A New Look for the Red Macroalga Palmaria palmata: A Seafood with Polar Lipids Rich in EPA and with Antioxidant Properties

Palmaria palmata is an edible red macroalga widely used for human consumption and valued for its high protein value. Despite its low total lipid content, it is rich in eicosapentaenoic acid (EPA). This seaweed has been scarcely explored with regard to its lipid composition. The polar lipids of seaweeds are nowadays recognized as important phytochemicals contributing to their add value valorization and providing support for claims of potential health benefits. The present study aimed to disclose the polar lipid profile of P. palmata, farmed in an integrated multi-trophic aquaculture (IMTA) through modern lipidomic approaches using high-resolution LC-MS and MS/MS and to screen for the antioxidant properties of this red macroalga. A total of 143 molecular species of lipids were identified, belonging to several classes of polar lipids, such as glycolipids, phospholipids, and betaine lipids. It is noteworthy that the most abundant lipid species in each class were esterified with eicosapentaenoic acid (EPA), accounting for more than 50% of the lipid content. The polar lipid extract rich in EPA showed antioxidant activity with an inhibition concentration (IC) of IC30 = 171 ± 19.8 µg/mL for α,α-diphenyl-β-picrylhydrazyl radical (DPPH●) and IC50 = 26.2 ± 0.1 µg/mL for 2,20-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS●+). Overall, this study highlights that P. palmata farmed in an IMTA framework can be a sustainable source of beneficial lipids with antioxidant activity. Moreover, this red macroalga can be exploited for future applications as a source of lipids rich in EPA for food and feed, nutraceuticals, and cosmetics.


Introduction
Over the last 20 years, macroalgae have ceased to be a trifle in western countries and have been considered as a nutritional and functional food, generating wealth for many coastal countries [1]. Red macroalgae (phylum Rhodophyta) are mainly used as a source of hydrocolloids (agar and carrageenans), generating a large turnover worldwide [2]. Nevertheless, the marketing of edible red macroalgae is also very important, as is the trade of Asian species of Porphyra sp., the most valued variety of seaweed in the world, generally recognized by its common Japanese name, nori. Recently, some of the species of the genus Porphyra were transferred to the genus Pyropia [3]. Food and agriculture organization of the united nations (FAO) data indicate that the production of Porphyra

Identification of the of Palmaria Palmata Lipidome
The lipid extracts of P. palmata represented an average yield of 1% by dry weight (255.6 ± 1.19 mg of biomass, 2.58 ± 0.19 mg lipid extract). The fatty acid composition of the lipid extract of P. palmata, identified by gas chromatography-mass spectrometry (GC-MS) ( Table 1), shows the prevalence of 20:5(n-3) EPA with a relative abundance of 51.68% ± 6.5. The saturated FA 16:0, 18:0, and 14:0 represented 24.32% ± 1.11, 12.45% ± 6.74, and 5.32 ± 0.44, respectively. The others FA identified had relative abundances of less than 5%. The GC-MS analysis also identified phytol compounds, a component of chlorophyll and vitamin E and K. Phytol is a precursor of vitamin E, an important nutritional supplement obtained commercially by isolation from natural sources [26]. Identification of the polar lipid profile at the molecular level was performed by high-resolution hydrophilic interaction liquid chromatography-mass spectrometry -HILIC-LC-MS and HILIC-LC-MS/MS. In total, 46 molecular species of glycolipids were identified, 1 molecular species of betaine lipids, 91 molecular species of phospholipids, and 6 molecular species of inositephosphoceramide lipids, representing a total of 144 species of lipids. The glycolipids identified included the acidic glycolipids sulfoquinovosyl diacylglycerol (SQDG) (  Table 3). The most abundant species in each class of glycolipids was assigned as SQDG (36:5) corresponding to SQDG (20:5/16:0), SQMG (14:0), DGDG (36:5) as DGDG (20:5/16:0), DGMG(16:0), MGDG (40:10) as MGDG (20:5/20:5), and MGMG (20:5). Table 2. Molecular species of glycolipids SQDGs and SQMGs identified by HILIC-ESI-MS as [M − H] − ions. C represents the total number of carbon atoms and N represents the total number of double bonds on the fatty acyl chains. The most abundant species in each class are highlighted in bold type. Fatty acids' sn-1 and sn-2 position is based on biosynthetic pathways [27][28][29].  Betaine lipids of P. palmata included only one species of diacylglyceroltrimethylhomoserine (DGTS) assigned as DGTS (32:5), with a molecular formula of C 42 H 80 O 7 N, and identified in the LC-MS spectra as positive [M + H] + ions. Its theoretical m/z is 710.5935 and the observed m/z was 710.5936 (0.14 ppm). Table 3. Molecular species of glycolipids MGDG, MGMG, DGDG, and DGMG identified by HILIC-ESI-MS as [M + NH4] + ions. C represents the total number of carbon atoms and N represents the total number of double bonds on the fatty acyl chains. The most abundant species in each class are highlighted in bold type. Fatty acids' sn-1 and sn-2 position is based on biosynthetic pathways [27][28][29]. The classes of phospholipids found in P. palmata included phosphatidylcholine (PC) ( Figure 3A (Table 5). Also, inositephosphoceramide lipids (IPC) were identified as negative [M + CH 3 COO] − ions ( Table 6)         The relative quantification of the identified species was calculated after integrating the peak area of each identified ion, normalized to the internal standard and divided by the sum of the normalized area of all species. Lipid species identified with a relative percentage of 0.5% or greater are shown in Figure 5.

Evaluation of Antioxidant Activity
The antioxidant potential of P. palmata polar lipid-rich extract was evaluated through free radical DPPH and ABTS + scavenging assays. While DPPH assay measures the ability of antioxidants to scavenge the DPPH generated in the organic phase, the ABTS + assay acts in the same way in the aqueous phase. The percentage of radical inhibition in the presence of extracts rich in polar lipids was calculated after 120 min. The percent inhibition is proportional to time and the lipid extract concentration, even though the response appeared to be non-linear, with better performance for the ABTS + assay (Figure 7). Results gathered in these assays showed that for the DPPH assay, the polar lipid-rich extract concentration providing 30% of inhibition (IC30) was 171 ± 19.8 µg/mL with a TE of 88.7 ± 7.7 Trolox µmol/g lipid. The polar lipid-rich extract concentration providing 50% inhibition (IC50) in the ABTS assay was 26.2 ± 0.1 µg/mL with a TE of 555.4 ± 28.1 Trolox µmol/g lipid. The reactivity and the capacity of the extract rich in polar lipids to scavenge these radical were very different. While for the ABTS assay it was possible to calculate 50% inhibition, in the case of the DPPH assay, the maximum percentage of inhibition reached nearly 30%.

Discussion
The consumption of long-chain PUFA, particularly n-3, may have long-term health benefits. Their positive effects include reduced cardiovascular disease, morbidity, and mortality [30][31][32]; enhanced visual and neurological development; and improved of inflammatory conditions, such as arthritis and asthma [33][34][35]. It is recognized that seafood is the main source of n-3 long-chain PUFAs, mainly EPA and docosahexaenoic acid (DHA). However, global overexploitation and depletion of marine fish stocks is of long-standing concern, as existing populations of wild and farmed species are unlikely sufficient to meet n-3 PUFA requirements for human consumption. In this context, P. palmata appears as an interesting source of EPA that can be supplied in a socio-ecologically sustainable way. Namely its production in IMTA systems compared to current wild harvesting practices might ensure the availability of biomass with increased stability of its nutritional value and biochemical profile, namely its polar lipids and its PUFA composition [36,37].
As described above (Tables 1-4), PL and GL of P. palmata are quite rich in EPA. Our work is consistent with previous studies that reported high levels of EPA in P. palmata compared to other edible seaweeds [8,38].
Phospholipids, in particular those rich in PUFA (namely EPA), contribute significantly to the added value of P. palmata. There is evidence that PLs are better delivers of PUFA than triglycerides (TGs) [39,40]. Several studies suggested that much of the dietary PLs fraction is integrated into high-density lipoprotein (HDL) in the intestine, later joining the plasma HDL pool [41][42][43]. Since PLs are associated with low toxicity, allowing their use for any route of administration, PL from P. palmata are potential candidates for food and nutraceutical formulations.
Phosphatidylcholine is the most abundant phospholipid class of P. palmata. Administration of PCs has been reported to be beneficial for senescence, cognitive function, inflammatory diseases, and plasma and hepatic lipid metabolism [44][45][46][47]. It can also be used as a choline supplier. Choline is a component of the vitamin B complex, essential for human nutrition and used by the body to produce acetylcholine, one of the major neurotransmitters of the nervous system, involved in neural networks associated with memory [48]. Choline supplementation is important for vegans and vegetarians, who are at a higher risk for choline deficiency. Some studies also linked high choline intake with reduced risk of breast and colorectal cancer [49][50][51]. Thus P. palmata PLs could be considered as a potential functional food or as an ingredient for the fortification of foods for nutraceutical applications.
Other potential uses of P. palmata PLs are either in the cosmetics or in the pharma industries. Cosmetic formulations use the emulsifying properties of PLs for skin moisturizing products [52]. Pharmaceutical applications of PLs rely on their ability to form liposomes after mixing in aqueous media, making them a potential drug delivery vector [52,53]. Therefore, P. palmata PLs can potentially be used as emulsifiers, as an n-3 FAs supplement, and as beneficial nutritional biomolecules. Macroalgae are also considered a promising source of value-added phytochemicals. Several studies have reported that glycolipids can also display bioactive activities, including antioxidant [54], anti-inflammatory [14], anti-proliferative [55], and anti-microbial [56] activities. Some of the glycolipids identified in P. palmata have already been described as having bioactive properties, such as SQDG(34:2) with antiviral activity against HCM-virus [16], SQMD(16:0) with antibacterial activity against Xanthomonas oryzae [57], and the SQDG carrier of EPA with anti-proliferative effect on the inhibition of human telomerase [12] and DNA polymerase α and β [58]. Additionally, a study by Banskota and collaborators [14] attributed the anti-inflammatory action of P. palmata to polar lipids, including SQDG(20:5;14:0), via the inhibitory activity of nitric oxide, highlighting the role of EPA. Moreover, a MGDG carrier of EPA was patented because of its anti-inflammatory action [59]. The search for natural antioxidants has long attracted the attention of researchers, since inflammation is associated with oxidative stress occurring in several diseases, including chronic and age-related ones [60,61]. Oxidative stress occurs when the production of radicals increases and exceeds the detoxification capacity of cellular antioxidant defense systems. Antioxidants are biomolecules that prevent, or delay damage to other molecules, by the presence of free radicals, because they have the ability to block or inhibit them directly or indirectly, or because they stimulate cellular antioxidant defenses [62]. Antioxidants from natural or organic sources aim not only to promote the development of new drugs but also to act in the prevention of inflammatory diseases [63]. Such biomolecules with antioxidant properties can be obtained from foods, including plants and marine vegetables [64,65]. There are some studies of seaweed lipid characterization using lipidomics based on the LC-MS approach that allowed the putative bioactivity and health benefits of seaweeds lipids to be highlighted, as well the exploitation of seaweeds as a functional food, as reported for Codium tomentosum, Porphyra dioica, Gracilaria sp, Chondrus crispus, Ulva rigida, Saccharina latissima, and Fucus vesiculosus [21][22][23][24][25]66,67]. However, seaweeds' lipids antioxidant potential was not explored.
The antioxidant activity of seaweeds has been mostly related to their phenolic compounds and pigment contents [68][69][70][71][72], whereas it is only now that lipids are beginning to deserve special attention. To the best of our knowledge, there is only one study of lipids' fraction antioxidant activity for the red and brown seaweeds Solieria chordalis ((C.Agardh) J. Agardh, 1842) and Sargassum muticum ((Yendo) Fensholt, 1955), respectively, whose antioxidant potential was lower than the one obtained in this study for P. palmata [19]. Overall, our results showed that the polar lipid-rich extract of P. palmata exhibits antioxidant proprieties, showing a higher percentage inhibition of DPPH and TE than those of the methanolic extract of P. palmata, mainly composed of phenolic compounds, as previously described [68]. It also showed significantly better antioxidant activity for the DPPH and ABTS + assays compared to the red seaweed Gracilaria manilaensis (Yamamoto & Trono, 1994) organic extract [73]. The polar lipid-rich extract of P. palmata still had a higher antioxidant potential for the ABTS + assay compared to a study of antioxidant activity using different organic solvents with phenolic content from the brown seaweed Sargassum serratifolium ((C. Agardh) C. Agardh, 1820) [74].

Seaweed Biomass
The biomass of Palmaria palmata ((Linnaeus) F.Weber & D.Mohr, 1805) was provided by a local aquaculture producer-ALGAplus-who farms seaweed integrated with finfish production (located at Ria de Aveiro coastal lagoon, mainland Portugal, 40 • 36 43"N, 8 • 40 43"W). Palmaria palmata is produced by vegetative propagation, in a controlled indoor system from June to September (approximately) and in an open-flow outdoor tank system during colder months. For this work, the red algae were harvested in May 2018, cleaned to remove the epiphytes, and washed using sterilized seawater. It was then dried at 25 • C in an air tunnel until 10% to 12% total moisture was reached. Five 250-mg aliquots were obtained from bulk production and were used for total lipid extraction.

Lipid Extraction
The biomass of P. palmata was cut into small pieces and ground in a mortar and pestle with liquid nitrogen until homogenized. The lipid extraction procedure was performed using a modified Bligh and Dyer protocol [23], mixing 250 mg of seaweed biomass (five replicates) with 2.5 mL of MeOH and 1.25 mL of CHCl 3 in a glass PYREX tube and homogenized by vortexing for 2 min and incubation in ice on a rocking platform shaker (Stuart equipment, Bibby Scientific, Stone, UK) for 2 h and 30 min. The mixture was centrifuged (Selecta JP Mixtasel, Abrera, Barcelona, Spain) for 10 min at 2000 rpm and the organic phase was collected in a new glass tube. The biomass residue was re-extracted twice with 2 mL of MeOH and 1 mL of CHCl 3 . To wash the lipid extract and induce phase separation, 2.3 mL of Milli-Q water was added to the final organic phase, followed by centrifugation for 10 min at 2000 rpm. The organic lower phase was collected in a new glass tube and dried under a nitrogen stream. Lipid extracts were then transferred to amber vials, dried again, weighed, and stored at −20 • C. Lipid content was estimated as a dry weight percentage.

Polar Lipid Analysis by Hydrophilic Interaction Liquid Chromatography Mass Spectrometry (HILIC-ESI-MS)
Lipid extracts were analyzed by mass spectrometry using hydrophilic interaction liquid chromatography (HILIC) on an Ultimate 3000 Dionex (Thermo Fisher Scientific, Bremen, Germany) with an autosampler coupled to a Q-Exactive hybrid quadrupole mass spectrometer (Thermo Fisher, Scientific, Bremen, Germany). The elution method was previously described [21,24,25] and applied with some modifications. The system is based on two mobile phases: Mobile phase A (25% water, 50% acetonitrile, and 25% methanol) and mobile phase B (60% acetonitrile and 40% methanol), both with 1 mM ammonium acetate. Firstly, 40% of mobile phase A was held isocratically for 8 min, followed by a linear increase to 60% of mobile phase A within 7 min, and maintained for 5 min. After that, conditions returned to the initial settings in 15 min (5 min to decrease to 40% of phase A and a re-equilibration period of 10 min prior to the next injection). A volume of 5 µL of each sample, containing 5 µg of lipid extract in CHCl 3 , 4 µL of phospholipid standards mix (dMPC-0.02 µg, dMPE-0.02 µg, LPC-0.02 µg, dPPI-0.08 µg, dMPG-0.012 µg, dMPS-0.04 µg, NPSM-0.02 µg, dMPA-0.08 µg, tMCL-0.08 µg), and 91 µL of starting eluent (60% B and 40% A), was introduced into the Ascentis Si column HPLC Pore column (15 cm × 1 mm, 3 µm, Sigma-Aldrich) with a flow rate of 40 µL min −1 at 30 • C. The mass spectrometer with Orbitrap ® technology was operated in simultaneous positive (electrospray voltage 3.0 kV) and negative (electrospray voltage -2.7 kV) modes with high resolution with 70,000 and AGC target of 1 × 10 6 , the capillary temperature was 250 • C, and the sheath gas flow was 15 U. The tandem mass spectrometry experiments were performed at a resolution of 17,500 and AGC target of 1 × 10 5 with one full scan mass spectrum and 10 data-dependent MS/MS scans. The cycles were repeated continuously throughout the experiments with the dynamic exclusion of 60 s and an intensity threshold of 1 × 10 4 . Normalized collision energy™ (CE) ranged between 25, 30, and 35 eV. Data acquisition was performed using the Xcalibur data system (V3.3, Thermo Fisher Scientific, USA). The identification of molecular species of polar lipids was based on the assignment of the molecular ions observed in the LC-MS spectra, typical retention time, mass accuracy, and MS/MS spectra information.

Data Analysis
In a first approach, the lipid species of P. palmata were identified using MZmine 2.32 software. This tool allows the analysis of raw data acquired in full MS by the integration of each lipid species peak, peak processing, and assignment against an in-house database. The validated peaks were within the time range of a MS full run and peaks with raw intensity lower than 1e4 were excluded. All the information originating from the MZmine software was confirmed based on the assignment of the molecular ions observed in the LC-MS spectra, typical retention time, exact mass accuracy, and MS/MS spectra information. Only exact mass accuracy with an error of less than 5 ppm was considered. The typical fragmentation of each lipid class in MS/MS spectra was taken into account according to what has already been described in the literature [25]. The normalization of the identified lipid species was performed by exporting integrated peak areas values (.csv file) and dividing the peak area value of each species by the peak area value of a standard lipid species with the closest retention time. The lipid species' relative abundance was calculated in terms of the percentage dividing the normalized value of each lipid species by the sum of all identified lipid species. Bar graphs were created using the software GraphPad Prism 8.0.1

2-Diphenyl-1-Picrylhydrazyl Radical Assay-DPPH Radical Scavenging Activity
The antioxidant scavenging activity against the α,α-diphenyl-β-picrylhydrazyl radical (DPPH ) was evaluated using a previously described method [75,76] applied with some modifications. A stock solution of DPPH in ethanol (250 µmol/L) 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) controlled by the SkanIT software version 3.2 (Thermo Scientific). To evaluate the radical stability, a volume of 150 µL of ethanol was added to 15 microplate wells followed by addition of 150 µL of DPPH diluted solution and an incubation period of 120 min, with absorbance measured at 517 nm every 5 min. For evaluation of the radical scavenging potential, a volume of 150 µL of P. palmata lipid extract (25, 50, 100, 250 µg/mL in ethanol) and 150 µL of Trolox standard solution (5, 12.5, 25, 37.5 µmol/L in ethanol) were placed in each well followed by addition of 150 µL of DPPH diluted solution, and again an incubation period of 120 min, with absorbance measured at 517 nm every 5 min. The control lipid extracts were also assayed by replacing 150 µL of DPPH diluted solution by 150 µL of ethanol. Radical reduction by hydrogen donor antioxidants was monitored by measuring the decrease in absorbance during the reaction, thereby quantifying radical scavenging, which is accompanied by a radical color change. All measurements were performed in triplicate on two different days. The % of DPPH radical remaining was determined according to: % DPPH remaining = (Abs samples after 120 min/Abs sample at the beginning of reaction) × 100.
The free radical-scavenging activity of samples was determined as the percentage of inhibition of DPPH radical according to: Inhibition% = ((Abs DPPH − (Abs samples − Abs control)) / Abs DPPH) × 100.
The concentration of samples capable of reducing 30% of DPPH radical after 120 min (IC30) were calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity is expressed as Trolox Equivalents (TE, µmol Trolox/g of sample), according to: TE = IC30 Trolox (µmol/L) × 1000/IC30 of samples (µg/mL). 4.8. 2,20-Azino-bis-3-Ethylbenzothiazoline-6-Sulfonic Acid Radical Cation Assay-ABTS Radical Scavenging Activity The antioxidant scavenging activity against the 2,20-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS + ) was evaluated using a previously described method [76,77] applied with some modifications. The ABTS radical solution (3.5 mmol/L) was prepared by mixing 10 mL of ABTS stock solution (7 mmol/L in water) with 10 mL of potassium persulfate K 2 S 2 O 8 (2.45 mmol/L in water) [77,78]. This mixture was kept for 12 h at room temperature and 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) controlled by the SkanIT software version 3.2 (Thermo Scientific). For an evaluation of the radical stability, a volume of 150 µL of ethanol was added to 15 microplate wells followed by addition of 150 µL of ABTS + diluted solution and an incubation period of 120 min, with absorbance measured at 734 nm every 5 min. For an evaluation of the radical scavenging potential, a volume of 150 µL of lipid extract (25, 50, 100, 250 µg/mL in ethanol) and 150 µL of Trolox standard solution (4,8,16,28,40,56 µmol/L in ethanol) were placed in each well followed by addition of 150 µL of ABTS + diluted solution, and a new incubation period of 120 min, with absorbance measurements at 734 nm every 5 min. The control lipid extracts were also assayed by replacing 150 µL of ABTS + diluted solution by 150 µL of ethanol. Radical reduction by hydrogen donor antioxidants was monitored by measuring the decrease in absorbance during the reaction, thereby quantifying radical scavenging, which is accompanied by a radical color change. All measurements were performed in triplicate on two different days. The % of ABTS radical remaining was determined according to: % ABTS remaining = (Abs samples after 120 min/Abs sample at the beginning of reaction) × 100.
The free radical-scavenging activity of samples was determined as the percentage of inhibition of ABTS radical according to: Inhibition% = ((Abs ABTS − (Abs samples − Abs control))/Abs ABTS) × 100.
The concentration of samples capable of reducing 30% of ABTS radical after 120 min (IC50) were calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity is expressed as Trolox Equivalents (TE, µmol Trolox/g of sample), according to: TE = IC50 Trolox (µmol/L) × 1000/IC50 of samples (µg/mL).

Conclusions
In this study, the use of a mass spectrometry-based approach identified 144 lipid species of P. palmata produced in an environmentally friendly IMTA system. Eicosapentaenoic acid was the most abundant fatty acid identified in P. palmata, with a relative abundance greater than 50%. The content of EPA may be one of the major factors in the antioxidant activity of the polar lipid-rich extract, which has been found to be superior to that of phenolic compounds described in previous studies. Nevertheless, our results may not be exclusive to lipids, and synergy with other molecules should be taken into account. These findings revealed the lipid profile of P. palmata and highlighted that when farmed under IMTA, P. palmata can be a stable and sustainable source of beneficial lipids. The findings reported in this study add value to the polar lipid-rich extract of P. palmata and open a window of opportunity for innovative biotechnological applications targeting algal-based products. This work is the beginning of polar lipid prospection in P. palmata biomass and its results should now be validated across other possible P. palmata sources.