extracts. The oxygen radical absorbance capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging ability (DPPH) methods are based on hydrogen atom transfer, while ferric reducing/antioxidant power (FRAP) method is based on electron transfer [11,65,66].
Previously reported methods [11,65] were used to measure the reducing activity as FRAP. The results were expressed using the Trolox standard calibration curve (y = 0.0013x, R 2 = 0.99) as micromoles of Trolox equivalents (µM TE). The inhibition of the free peroxyl radicals, measured as ORAC [11], was measured for the samples in 1:100 dilution, and the results were expressed using the Trolox standard calibration curve (y = 22.842x + 26.473, R 2 = 0.99) as µM TE. DPPH radical inhibition was expressed as a percentage of inhibition and measured according to the previously reported methods [11,67].

Statistical Analyses
The relationship between the dominant volatiles (>2%) of fresh and dried C. spongiosus samples was determined via principal component analysis (PCA) using the software STATISTICA ® (version 13, StatSoft Inc., Tulsa, OK, USA). Before analyses, the data (average percentage of peak areas of the dominant volatiles) were log-transformed [9].

Conclusions
HS-SPME showed that aliphatic compounds dominated in all samples. Hydrocarbons were more than twice as high in the dry samples (except in May). Aliphatic alcohols were present in large amounts and were more abundant in the fresh samples. Hexanal, heptanal, nonanal, and tridecanal formed the majority of aldehydes. Aliphatic ketones were more abundant in the fresh samples. Benzene derivatives were predominant in the fresh samples of May and August. Mono-and sesquiterpenes (mainly (E)-verbenol and p-cymen-8-ol) were the most abundant in the May dry sample.
Seasonal variations in antioxidant activity of C. spongiosus extracts obtained with MAE and UAE were confirmed. All MAE extracts showed higher activity. The highest peroxyl radical inhibition and reducing activity was observed in the May samples. Therefore, the ethanolic extract from May was analysed by UHPLC-ESI-HRMS. A total of 17 fatty acid derivatives, 9 pigments and derivatives, and 2 steroid derivatives were found. The highest content of pheophorbide a and fucoxanthin and the presence of other pigment derivatives could be related to the observed antioxidant activity.
Further research should focus on exploring the seasonal variations of the non-volatile compounds of this alga and the potential use of the extracts obtained.