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Polar Lipids Composition, Antioxidant and Anti-Inflammatory Activities of the Atlantic Red Seaweed Grateloupia turuturu

Grateloupia turuturu Yamada, 1941, is a red seaweed widely used for food in Japan and Korea which was recorded on the Atlantic Coast of Europe about twenty years ago. This seaweed presents eicosapentaenoic acid (EPA) and other polyunsaturated fatty acids (PUFAs) in its lipid fraction, a feature that sparked the interest on its potential applications. In seaweeds, PUFAs are mostly esterified to polar lipids, emerging as healthy phytochemicals. However, to date, these biomolecules are still unknown for G. turuturu. The present work aimed to identify the polar lipid profile of G. turuturu, using modern lipidomics approaches based on high performance liquid chromatography coupled to high resolution mass spectrometry (LC–MS) and gas chromatography coupled to mass spectrometry (GC–MS). The health benefits of polar lipids were identified by health lipid indices and the assessment of antioxidant and anti-inflammatory activities. The polar lipids profile identified from G. turuturu included 205 lipid species distributed over glycolipids, phospholipids, betaine lipids and phosphosphingolipids, which featured a high number of lipid species with EPA and PUFAs. The nutritional value of G. turuturu has been shown by its protein content, fatty acyl composition and health lipid indices, thus confirming G. turuturu as an alternative source of protein and lipids. Some of the lipid species assigned were associated to biological activity, as polar lipid extracts showed antioxidant activity evidenced by free radical scavenging potential for the 2,2′-azino-bis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS●+) radical (IC50 ca. 130.4 μg mL−1) and for the 2,2-diphenyl-1-picrylhydrazyl (DPPH●) radical (IC25 ca. 129.1 μg mL−1) and anti-inflammatory activity by inhibition of the COX-2 enzyme (IC50 ca. 33 µg mL−1). Both antioxidant and anti-inflammatory activities were detected using a low concentration of extracts. This integrative approach contributes to increase the knowledge of G. turuturu as a species capable of providing nutrients and bioactive molecules with potential applications in the nutraceutical, pharmaceutical and cosmeceutical industries.


Introduction
Seaweeds play a central role in oceans food webs, as habitats for marine species that support biodiversity and as bioremediators through nutrient assimilation from seawater, all these features granted them a renewed attention in the blue bioeconomy [1][2][3]. Beyond the context of natural systems, seaweeds have been traditionally used as food in Asian countries and, in recent decades, the use of seaweeds as food has been increasing in the potential of Grateloupia extracts has generated more interest due to their beneficial effects in the prevention of chronic diseases and aging. A supplementation with 20% of G. turuturu biomass increased the longevity of Drosophila melanogaster, while a formula containing 100% G. turuturu achieved the highest antigenotoxic potential against streptonigrin-induced genotoxicity [33]. Grateloupia turuturu mixed diet is a promising complementary source of lipids in abalone aquaculture [38], thus reinforcing the potential use of this seaweed as a source of nutritious lipids in feed applications.
In order to increase our knowledge of this seaweed, in the present study, we evaluated the nutritional value of G. turuturu by performing a thorough characterization of the polar lipidome using LC-MS and fatty acid profiling using GC-MS, followed by calculation of health lipid indices. The antioxidant activities of the lipid extracts were carried out by evaluating their potential for scavenging free radicals against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azino-bis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS) radicals and the anti-inflammatory activity of the lipid extracts was evaluated based on the capacity to inhibit the cyclooxygenase-2 (COX-2) enzyme.

Lipids Content and Carbon/Nitrogen Ratio
The total lipid content of G. turuturu was 0.88% ± 0.25% of dry weight biomass (DW). The total content of lipid, carbon, nitrogen and protein, along with the C/N ratio of G. turuturu, are presented in Table 1. The tissue carbon-to-nitrogen ratio (C/N) accounted for 8.30 ± 0.19. The protein content accounted for 26.26% ± 0.69% DW. Table 1. Content of lipid, tissue carbon, tissue nitrogen and proteins expressed in g 100 g −1 of dry weight biomass (DW) and carbon-to-nitrogen ratio (C/N) of the Grateloupia turuturu biomass (means ± SD: a n = 5, b n = 3).

Lipidome of Grateloupia turuturu
A total of 205 lipid species (m/z values) were identified, with these being distributed over glycolipids (74 species), phospholipids (109 species) and betaine lipids (

Glycolipids Profile of Grateloupia turuturu
The glycolipids species (GLs) identified in G. turuturu included the galactolipid classes MGDG (16 lipid species), DGDG (13 lipid species), MGMG (13 lipid species) and DGMG (4 lipid species) and the classes of the anionic sulfolipid SQDG (20 lipid species) and SQMG (8 lipid species). Galactolipids were identified by LC-MS as [M + NH 4 ] + ions and sulfolipids as [M − H] − ions. The identification of the lipids species is provided in Table 2  (Supplementary Materials, Table S1, Figures S2 and S3). The relative percentage of each lipid species within each specific class was calculated and the graphical representation is shown in Figure 2.      Glycolipids species were mainly esterified with C14:0, C16-, C18-and C20-fatty acyl chains and the unsaturation degree of the lipids species (DoU) ranged between 0 and 5 for lyso-GLs (MGMG, DGMG and SQMG), 1 and 10 for MGDG and 0 and 10 for the DGDG and SQDG classes ( Table 2). Our data showed that the more abundant lipid species had a total number of 36-

Betaine Lipids Profile of Grateloupia turuturu
The betaine lipids diacylglyceryl-N,N,N-trimethyl-homoserine (16 DGTS lipid species) and monoacylglyceryl-N,N,N-trimethyl-homoserine (6 MGTS lipid species) were identified in G. turuturu. Lipid species were identified using LC-MS spectra as [M + H] + ions, as described in Table 3. The RA of lipids species within each of the aforementioned classes is shown in Figure 3 and Supplementary Materials, Figure S4. The fragmentation pattern observed in the MS/MS spectra of these lipids is shown in the Supplementary Materials (Table S1, Figure S4b

Phospholipids Profile of Grateloupia turuturu
Ten lipid classes were identified in the phospholipids (PLs) of G. turuturu: phosphatidylcholine (PC, 28 lipid species) and lyso-PC (LPC, 13 lipid species), phosphatidylethanolamine (PE, 14 lipid species) and lyso-PE (LPE, 9 lipid species), phosphatidylglycerol (PG, 15 lipid species) and lyso-PG (LPG, 9 lipid species), phosphatidic acid (PA, 6 lipid species) and lyso-PA (LPA, 1 lipid species) and phosphatidylinositol (PI, 5 lipid species) and lyso-PI (1 LPI species), as summarized in the Table 4. LC-MS spectra of these phospholipid classes and MS/MS data are shown in the Supplementary Materials (Table S1, Figures S5-S9). The relative percentage of each lipid species within each specific class is shown in Figure 4. The majority of PLs species included C14:0, C16-, C18-and C20-fatty acyl chains. The DoU of lipid species varied between 0 and 11, in the case of PC, between 0 and 8 in PE, between 0 and 9 in PG, between 5 and 9 in PA (that included C16-and C20-fatty acyl chains), between 0 and 10 in PI (that included C16-and C18-fatty acyl chains), between 0 and 6 in LPC and between 0 and 5 in LPE and LPG. Regarding the lyso-lipids LPI and LPA, the only lipid species identified were LPI ( Figure S10 in the Supplementary Materials. The eight lipid species identified included saturated-and unsaturated-sphingosine-bases and saturated and unsaturated hydroxylated acyl chains. The most abundant lipid species included 42carbons PI-Cer. The most abundant PI-Cer (t42:2h) at m/z 920.6240 contained the long-chain base 18:1-phytosphingosine (trihydroxy sphingoid base) and the long monounsaturated hydroxy-fatty acid having 24-carbons (PI-Cer (t18:1/h24:1).

Antioxidant Activity of the Lipid Extracts of Grateloupia turuturu
A dose-dependent increase in the scavenging capacity was observed for all concentrations tested ( Figure 5). For ABTS, a 50% inhibition (IC 50 ) of the ABTS •+ radical was achieved at a concentration of 130.4 ± 52.4 µg mL −1 , which represents a TE of 7.3 ± 3.7 µmol g −1 .
In the DPPH assay, the IC 50 value could not be calculated because the percentage of antioxidant activity was less than 50%, so the IC 50 values of the extracts were outside the tested concentration range. Thus, a 25% inhibition (IC 25 ) of DPPH • was obtained at a concentration of 129.1 ± 58.7 µg mL −1 , representing a TE of 83.2 ± 39.6 µmol g −1 . The best radical scavenging capacity was achieved at a concentration of 250 µg mL −1 in both ABTS (89.79% ± 8.22%) and DPPH (52.21% ± 19.57%).

Antioxidant Activity of the Lipid Extracts of Grateloupia turuturu
A dose-dependent increase in the scavenging capacity was observed for all concentrations tested ( Figure 5). For ABTS, a 50% inhibition (IC50) of the ABTS •+ radical was achieved at a concentration of 130.4 ± 52.4 μg mL −1 , which represents a TE of 7.3 ± 3.7 μmol g −1 . In the DPPH assay, the IC50 value could not be calculated because the percentage of antioxidant activity was less than 50%, so the IC50 values of the extracts were outside the tested concentration range. Thus, a 25% inhibition (IC25) of DPPH • was obtained at a concentration of 129.1 ± 58.7 μg mL −1 , representing a TE of 83.2 ± 39.6 μmol g −1 . The best radical scavenging capacity was achieved at a concentration of 250 μg mL −1 in both ABTS (89.79% ± 8.22%) and DPPH (52.21% ± 19.57%).

Cyclooxygenase 2 (COX 2) Enzyme Inhibitory Capacity of the Lipids Extracts of Grateloupia turuturu
All the concentrations of lipid extracts tested showed the capacity to inhibit COX-2 activity in vitro, although showing only a dose-dependent response between 12.5 and 50 μg mL −1 of lipid extract ( Figure 6). However, a concentration of ca. 33 μg mL −1 was able to inhibit the PG2 production by 50%, demonstrating that the extract has a potential antiinflammatory activity.

Cyclooxygenase 2 (COX 2) Enzyme Inhibitory Capacity of the Lipids Extracts of Grateloupia turuturu
All the concentrations of lipid extracts tested showed the capacity to inhibit COX-2 activity in vitro, although showing only a dose-dependent response between 12.5 and 50 µg mL −1 of lipid extract ( Figure 6). However, a concentration of ca. 33 µg mL −1 was able to inhibit the PG2 production by 50%, demonstrating that the extract has a potential anti-inflammatory activity.

Antioxidant Activity of the Lipid Extracts of Grateloupia turuturu
A dose-dependent increase in the scavenging capacity was observed for all concentrations tested ( Figure 5). For ABTS, a 50% inhibition (IC50) of the ABTS •+ radical was achieved at a concentration of 130.4 ± 52.4 μg mL −1 , which represents a TE of 7.3 ± 3.7 μmol g −1 . In the DPPH assay, the IC50 value could not be calculated because the percentage of antioxidant activity was less than 50%, so the IC50 values of the extracts were outside the tested concentration range. Thus, a 25% inhibition (IC25) of DPPH • was obtained at a concentration of 129.1 ± 58.7 μg mL −1 , representing a TE of 83.2 ± 39.6 μmol g −1 . The best radical scavenging capacity was achieved at a concentration of 250 μg mL −1 in both ABTS (89.79% ± 8.22%) and DPPH (52.21% ± 19.57%).

Cyclooxygenase 2 (COX 2) Enzyme Inhibitory Capacity of the Lipids Extracts of Grateloupia turuturu
All the concentrations of lipid extracts tested showed the capacity to inhibit COX-2 activity in vitro, although showing only a dose-dependent response between 12.5 and 50 μg mL −1 of lipid extract ( Figure 6). However, a concentration of ca. 33 μg mL −1 was able to inhibit the PG2 production by 50%, demonstrating that the extract has a potential antiinflammatory activity.

Discussion
Grateloupia turuturu is widely consumed in Asia [39] and its nutritional value is evidenced by the high proportion of proteins and n-3 fatty acid EPA (C20:5, n-3) that it displays [13,40,41]. In Europe, G. turuturu is yet to be explored, despite having EPA as one of its main fatty acids in the lipid pool, much as Palmaria palmata, a red seaweed commercially used and appreciated in western countries [42].
Grateloupia turuturu, studied in the present work, contained 26.26 ± 0.69 g 100g −1 DW of protein, in line with the levels reported for other red seaweeds [13,14]. This value is close to the maximum concentration indicated by Denis et al. for the specimens sampled on the Brittany coast [13]. The total lipid content of G. turuturu was determined and accounted for 0.88 ± 0.25 g 100 g −1 of biomass in dry weight (DW). This result is consistent with data from the literature (ca. 0.7%, up to 4.0% DW) [14,17,43,44] and in line for that of other red seaweeds [28,[45][46][47]. Low fat and high protein content make red seaweeds a promising alternative for non-animal proteins [48,49]. In addition, the obtained C/N ratio of 8.30 ± 0.19 (Table 1) represents a value quite similar to those of nitrophilic red seaweeds, values below the proposed critical limit of 10 [50]. This value expresses a balanced nutrient status of G. turuturu [51,52] and corresponds to the trend high protein-low lipid relationship as found in our study.
The nutritional value of G. turuturu was also demonstrated by the profiling of FAs and the calculation of nutritional indices ( Table 5). The saturated FA level was lower than that previously described for G. turuturu from Brittany [13] and within the range reported by Hotimchenko for G. turuturu sampled in the Sea of Japan [44]. Palmitic acid (C16:0) was the most abundant FA (ca. 22.03%) followed by C20:5n-3 (ca. 20.86%) ( Table 5), as previously described by Rodrigues et al. [15] and Hotimchenko [44], but different from those reported by Denis et al. [13] and Kendel et al. [16,19], who obtained a 2.0-fold relationship of C16:0 versus C20:5n-3 FA in G. turuturu. This difference was attributed to both environmental conditions and genetic status of the seaweed [13].
Eicosapentaenoic acid is the main PUFA in the lipid extract of G. turuturu (5.7 g per 100 g −1 of lipid extract (ca. 0.06 g of EPA per 100 grams of DW biomass, or ca. 0.01 g of EPA per 100 grams of fresh biomass)), that can be proximate to certain n-3 in fish such as dogfish [53]. According to U.S. Food and Drug Administration (FDA, https://www.fda.gov/food, accessed on 13 May 2021), new recommendations for the consumption of EPA and DHA claim that a daily dose of, at least, 0.8 g of EPA and DHA (combined total) provides a beneficial effect on health. Taking into account the EPA content of the lipid extract of G. turuturu, ca. 14 g of lipid extract (1.4 times a tablespoon) would provide proximately 0.8 g of EPA per serving. The nutritional value of G. turuturu extracts was corroborated by the PUFA/SFA ratio calculated in the present work, as it was greater than the recommended threshold 0.45, as well as by its n-6/n-3 ratio (<1). These values are consistent with those previously reported in Grateloupia spp. [15] and other red seaweeds [46] and have been associated with human health benefits, such as the prevention of noncommunicable and cardiovascular diseases [54,55]. The nutritive index (NVI) obtained (0.77 ± 0.03) was clearly driven by the higher proportion of C16:0, compared to C18:0 and C18:1 FA. The calculated AI and TI were < 1 and < 0.5, respectively, as recommended to be protective against coronary artery disease [56]. The values of AI and TI were within the reported for the red seaweeds Porphyra dioica [29] and P. palmata [46] and marine fish [57]. The ratio between hypocholesterolemic and hypercholesterolemic fatty acids (h/H index) is also within the values reported for marine fish [57]. Finally, the quality of lipid extract and the stability of PUFA included in food and its vulnerability to oxidation, evaluated by calculating the peroxidizability index (PI percentage), achieved a value of 218.42 ± 30.47%, thus expressing a balanced relation between the required amount and type of PUFA that guarantees a protective effect for coronary disease. Therefore, G. turuturu can be used in the food industry applications contributing to dietary recommendations of SFA and PUFA intake (http://www.fao.org, accessed on 13 May 2021). Polar lipids from seaweeds are considered high-value lipids with beneficial health effects fostering seaweed valorization. The lipidome of G. turuturu was identified here for the first time using an approach based on liquid chromatography-mass spectrometry, which allowed the identification of 205 lipid species distributed over glycolipids, betaine lipids, phospholipids and phosphoinositol ceramides PI-Cer (Tables 2-4). The complex lipids (polar lipids) from seaweeds are gaining a new interest because they are the key carriers of PUFAs and feature bioactive properties [58,59]. The classes of glycolipid were previously identified for G. turuturu [19] and G. jilicina [60] by using preparative thin-layer chromatography and the PL was determined by HPLC coupled to an evaporative light scattering detector [16]. Betaine lipids and PI-Cer classes were identified in the lipidome of G. turuturu for the first time in the present study but have already been described in other red seaweeds [26], such as Porphyra dioica [29] and Palmaria palmata [46]. It is also worth referring that PI-Cer lipids are considered to be biomarkers of Rhodophyta [46,61,62].
Several glycolipids and phospholipids identified in the G. turuturu lipidome were esterified to EPA, including the most abundant lipid species in most classes, as described for P. palmata [46]. Marine polar lipids are considered to be important carriers of n-3 fatty acids with higher n-3 PUFA levels than triglycerides (TAG) [63,64]. The bioavailability of PUFAs comprising n-3 FAs is also considered to be higher when these FAs are esterified into polar lipids, namely PL, compared to TAG [65]. In addition, polar lipids such as glycolipids and phospholipids from seaweeds are recognized by their wide range of bio functionalities [66] and the interest in these healthy and bioactive molecules is growing. Polar lipids can be used as an ingredient for food fortification in PUFA and in functional food, or used as emulsifying agents in the food industry [63][64][65][66].
In this study, the antioxidant and cyclooxygenase-2 inhibitory actions of the lipid extract of G. turuturu were tested. The concentrations necessary to inhibit the activity of the DPPH radical by 20% are of the same order as that reported for G. gracilis (IC 20 119.5 ± 1.8 µg mL −1 ) and P. palmata (IC 20 119.6 ± 8.0 µg mL −1 ) [67]. Our results reveal that a polar lipid extract of G. turuturu features antioxidant activity at low concentrations. The scavenging activity of G. filicina extracts, obtained using other organic solvent systems (e.g., ethyl acetate, chloroform, or acetone) [35], was a 50% inhibition achieved at higher concentrations (2 mg mL −1 of extract). In the same study, chloroform extracts had higher antioxidant activity than commercial antioxidants, such as BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene) and a-tocopherol [35]. Overall, G. turuturu lipid extracts exhibit antioxidant activity and can be used as natural antioxidants for food and feed, as a dietary supplement to nutraceuticals, or as an active ingredient in functional foods or cosmeceuticals promoting health and prevention against damages caused by free radicals. Other potential applications include food processing industries to prevent oxidation and replace synthetic antioxidants. The antioxidant effects of natural products are necessary both for health and wellbeing to counter oxidative stress [68,69], but also for the preservation and packaging of food to increase shelf life through reduction of lipid peroxidation [70].
The ability of lipid extracts from G. turuturu to inhibit COX-2 and, thus, reducing the formation of PGH2, was herein demonstrated reaching 50% inhibitory effect at 33 µg mL −1 (Figure 6). Our findings show that these extracts were more effective than those from other red seaweeds, such as P. palmata and P. dioica, which achieved more than 80% inhibition of COX-2 using 500 µg mL −1 of lipid extract [67]. Cyclooxygenase-2 (COX-2) is a key enzyme in fatty acid metabolism that is upregulated in inflammation. COX-2 is induced by proinflammatory cytokines and enhances the synthesis of prostaglandins, which stimulates inflammation response [54]. Targeted inhibition of COX-2 is a promising approach to inhibit inflammation, with phytonutrients and phytochemicals holding the potential to act in this regulation [71]. These results are in line with those reported by Yang et al. [37], who showed the anti-inflammatory effect of ethyl acetate extracts from G. elliptica on the inhibition of prostaglandin E2 (PGE2) in a macrophages cell line (IC 50 values of the same order found in this study). In the same work, inhibition of the production of pro-inflammatory mediators such as nitric oxide (NO) was also reported. Even though no lipid species have so far been attributed to this specific activity, several lipid species that were well-represented in the lipid extracts of G. turuturu analyzed in the present work (MGDG and DGDG contained ( [72,73]. These lipid species were isolated from extracts of red seaweeds Chondrus crispus and P. palmata. All of those lipid species contained C20:5(n-3) FA and showed higher activity than the free FA C20:5(n-3), suggesting that a higher bioactivity was likely due to the polar head of these molecules. These characteristics support per se the growing interest in polar lipids from seaweeds, principally those originating from red seaweeds [67]. Particularly, GLs bearing n-3 PUFA have been related to bioactive properties, such as antibacterial, antitumoral and antiviral activities, enhancing the pharmacological potential of these compounds [58]. Recent research associated with galactolipids "takes us back to our origins" by reminding that eating vegetables provides access to n-3 FA and contributes to the balance anti-inflammatory (n-3) and pro-inflammatory (n-6) fatty acids [74]. It is known that PLs display anti-inflammatory, anti-oxidant, anti-fibrogenic, anti-apoptotic, membrane-protective and lipid-regulating effects, with a positive impact on several diseases, apparently without significant side effects [63]. Although the market for marine PLs is still in its early stages, there is a growing trend to use marine PLs to supply n-3 PUFA for the global food and dietary supplements market [63]. All beneficial effects of marine lipids should be considered venues for future research.
In summary, G. turuturu lipids can be used in the food industry, using the seaweed biomass as an ingredient for food fortification and functional foods (e.g., dairy products or supplements), or by using the lipid extracts as ingredients for nutraceutical, pharmaceutical and cosmeceutical applications, providing valuable products to be explored for commercialization [75]. Lipidomics can therefore be used for screening the biomass with the best composition in polar lipids richer in PUFA and with the most promising bioactivities, allowing the selection of the seaweed strains for commercial exploitation [6,29].
Overall, the findings reported in the present work add value to G. turuturu and contribute to the development of blue bioeconomy.

Collection of Macroalgae and Preparation of Biomass
The specimens of the red seaweed G. turuturu Yamada were hand-harvested in Ria de Aveiro coastal lagoon (Gafanha da Nazaré, Portugal, 40 • 39 N, 8 • 43 W) in February 2017 (winter). Samples were rinsed with fresh water, cleaned of epiphytes, if present, and oven-dried at 30 • C in an air tunnel (up to ca. 13% moisture content was achieved). Biomass of at least 5 specimens was used, five replicates of ca. 200 mg to extract total lipids and three replicates of 2 mg to determine the C/N ratio.

Proximate Elemental Composition Analysis
Elemental analysis (C and N) (2 mg × 3 replicates) was performed using an elemental analyzer (Leco TruspecMicro CHNS 630-200-200) at the combustion furnace temperature of 1075 • C and an afterburner temperature of 850 • C. The carbon-to-nitrogen ratio (C/N) was calculated through tissue C (g 100 g −1 dry weight biomass, DW)/tissue N content (g 100 g −1 DW) ratio (excluding moisture from sample weight). The nitrogen-protein conversion factor 6.25 was used to estimate protein content.

Lipid Extraction
The lipid content was determined by gravimetry. Lipids were extracted from weighed samples (200 mg each, for a total of 5 replicates) by adding 3 mL of methanol: chloroform mixture (2:1, v/v) to each sample replicate in glass tubes with Teflon-lined screw caps (modified Bligh and Dyer method used in the Marine Lipidomics Laboratory [28,71]). The organic phase was collected first and the remaining biomass residue was re-extracted twice. Water (2 mL) and chloroform (2 mL) were added to the collected total organic phase and the lower organic phase was collected for drying under a stream of nitrogen gas. The content of total lipid extract was estimated by gravimetry and stored at -20 • C, under a nitrogen atmosphere, before analysis. The five replicates were analyzed by LC-MS.

Hydrophilic Interaction Liquid Chromatography−High Resolution Mass Spectrometry (HILIC−MS) and Tandem Mass Spectrometry (MS/MS)
Analysis of lipid extracts was performed in a high-performance liquid chromatography (HPLC) Ultimate 3000 Dionex (Thermo Fisher Scientific, Bremen, Germany) system with an autosampler and coupled online to the Q-Exactive ® hybrid quadrupole Orbitrap ® mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). An Ascentis Si column (150 mm × 1 mm, 3 µm, Sigma-Aldrich, St. Louis, MO, USA) and a two-phases-solvent system (mobile phase A, acetonitrile/methanol/water (50:25:25, v/v/v) with 1 mM ammonium acetate; mobile phase B, acetonitrile/ methanol (60:40, v/v) with 1 mM ammonium acetate) were employed for the separation of lipids by HILIC-chromatography, as previously described [71]. The injection volume was 5 µL of each sample containing 10 µg of lipid extract, a volume of 4 µL of a mixture of phospholipid standards mix, described in da Costa et al. (2020) [71], and 86 µL of mobile phase B.
The MS employed was equipped with Orbitrap technology and was operated using a positive/negative switching toggle between positive (electrospray voltage of 3.0 kV) and negative (electrospray voltage of 2.7 kV) ion modes, with a capillary temperature of 250 • C and sheath gas flow of 15 arbitrary units (a.u.). Mass spectra were acquired using data-depending acquisition (DDA) mode, with cycles of one full-scan mass spectrum (mass resolving power of 70,000 full width at half-maximum, automatic gain control target of 1 × 10 6 , 200-1600 m/z scan range) and ten data-dependent MS/MS scans (resolution of 17,500 width at half-maximum and automatic gain control target of 1 × 10 5 ) with the dynamic exclusion of 60 s and intensity threshold of 1×10 4 , repeated continuously throughout the experiments. Tandem MS fragmentation was performed by higher-energy collisional dissociation (HCD), using stepped normalized collision energy ranging between 25, 30 and 35 eV. Data acquisition was carried out using the Xcalibur data system (V3.3, Thermo Fisher Scientific, MA, USA).

Data Analysis and Lipids Identification
Data acquisition was performed using the Xcalibur data system (V3.3, Thermo Fisher Scientific, Waltham, MA, USA). Peak integration and HPLC-MS data assignments were performed using MZmine 2.42 and the predefined parameters for data processing, as described in similar approaches published by the Marine Lipidomics Group [30]. Lipid identification was performed by matching the LC retention time (shown in the total ion chromatograms, Supplementary Materials, Figure S1), with the assignment of the molecular ions observed in the LC-MS spectra ( Figures S2-S8), with an accuracy of mass measurements < 5 ppm (Tables 2-4). Additionally, manual analysis of MS/MS spectra confirmed the identity of the polar head and fatty acyl composition of most of the identified lipid the molecular species (Supplementary Materials, Table S1 and Figures S2-S10). Typical fragmentation rules used to generate the assignments were included [71]. The normalization of lipid species was completed by dividing the exported values of integrated peak areas of each lipid species by the value of the peak area of a standard lipid species with the closest retention time.

Analysis of Fatty acid by Gas Chromatography-Mass Spectrometry (GC-MS)
Fatty acid methyl esters (FAMEs) were prepared by an alkaline well-established methodology [28,71,76]. A 70 µg aliquot of the lipids extracted from each sample was derivatized in a screw cap glass tube. Transesterification was performed by transferring 1 mL of a standard of methyl nonadecanoate (C19:0, 74208, Sigma-Aldrich, St. Louis, MO, USA) solution in hexane (1.27 µg mL −1 ) and 200 µL of KOH 2 M methanol. An aliquot of 600 µL of the upper phase was collected, dried under a stream of nitrogen gas and, subsequently, 120 µL of hexane was added to prepare the final solution for GC-MS analysis.
A 2 µL subsample of the FAME solution in hexane obtained from the derivatization was injected in an Agilent 8860 GC System gas chromatograph with GC 5977B Network Mass Selective Detector operating at 70 eV at 250 • C and equipped with a DBFFAP (Agilent 123-3232, 30 m × 320 µm × 0.25 µm) column. GC-MS was equipped with an auto sampler with a splitless injector at 220 • C. The separation of FAME was carried out with helium being used as the carrier gas (constant flow rate of 1.4 mL min −1 ) and using a temperature program for the column starting at 80 • C during 2 min and increasing to 160 • C at 25 • C/min, heating up to 210 • C at 2 • C/min, then to 225 • C at 20 • C/min and holding for 20 min. The system employed includes a Mass Selective Detector operating in Electron Ionization (EI) mode at 70 eV and scanning the mass range m/z 50-550 in a 1 s cycle in a full scan mode acquisition. Analyses were always replicated (at least n = 3). Methyl esters were identified using the software Agilent MassHunter Qualitative10.0, supported by NIST2014 mass spectral library, by comparing their retention time and MS spectra with those of Sigma-Aldrich standards (37 Component FAME Mix, Sigma-Aldrich) and by MS spectra comparison with online databases (AOCS lipid library). Quantitative analysis of FA was achieved from calibration curves of each methyl ester of FA from a FAME mixture (Supelco 37 Component FAME Mix, CRM47885, Sigma Aldrich, St. Louis, MO, USA), analyzed by GC-MS under the same conditions of extracts, with results being expressed as µg mg −1 of extract and µg g −1 of dry biomass. The relative amounts of FAs were calculated using the ratio of the amount of each FAME and the sum of all FAMEs identified; results are expressed as means (%, w/w). Nutritional, health and quality indices nutritive value (NVI), atherogenic (AI), thrombogenic (TI), hypocholesterolemic/hypercholesterolemic (h/H) and peroxidizability indices (PI) were determined according to the literature [56].

2,2 -Azino-bis-3-Ethylbenzothiazoline-6-Sulfonic Acid Radical Cation Assay-ABTS Radical Scavenging Activity
The antioxidant scavenging activity against the 2,2 -azino-bis-3-ethyl benzothiazoline-6sulfonic acid radical cation (ABTS •+ ) was evaluated using a previously described method [77]. The ABTS radical solution (3.5 mmol L −1 ) was prepared. This mixture was kept for 16 h in the dark at room temperature; then, it was diluted in ethanol to obtain an absorbance value of~0.9, measured at 734 nm using a UV-vis spectrophotometer (Multiskan GO 1.00.38, Thermo Scientific, Hudson, NH, USA). Radical stability was determined as reported by Santos et al. [77]. For an evaluation of the radical scavenging potential, a volume of 150 µL of each lipid extract of G. turuturu (12.5-250 µmol L −1 in ethanol, n = 4), or 150 µL of Trolox standard solution (10-75 µmol L −1 ), was placed in each well, followed by the addition of 150 µL of ABTS •+ diluted solution. Control lipid assays were prepared by replacing 150 µL of ABTS •+ diluted solution with 150 µL of ethanol. The % of the ABTS radical remaining was determined according to Equation (1); free radical-scavenging activity of samples was calculated as the percentage of inhibition of the ABTS radical (Equation (2)). The concentration of samples reducing 50% of the ABTS radical after 120 min (IC 50 ) were calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity was expressed as Trolox Equivalents (TE, µmol Trolox/g of sample) according to Equation ( The antioxidant scavenging activity against the 2,2-diphenyl-β-picrylhydrazyl radical (DPPH • ) was evaluated using a previously described method [77]. A stock solution of DPPH • in ethanol (250 µmol L −1 ) was prepared and diluted to provide a working solution with an absorbance value of~0.9, measured at 517 nm using a UV-Vis spectrophotometer (Multiskan GO 1.00.38, Thermo Scientific, Hudson, NH, USA). The evaluation of the radical stability was determined as previously reported [77]. For evaluation of the radical scavenging potential, a volume of 150 µL of each lipid extract of G. turuturu (12.5-250 µmol L −1 , n = 4), or 150 µL of Trolox standard solution (10-75 µmol L −1 ), was placed in each well, followed by the addition of 150 µL of a DPPH • diluted solution and, again, an incubation period of 120 min before measuring absorbance at 517 nm every 5 min. The % of the DPPH radical remaining was calculated according to Equation (4); free radicalscavenging activity of samples was determined as the percentage of inhibition of the DPPH radical (Equation (5)); the concentration of samples reducing 25% of the DPPH radical after 120 min (IC 25 ) was calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity expressed, as TE (µmol Trolox g −1 of sample), was determined (Equation (6)). The significant differences (p < 0.05) calculated by Kruskal-Wallis with Dunn's multiple comparisons were used to analyze the results between groups (GraphPad Prism 8).

Cyclooxygenase 2 (COX 2) Enzyme Inhibitory Capacity of Lipid Extract
The inhibition potential against COX-2 was carried out by enzyme immunoassay (EIA) kit (catalogue No. 701080, Cayman Chemical Company, Ann Arbor, MI, USA), as described by the manufacturer (https://www.caymanchem.com/pdfs/701080.pdf, accessed on 6 March 2021) [67,71]. Lipid extracts were dissolved in 100% DMSO and five concentrations ranging between 12.5 and 250 µg mL −1 were tested. The amount of prostaglandin F2α generated from AA in the cyclooxygenase reaction was determined by spectrophotometry at 412 nm using a Multiskan GO 1.00.38 (Thermo Scientific, Hudson, NH, USA). The results are expressed as a percentage of inhibited COX-2. The significant differences (p < 0.05) calculated by Kruskal-Wallis with Dunn's multiple comparisons were used to analyze the results between groups (GraphPad Prism 8).

Conclusions
The present work provides the first insight on the G. turuturu lipidome, its nutritional value and its anti-oxidant and anti-inflammatory potential. The lipid and protein composition of this red seaweed, the profiles of fatty acids and lipid species and the health lipid indices determined demonstrated the nutritional value of G. turuturu. The biomass of this red seaweed can therefore be considered as a potential source for food, feed and other high-end uses. For industrial applications, structural characterization efforts are essential for the valorization of these lipids. The antioxidant free radical scavenging potential and cyclooxygenase-2 inhibitory activity were investigated and activities were achieved at low concentrations of extracts. The lipid extracts of G. turuturu constitute an interesting offer of natural and effective molecules to fight against pathological complications linked to free radicals and to be used in industry. Overall, our findings will likely inspire future studies targeting G. turuturu as a source of nutritive biomass and bioactive molecules for nutraceutical, pharmaceutical and cosmeceutical applications.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/md19080414/s1, Table S1: Typical fragmentation patterns observed by Tandem Mass Spectrometry used to identify the head group and fatty acyl composition of polar lipids structures from Grateloupia turuturu (NL-neutral loss; PI-product ion), Figure S1