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Unraveling the Lipidome and Antioxidant Activity of Native Bifurcaria bifurcata and Invasive Sargassum muticum Seaweeds: A Lipid Perspective on How Systemic Intrusion May Present an Opportunity
Unraveling the Lipidome and Antioxidant Activity of Native Bifurcaria bifurcata and Invasive...
Santos, Fábio;Monteiro, João P.;Duarte, Daniela;Melo, Tânia;Lopes, Diana;da Costa, Elisabete;Domingues, Maria Rosário
2020-07-21 00:00:00
antioxidants Article Unraveling the Lipidome and Antioxidant Activity of Native Bifurcaria bifurcata and Invasive Sargassum muticum Seaweeds: A Lipid Perspective on How Systemic Intrusion May Present an Opportunity 1 ,y 1 , 2 ,y 1 1 , 2 1 , 2 Fábio Santos , João P. Monteiro , Daniela Duarte , Tânia Melo , Diana Lopes , 1 , 2 1 , 2 , Elisabete da Costa and Maria Rosário Domingues * Mass Spectrometry Centre, LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Santiago University Campus, 3810-193 Aveiro, Portugal; fabiofs379@gmail.com (F.S.); jpspmonteiro@yahoo.com (J.P.M.); danieladuarte98@ua.pt (D.D.); taniamelo@ua.pt (T.M.); dianasalzedaslopes@ua.pt (D.L.); elisabetecosta@ua.pt (E.d.C.) CESAM—Centre for Environmental and Marine Studies, Department of Chemistry, University of Aveiro, Santiago University Campus, 3810-193 Aveiro, Portugal * Correspondence: mrd@ua.pt; Tel.: +351-234-370-692 y Both authors share first co-authorship. Received: 8 June 2020; Accepted: 17 July 2020; Published: 21 July 2020 Abstract: Brown seaweeds are known to present components with appealing bioactive properties eliciting great interest for industrial applications. However, their lipid content is generally disregarded beyond their fatty acid (FA) composition. This study thoroughly characterized the lipid profile of two brown seaweeds collected from Portuguese coast, the native Bifurcaria bifurcata and the invasive Sargassum muticum species, and bioprospecting for antioxidant activity. An integrated state-of-the-art approach including gas chromatography-mass spectrometry (GC–MS) and liquid chromatography-mass spectrometry (HILIC–ESI-MS/MS), allowed a comprehensive picture of FA and polar lipid content. Polar lipid profile of B. bifurcata and S. muticum included 143 and 217 lipid species respectively, distributed between glycolipids, phospholipids, and betaine lipids. Some of the lipid species found have been assigned biological activity and contain of n-3 and n-6 FA. Sargassum muticum presented the highest n-3 FA content. Low concentrations of extracts of both seaweeds displayed antioxidant activity, with S. muticum presenting more promising results. These findings contribute to the nutritional and industrial exploitation of both seaweeds, highlighting their relevance as viable sources of bioactive and added-value compounds. Sargassum muticum presented interesting lipid composition and bioactivity, which may represent an accessible opportunity for the exploitation of this invasive seaweed, especially taking advantage of Sargassum blooms. Keywords: bioactivity; lipidomics; mass spectrometry; nutritional quality; polyunsaturated fatty acids; macroalgae; antioxidant 1. Introduction Over the last decades consumer priority turned its focus to lifestyle, healthiness, and well-being, without neglecting environmental and sustainability concerns [1]. These concerns, along with the increasing demand for natural compounds and functional foods, justified a new look at the composition of seaweeds, since it is well recognized that these marine resources are a natural and sustainable source of natural compounds [2,3]. This new paradigm led to an increasing interest in seaweed utilization for food, cosmetic, agricultural, pharmaceutical, biomedical, and nutraceutical applications [4,5]. Antioxidants 2020, 9, 642; doi:10.3390/antiox9070642 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 642 2 of 20 The Portuguese coast hosts 1909 dierent marine algae, 243 of which belong to Phaeophyceae group [6], where native seaweeds like Bifurcaria bifurcata (R. Ross, 1958) and invasive species like Sargassum muticum ((Yendo) Fensholt, 1955) share the same habitats. Bifurcaria bifurcata inhabits Atlantic Ocean and can be found from southern limit of Morocco to north-western Ireland [7,8]. As for S. muticum, from the shorelines of Gulf of Mexico in the Atlantic Ocean, it was pushed by maritime currents into the North Atlantic, composing the so-called Sargasso Sea [9]. In 2018 the total amount of S. muticum biomass was estimated to attain a whopping amount of 20 million tones, impacting activities like sailing or fishing and causing great environmental disturbances in beaches and reef lagoons [9,10]. Sargassum muticum invasion into new territories brought subsequent changes to local native species, causing the decrease in the occurrence of some native perennial seaweeds [11,12], or even leading to the loss of native genotypes [13,14]. Moreover, it has been suggested that invasive seaweeds have the potential to change epifaunal communities, and therefore alter the dynamics of entire ecosystems [15]. Until now there is no permanent method to remove this invasive species, nevertheless several eorts to fully take advantage of this biomass have been proposed [16,17]. In fact, invasive seaweeds such as S. muticum are potentially interesting and a viable natural resource for a global marine-derived drugs market, predicted to represent $21,955.6 billion by 2025 [18]. Brown seaweeds have been studied mostly due to the intrinsic properties of their polysaccharides, which are largely used by the hydrocolloid industry [19,20], but they also have been explored in the cosmetic and textile industries, and probed for biomedical and pharmaceutical applications [17,21]. Other compounds such as proteins, minerals, pigments, polyphenols, and polyunsaturated fatty acids (PUFA) justified the interest for distinct applications [22,23]. The total lipid content in brown seaweeds accounts for ca. 8% of dry weigh (DW) biomass and is commonly characterized in terms of fatty acid composition and recognized for its high content in PUFA. Lipid fractions from brown seaweeds, such as B. bifurcata and S. muticum, include an interesting content in PUFA eicosapentaenoic acid (20:5 n-3), docosahexaenoic acid (22:6 n-3), octadecatetraenoic acid (18:4 n-3), -linolenic acid (18:3 n-3), and eicosatetraenoic acid (20:4 n-6) [7,24–27]. This is especially important because n-3 PUFA are claimed to be beneficial for the prevention of cardiovascular diseases and other chronic diseases [21,28,29]. Antioxidant eects are among the most searched bioactivities for natural products, including lipids [30,31]. Natural antioxidant products, with radical scavenging activity and capacity to neutralize reactive oxygen species (ROS) protect cells from ROS-induced cellular damage [30–33], thereby reducing the risk of diseases associated with oxidative stress [34,35]. Seaweed antioxidants are sustainable alternatives to synthetic antioxidants, in line with consumer preference towards natural substances, and can be use as functional ingredients in food or in cosmetics products [25]. In general, in vitro studies have been mainly devoted to the survey of lipophilic compounds from seaweeds such as carotenoids, some polyphenols, and flavonoids exhibiting antioxidant activity [21,31]. Particularly, B. bifurcata dichloromethane extract, mainly composed of diterpenes, fatty acids (FA), and sterols, showed antioxidant, anti-inflammatory, and antibacterial activities [24] and provided interesting information for the further exploitation of this seaweed. Moreover, active components of the ethyl acetate, ethanol, and methanol extracts of S. serratifolium showed radical scavenging activities [31], while chloroform/methanol (1/1, v/v) polar lipids rich extract from S. muticum demonstrated radical scavenging activity [33]. Lipid extracts from brown algae containing glycolipids (GL) showed the capacity to suppress ROS in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages [36], that were positively correlated with the oxidative stability of eicosapentaenoic acid (20:5 n-3) and stearidonic acid (18:4 n-3) PUFA in their GL forms, which encouraged further studies in seaweed polar lipids as source functional lipids. The lipid content of brown seaweeds has usually been addressed by FA profiling. However, FA in seaweeds are mainly included in polar lipids related to GL, phospholipids (PL), and betaine lipids, that have been pinpointed by using low tech methods [37,38]. The characterization of detailed lipidomic signatures using omics approaches for brown seaweeds was only performed for Fucus vesiculosus [39] and Saccharina latissima [40] and has never been performed for these species. Antioxidants 2020, 9, 642 3 of 20 The goal of this work was to characterize lipid extracts from of B. bifurcata and S. muticum in terms of polar lipid molecular components, the polar lipdome, and antioxidant activity, and to attempt to relate composition with bioactivity, by signaling components with previously reported biological activity. Therefore, we performed an in-depth analysis of the lipid composition of B. bifurcata and S. muticum through a liquid chromatography coupled to high resolution mass spectrometry approach. The survey of antioxidant activity of total lipids extracts from B. bifurcata and S. muticum was conducted through their free radical scavenging potential against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals. Overall, this work will contribute to the valorization of these two species, highlighting the potential of turning S. muticum biomass in particular into natural high-value products and commercially viable resources. 2. Materials and Methods 2.1. Reagents Dichloromethane (CH Cl ), methanol (MeOH), and acetonitrile (high-performance liquid 2 2 chromatography—HPLC, grade) were purchased from Fisher Scientific Ltd. (Loughborough, UK). Milli-Q water (Synergy, Millipore Corporation, Billerica, MA, USA) was used. Phospholipid standards: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dMPC), 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (dMPE), 1,2- dimyristoyl-sn-glycero-3-phospho-(1 -rac-)glycerol (dMPG), 1,2- dipalmitoyl-sn-glycero-3- phosphatidylinositol (dPPI), 1–nonadecanoyl-2-hydroxy-sn-glycero-3- phosphocholine (LPC), dimyristoyl phosphatidic acid (dMPA), and N-heptadecanoyl-D-erythro- sphingosine (Cer (d18:1/17:0)) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Aldrich (Milwaukee, WI). 2,2’- Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was obtained from Fluka (Buchs, Switzerland). Ammonium acetate and 6-hydroxyl-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich (St Louis, MO, USA). All other reagents and chemicals used were of the highest grade of purity and were purchased from major commercial sources. 2.2. Sampling Bifurcaria bifurcata and S. muticum samples were collected in Aguda beach, Porto, Portugal 0 0 (Portugal, 41 2 38” N, 8 39 10” W) on 18 April 2018 (spring). Biomass samples (composite sampling of at least five specimens were used) were washed thoroughly with fresh water to remove epiphytes, eliminate salt, sand, or shells and freeze-dried. Samples were maintained at 80 C for posterior analyses. 2.3. Lipid Extraction The biomass samples were extracted using a modified Bligh and Dyer method [41]. Samples were weighed (250 mg, a total of five replicates for each seaweed) and transferred to glass tubes with Teflon-lined screw caps. Total lipid extraction was performed by adding 3.75 mL of a mixture methanol:dichloromethane (2:1) to each replicate sample. After individual homogenization during 2 min using a vortex and 1 min of sonication, samples were incubated on ice on a rocking platform shaker (Stuart Scientific STR6, Bibby, UK) for 2 h and 30 min. The mixture was centrifuged at 392 g for 10 min (Pro-Analytical series, UK), and the organic phase was collected to a new glass tube. The remaining biomass residue was re-extracted three times with 3 mL of a mixture methanol:dichloromethane (2:1). Water (2 mL) was then added to the total collected organic phase and tubes were centrifuged again at 392 g for 10 min, and the lower organic phase was recovered. The remaining organic solvent was dried under a nitrogen gas stream and the total lipid extract content was estimated by gravimetry. Lipid extracts were stored at 20 C, under nitrogen atmosphere until their use in liquid chromatography-mass spectrometry (LC–MS), gas chromatography-mass spectrometry (GC–MS) and bioactivity analyses [39,42]. Antioxidants 2020, 9, 642 4 of 20 2.4. Phospholipid and Glycolipid Quantification Phospholipids quantification was performed using modified Bartlett and Lewis method as previously described [43]. At least, five replicates of 100 L of lipid extract in dichloromethane (1 mg mL ) were used. Absorbance of standards and samples was measured on a microplate UV-Vis spectrophotometer (Multiskan GO, Thermo Scientific, Hudson, NH, USA). Phospholipids were calculated as P x 25. Glycolipids quantification was performed by calculating the hexose content (% glucose) by the orcinol colorimetric method as described previously (CyberLipids, [43]). The amount of sugar was estimated from a calibration curve prepared by performing the reaction with defined amounts of glucose (up to 40 g, from an aqueous solution containing 2 mg mL of sugar) At least five replicates of 50 L of lipid extract in dichloromethane (1 mg mL ) were used. The factor 100/35 was chosen to convert glucose to GL [43,44]. 2.5. Fatty Acid Analysis by Gas Chromatography-Mass Spectrometry (GC–MS) Fatty acid methyl esters (FAME) were prepared from total lipid extracts by alkaline transesterification using a methanolic solution of potassium hydroxide (2.0 M), according to the Aued-Pimentel-based methodology, as described for seaweeds [43]. Therefore, 1 mL of internal standard (1 g mL of methyl nonadecanoate (C19:0) in n-hexane) was added to 30 g of total lipid extract in dichloromethane (1 mg mL ), followed by 200 L of potassium hydroxide (2.0 M), prepared in methanol. After 2 min of vortexing, 2 mL of NaCl aqueous solution (10 g L ) was added. The mixture was centrifuged at 392 g for 5 min, and 600 L of organic phase were collected and dried under a nitrogen gas stream. For GC–MS analysis, the derivatized extract was diluted in 60 L of hexane. Sample volumes of 2.0 L of the hexane solution containing FAME were analyzed by GC-MS on an Agilent Technologies 6890 N Network (Santa Clara, CA, USA) equipped with a DB-FFAP column with the following specifications: 30 m of length, 0.32 mm internal diameter, and 0.25 m film thickness (123-3232, J&W Scientific, Folsom, CA, USA). The GC equipment was connected to an Agilent 5973 Network Mass Selective Detector operating with an electron impact mode at 70 eV and scanning the range m/z 50–550 in a 1 s cycle in a full scan mode acquisition. The oven temperature was programed at an initial temperature of 80 C for 3 min followed by a linear increase to 160 C and 10 min at this temperature. Helium was used as carrier gas at flow rate of 1.4 mL min . Two analytical replicates of four lipid extracts (total of eight replicates, n = 2 4) were injected into the equipment. The identification of each FA was performed considering the retention times (Figure S1) and similarity to MS spectra of FA standards (Supelco 37 Component Fame Mix, Sigma-Aldrich, St. Louis, MO, USA) and available spectra in the Wiley 275 library and AOCS Lipid Library. The relative amounts of FA were calculated by the percent relative area method with proper normalization using C19:0 as internal standard, considering the sum of all relative areas of the identified FA. Results were expressed as means standard deviation (SD). 2.6. Polar Lipid Analysis by Hydrophilic Interaction Liquid Chromatography-High Resolution Mass Spectrometry (HILIC MS) and Tandem Mass Spectrometry (MS/MS) Lipid extracts analysis was performed in a 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). The solvent system consisted of two mobile phases as follows: mobile phase A (acetonitrile:methanol 60:40 (per volume) with 2.5 mM ammonium acetate) and mobile phase B (acetonitrile:methanol:water 50:25:25 (per volume) with 2.5 mM ammonium acetate). Initially, 90% of mobile phase A was held isocratically for 2 min, followed by a linear decrease to 10% of A within 13 min, after which settings were maintained for 2 min and then returned to the initial conditions in 3 min, followed by a re-equilibration period of 10 min prior to the next analysis. A volume of 5 L of each sample, containing 5 g of lipid extract, a volume of 4 L of PL standards mix (dMPC—0.02 g, dMPE—0.02 g, lysophosphatidylcholine (LPC)—0.02 g, dPPI—0.08 g, dMPG—0.012 g, dMPA—0.08 g, Cer—0.04 g) and 91 L of eluent Antioxidants 2020, 9, 642 5 of 20 (10% of mobile phase A and 90% of mobile phase B) were mixed and introduced into the Ascentis Si column (10 cm 1 mm, 3 m, Sigma-Aldrich) with a flow rate of 50 L min and at 35 C. The mass spectrometer was operated simultaneously in positive (electrospray voltage 3.0 kV) and negative (electrospray voltage 2.7 kV) modes with a resolution of 70,000 and automatic gain control (AGC) target of 1 10 , the capillary temperature was 250 C and the sheath gas flow was 15 U. In MS/MS experiments, a resolution of 17,500 and AGC target of 1 10 were used. Cycles consisted of one full scan mass spectrum and 10 data-dependent MS/MS scans were repeated continuously throughout the experiments with the dynamic exclusion of 60 s and intensity threshold of 2 10 [39,42]. Normalized collision energy (CE) ranged between 20, 25, and 30 eV. Four replicates (corresponding to four lipid extracts, n = 4) were analyzed. 2.7. Data Analysis Data acquisition was carried out using the Xcalibur data system (V3.3, Thermo Fisher Scientific, Waltham, MA, USA). Peak integration and assignments of HPLC–MS data were performed using MZmine 2.39. The software was used for filtering and smoothing, peak detection, peak processing, and assignment against an in-house database. The validated peaks were within the time range of a MS full run. All the peaks with intensity lower than 1 10 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. MS/MS spectra were performed to confirm the identity of the molecular species as previously described by Marine Lipidomics Laboratory group [39,45,46]. The identification of molecular species of polar lipids was based on the LC retention time (Tables S1 and S2, Figure S2). 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 resulting data matrix with normalized areas of all species was used to relative quantitation of each lipid species per class, by dividing each normalized peak area by the sum of all normalized peak areas of the lipid species (ions) assigned in each class and multiplying by 100 to get the relative abundance in percentage. Data was exported into software GraphPad Prism 8.0 to design bar graphs. Identified lipid species were subjected to further analysis for determination of common and unique species through Venn diagram representation using the open source jvenn [47]. We defined “unique” when a lipid species was found only in B. bifurcata or S. muticum datasets. “Common” lipid species are those that have been found in samples from both seaweeds (Table S3). 2.8. 2.2´-Azino-bis-3-Ethylbenzothiazoline-6-Sulfonic Acid Radical Cation (ABTS) Assay Radical Scavenging Activity The antioxidant scavenging activity against 2,2´-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS ) was evaluated using a previously described method [42] with some modifications. The ABTS radical solution (3.5 mmol L ) was prepared by mixing 10 mL of ABTS stock 1 1 solution (7 mmol L in water) with 10 mL of potassium persulfate K S O (2.45 mmol L in water). 2 2 8 This mixture was kept for 12–16 h in the dark at room temperature and was diluted in ethanol to obtain an absorbance value of0.9 measured at 734 nm using a UV-vis spectrophotometer (Multiskan GO 1.00.38, Thermo Scientific, Hudson, NH, USA). For an evaluation of the radical stability, a volume of 150 L of ethanol was added to 12 microplate wells followed by addition of 150 L 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 L of S. muticum (5–250 mol L in ethanol) and B. bifurcata (25–250 mol L in ethanol) lipid extracts and 150 L of Trolox standard 1 + solution (10–75 mol L ) were placed in each well followed by addition of 150 L of ABTS diluted solution, and absorbance was measured at 734 nm every 5 min during a total incubation period of 120 min. Control lipid assays were performed by replacing 150 L of ABTS diluted solution by 150 L of ethanol. Radical reduction was monitored by measuring the decrease in absorbance during Antioxidants 2020, 9, 642 6 of 20 the reaction, thereby quantifying radical scavenging activity. All measurements were performed in triplicate. The % of ABTS radical remaining was determined according to Equation (1): % ABTS remaining = (Abs samples after incubation time/Abs sample at the beginning of reaction) 100 (1) The free radical-scavenging activity of samples was determined as the percentage of inhibition of ABTS radical according to Equation (2): % Inhibition = ((Abs ABTS (Abs samples Abs control))/Abs ABTS) 100 (2) The concentration of samples reducing 50% of ABTS radical after 120 min (IC ) were calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity, expressed as Trolox Equivalents (TE, mol Trolox/g of sample), was calculated according to Equation (3): 1 1 TE = IC Trolox (mol L ) 1000/IC of samples (g mL ) (3) 50 50 2.8. 2.2-Diphenyl-1-Picrylhydrazyl Radical Assay (DPPH) Radical Scavenging Activity The antioxidant scavenging activity against ,-diphenyl- -picrylhydrazyl radical (DPPH ) was evaluated using a previously described method applied with some modifications [42,47]. A stock solution of DPPH in ethanol (250 mol L ) was prepared and diluted to provide a working solution with an absorbance value of0.9 measured at 517 nm using a UV-Vis spectrophotometer (Multiskan GO 1.00.38, Thermo Scientific, Hudson, NH, USA). To evaluate the radical stability, a volume of 150 L of ethanol was added to 12 microplate wells followed by addiction of 150 L 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 L of S. muticum and B. bifurcata lipid extracts 1 1 (25–250 mol L ) and 150 L of Trolox standard solution (10–75 mol L ) were placed in each well followed by addition of 150 L of DPPH diluted solution, and again an incubation period of 120 min was followed with absorbance being measured at 517 nm every 5 min. Control lipid assays were performed by replacing 150 L of DPPH diluted solution by 150 L of ethanol. Radical reduction was monitored by measuring the decrease in absorbance during the reaction, thereby quantifying radical scavenging activity. All measurements were performed in triplicate. The % of DPPH radical remaining was determined according to Equation (4): % DPPH remaining = (Abs samples after incubation time/Abs sample at the beginning of reaction) 100 (4) The free radical-scavenging activity of samples was determined as the percentage of inhibition of DPPH radical according to Equation (5): % Inhibition = ((Abs DPPH (Abs samples Abs control))/Abs DPPH) 100 (5) The concentration of samples reducing 50% of DPPH radical after 120 min (IC ) was calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. For B. bifurcata the IC was calculated instead of IC . The activity expressed, as TE (mol Trolox/g of 25 50 sample), was calculated according to Equation (3) (for S. muticum) and Equation (6) (for B. bifurcata): 1 1 TE = IC Trolox (mol L ) 1000/IC of samples (g mL ) (6) 25 25 Antioxidants 2020, 9, 642 7 of 20 3. Results 3.1. Total Lipid Content The total lipid content of the two brown seaweeds, B. bifurcata and S. muticum, was estimated by gravimetry after lipid extraction procedure. The average lipid content (expressed as g/100 g of dry weigh biomass, DW) of B. bifurcata was 7.20 0.31 g/100 g while that of S. muticum was lower, representing 2.80 0.06 g/100 g. Bifurcaria bifurcata presented a total of 0.12 0.03 g/100 g of PL and 1.01 0.07 g/100 g of GL. Sargassum muticum presented higher content of both PL and GL in lipid extracts, with 0.25 0.03 g/100 g and 1.31 0.04 g/100 g, respectively. 3.1.1. Bifurcaria bifurcata and S. muticum Fatty Acid Profiles The FA profile of the extracts obtained from B. bifurcata and S. muticum were determined by GC–MS analysis of the FAME obtained after alkaline transmethylation [45] (Table 1). In the case of B. bifurcata, the fatty acid profile included 16 FA species, and the most abundant was 16:0 (30.07% 2.78%), followed by 20:4 n-6 (14.28% 0.72%) and 18:1 n-9 (12.12% 1.22%). The lipid fraction of this seaweed is rich in saturated fatty acids (SFA) (46.09% 4.30%), with 16:0 and 18:0 being the major contributors for total SFA. The monounsaturated fatty acids (MUFA) included 16:1, 18:1 n-9, and 20:1, and the PUFA included FAs 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:3, 20:4 n-6, and 20:5 n-3 (Table 1). The results obtained for the S. muticum allowed identifying 20 dierent FA (Table 1). The most abundant was 16:0 with a relative content of 24.18% 0.48%, followed by 20:4 n-6 (12.53% 0.72%) and 20:5 n-3 (9.71% 0.34%). This brown seaweed exhibited a FA profile rich in PUFA, with 16:2, 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:2, 20:3, 20:4 n-6, and 20:5 n-3 as the major contributors for total PUFA abundance. MUFA such as 16:1, 18:1 n-9, 20:1, and 22:1, and SFA such as 14:0, 15:0, 16:0, 18:0, 20:0, 22:0 were also identified (Table 1). PUFA content of B. bifurcata was lower than that of S. muticum. Table 1. Fatty acid profile of Bifurcaria bifurcata and Sargassum muticum determined by gas chromatography-mass spectrometry (GC–MS) analysis fatty acid methyl esters (FAME) and expressed as relative abundance mean (%) + SD, n = 8. Fatty Acid B. bifurcata (%) S. muticum (%) 14:0 4.04 1.18% 3.00 0.17% 15:0 0.90 0.36% 0.77 0.05% 16:0 30.07 2.78% 24.18 0.48% 16:1 2.84 0.56% 2.34 0.43% 16:2 – 1.65 0.86% 18:0 10.42 7.06% 3.29 0.33% 18:1 n-9 12.12 1.22% 7.91 0.14% 18:2 n-6 3.22 0.45% 5.61 0.26% 18:3 n-6 0.37 0.26% 0.67 0.09% 18:3 n-3 3.84 0.51% 7.07 0.07% 18:4 n-3 5.02 0.73% 8.03 0.15% 20:0 0.67 0.18% 0.64 0.12% 20:1 2.23 0.33% 1.56 0.10% 20:2 – 1.46 0.14% (a) (a) 20:3 4.08 1.31% 3.57 0.33% 20:4 n-3 1.19 0.20% 0.72 0.09% 20:4 n-6 14.28 0.72% 12.53 0.72% 20:5 n-3 4.72 0.26% 9.71 0.34% 22:0 – 0.74 0.10% 22:1 – 4.56 1.05% SFA 46.09 4.30% 32.62 0.92% MUFA 17.19 1.95% 16.36 0.49% PUFA 36.72 2.55% 51.02 4.79% (n-3) 14.77 1.28% 25.53 0.49% (n-6) 17.87 0.74% 18.81 0.94% n-6/n-3 1.22 0.09 0.74 0.03 (a)With contribution of unknown compound. (–) not detected. Antioxidants 2020, 9, 642 8 of 20 3.1.2. Bifurcaria bifurcata and S. muticum Polar Lipid Profiles The polar lipid profiles of B. bifurcata and S. muticum were characterized at the molecular level by high resolution HILIC-LC–MS and MS/MS analysis. In the case of B. bifurcata, this approach allowed identifying 143 lipid species, from nine classes of polar lipids. Polar lipid profile included 68 GL species distributed into three GL classes, 44 PL species distributed into four PL classes and 31 betaine lipids species distributed into two classes (Table 2). Considering GL profile, in B. bifurcata, the GL classes identified included the acidic glycolipid sulfoquinovosyl diacylglycerol class (SQDG), as well as the neutral glycolipids digalactosyl diacylglycerol (DGDG) and monogalactosyl diacylglycerol (MGDG) classes (Table 2 and Table S1 from Supplementary material) but no lyso-forms were identified. Table 2. Polar lipid classes identified by hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC LC–MS) in total lipid extracts of B. bifurcata and S. muticum with the indication of the total number of lipid species identified in each class and the major lipid species per class. Bifurcaria bifurcata Sargassum muticum Lipid Classes Lipid Species Lipid Species Major Species Major Species Number Number MGDG 26 MGDG (34:1) 35 MGDG (38:9) MGMG – – 10 MGMG (18:4) DGDG 14 DGDG (38:9) 17 DGDG (38:9) SQDG 28 SQDG (34:1) 32 SQDG (34:1) LPC – – 3 LPC (16:0) PC 3 PC (30:3) 10 PC (30:3) LPE – – 2 LPE (20:4) PE 24 PE (40:8) 34 PE (40:8) LPG – – 1 LPG (16:0) PG 10 PG (34:1) 15 PG (34:4) PI 7 PI (38:8) 8 PI (34:2) DGTS 5 DGTS (34:1) 13 DGTS (34:2) DGTA 26 DGTA (36:4) 37 DGTA (36:4) Glycolipids 68 – 94 – Phospholipids 44 – 73 – Betaine lipids 31 50 – Total 143 217 – (–) not detected, MGDG: monogalactosyl diacylglycerol; MGMG: monogalactosyl monoacylglycerol; DGDG: digalactosyl diacylglycerol; SQDG: sulfoquinovosyl diacylglycerol; LPC: lysophosphatidylcholine; PC: phosphatidylcholine; LPE: lysophosphatidylethanolamine; PE: phosphatidylethanolamine; LPG: lysophosphatidylglycerol; PG: phosphatidylglycerol; PI: phosphatidylinositol; DGTS: diacylglyceroltrimethylhomoserine; DGTA: diacylglyceroltrimethyl- -alanine. In the S. muticum lipid extracts a total of 217 polar lipid species were identified, distributed into 13 lipid classes. A total of 94 species of GL distributed into four dierent classes were identified, as well as 73 PL species distributed into seven classes, and 50 betaine lipid species distributed in two classes (Table 2). The criteria for identifying the lipid species included accuracy of the mass measurements (<5 ppm), the LC retention time and the characteristics of the MS/MS spectra for each lipid class. In what concerned GL profile, the classes of SQDG, of DGDG, and MGDG were identified in both algae, while lyso forms monogalactosyl monoacylglycerol (MGMG) were only seen in S. muticum (Table 2 and Table S2 from Supplementary material). These acidic SQDGs were identified as [M H] and [M + NH ] in LC-MS spectra and typical fragmentation observed in LC–MS/MS spectra is shown in Figure S3a,b in the Supplementary material, respectively. Overall, 28 and 32 lipid species of SQDG were identified for B. bifurcata and S. muticum, respectively (Figure 1, Tables S1 and S2). The most abundant SQDG was assigned as SQDG (34:1) at m/z 819.5, for both seaweed species, being identified as SQDG (14:0/20:1), SQDG (16:0/18:1), and SQDG Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 20 In what concerned GL profile, the classes of SQDG, of DGDG, and MGDG were identified in both algae, while lyso forms monogalactosyl monoacylglycerol (MGMG) were only seen in S. muticum (Table 2 and Table S2 from Supplementary material). − + These acidic SQDGs were identified as [M − H] and [M + NH4] in LC-MS spectra and typical fragmentation observed in LC–MS/MS spectra is shown in Figure S3a,b in the Supplementary material, respectively. Overall, 28 and 32 lipid species of SQDG were identified for B. bifurcata and S. muticum, respectively (Figure 1, Tables S1 and S2). The most abundant SQDG was assigned as SQDG Antioxidants 2020, 9, 642 9 of 20 (34:1) at m/z 819.5, for both seaweed species, being identified as SQDG (14:0/20:1), SQDG (16:0/18:1), and SQDG (16:1/18:0) in B. bifurcata (Figure 1a, Table S1), and as SQDG (16:0/18:1) in S. muticum (16:1 (Figure /18:0) 1bin , Table S2). B. bifurcata (Figure 1a, Table S1), and as SQDG (16:0/18:1) in S. muticum (Figure 1b, Table S2). Figure 1. Relative abundance (RA) of lipid species of the sulfoquinovosyl diacylglycerol (SQDG) class Figure 1. Relative abundance (RA) of lipid species of the sulfoquinovosyl diacylglycerol (SQDG) class identified in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentages after identified in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentages after dividing the normalized peak area of each lipid species to the sum of normalized peak areas for all the dividing the normalized peak area of each lipid species to the sum of normalized peak areas for all lipid species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the lipid species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) the number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with indicate the number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The higher RA was SQDG (34:1) for both seaweeds. species with higher RA was SQDG (34:1) for both seaweeds. The neutral GL were detected in the positive LC–MS spectra as [M + NH4]+ ions (Tables S1 and The neutral GL were detected in the positive LC–MS spectra as [M + NH4]+ ions (Tables S1 and S2) and the typical fragmentation obtained in the LC–MS/MS spectra of MGDG, MGMG, and DGDG species S2) and t ash [M e ty + pNH ical fr ] agm are e shown ntation ob in Figur taine ed S4 in t for he LC MGDG –Mand S/MS MGMG spectra of and MG Figur DG e , S5 MG for MG DGDG , and spec DGDG ies. species as [M + NH4] are shown in Figure S4 for MGDG and MGMG and Figure S5 for DGDG species. Considering the neutral GL, 26 species of MGDG (Figure 2a and Table S1) and 14 of DGDG (Figure 3a and Consider Table ing S1) the neutr were identified al GL, in 26 spec B. bifur ies cata of MGDG and 35 lipid (Figure 2a a species of nd MGDG Table S1 (Figur ) and 14 e 2b of and DGDG Table (S2), Figure 10 3a and Table S1) were identified in B. bifurcata and 35 lipid species of MGDG (Figure 2b and Table of MGMG (Figure 2c and Table S2), and 17 of DGDG (Figure 3b and Table S2) were identified in S. muticum S2), 10 of . In MG B. MG (F bifurcata igur the e 2cmost and T abundant able S2), an MGDG d 17 owas f DGDG (Fig MGDG (34:1), ure 3b at and T m/z a 774.6, ble S2and ) weassigned re identifie as d in S. muticum. In B. bifurcata the most abundant MGDG was MGDG (34:1), at m/z 774.6, and assigned MGDG (16:0/18:1), while in S. muticum the most abundant was MGDG (38:9) at m/z 814.5, assigned as MGDG as MGDG (20:5 (16: /18:4) 0/18:(Figur 1), whe il2 e in , Tables S. muS1 ticu and m the most abundant was S2). The DGDG (38:9) at Mm G/DG (38:9) at z 976.6 was the m/zmost 814.5, abundant assigned as MGDG (20:5/18:4) (Figure 2, Tables S1 and S2). The DGDG (38:9) at m/z 976.6 was the most DGDG in both seaweed species and was identified as DGDG (20:5/18:4) also for both (Figure 3, Tables S1 aband unda S2). nt D The GDG most in b abundant oth seawe MGMG ed spein cies S. and w muticum as ident was detected ified as DG at mDG /z 530.3 (20:and 5/18: was 4) al assigned so for bo as th (Figure 3, Tables S1 and S2). The most abundant MGMG in S. muticum was detected at m/z 530.3 and MGMG (18:4) (Figure 2c and Table S2). The profile for each GL class is represented in Figures 2 and 3, showing was assig the ned relative as MGabundance MG (18:4) (F ofig the ure 2c lipid an species d Table S2 per ). class. The profile for each GL class is represented in Figures 2 and 3, showing the relative abundance of the lipid species per class. Betaine lipids identified in B. bifurcata and S. muticum were assigned to the diacylglyceroltrimethyl homoserine (DGTS) and diacylglyceroltrimethyl- -alanine (DGTA) classes, which were identified in the LC–MS spectra in positive ion mode, as [M + H] ions. The DGTA class is a structural isomer of DGTS and both were discriminated on the basis of having dierent retention times [39]: DGTA molecular species eluted at RT 9 min while DGTS molecular species are eluted at RT 6 min. Overall, five lipid species of DGTS (Figure 4a, Table S1) and 26 lipid species of DGTA (Figure 4c and Table S1) were found in B. bifurcata, and 13 lipid species of DGTS (Figure 4b, Table S2) and 37 lipid species of DGTA (Figure 4d, Table S1) were present in S. muticum. The predominant DGTS species in B. bifurcata was detected at m/z 738.6, corresponding to DGTS (34:1) (Figure 4a and Table S1) while in S. muticum the predominant DGTS was detected at m/z 736.6, corresponding to DGTS (34:2), (Figure 4b, Table S2). DGTA assigned as DGTA (36:4) at m/z 760.6 had similar predominance for both seaweed species (Figure 4c,d, and Tables S1 and S2). The structural features of betaine lipids were confirmed through the identification of the typical product ions and fragment pathways observed in the LC–MS/MS spectra (Figure S6). Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 20 Antioxidants 2020, 9, 642 10 of 20 Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 20 Figure 2. Relative abundances (RA) of lipid species of monogalactosyl diacylglycerol (MGDG) Figure 2. Relative abundances (RA) of lipid species of monogalactosyl diacylglycerol (MGDG) identified identified in B. bifurcata (a) and S. muticum (b) and of monogalactosyl monoacylglycerol (MGMG) Figure 2. Relative abundances (RA) of lipid species of monogalactosyl diacylglycerol (MGDG) in B. bifurcata (a) and S. muticum (b) and of monogalactosyl monoacylglycerol (MGMG) identified in S. identified in S. muticum (c). The results are expressed as relative percentage after dividing the identified in B. bifurcata (a) and S. muticum (b) and of monogalactosyl monoacylglycerol (MGMG) muticum (c). The results are expressed as relative percentage after dividing the normalized peak area of normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid species identified in S. muticum (c). The results are expressed as relative percentage after dividing the each lipid species to the sum of normalized peak areas for all the lipid species within the class obtained within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid species after LC–MS analysis. Numbers in parentheses (C:N) indicate the number of carbon atoms (C) and of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number double bounds (N) in the fatty acid side chains. The species with higher RA were MGDG (34:1) for B. were MGDG (34:1) for B. bifurcata and MGDG (38:9) and MGMG (18:4) for S. muticum. of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA bifurcata and MGDG (38:9) and MGMG (18:4) for S. muticum. were MGDG (34:1) for B. bifurcata and MGDG (38:9) and MGMG (18:4) for S. muticum. Figure 3. Relative abundances (RA) of lipid species of digalactosyl diacylglycerol (DGDG) glycolipids identified in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after Figure 3. Relative abundances (RA) of lipid species of digalactosyl diacylglycerol (DGDG) glycolipids dividing the normalized peak area of each lipid species to the sum of normalized peak areas for all the identified in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after Figure 3. Relative abundances (RA) of lipid species of digalactosyl diacylglycerol (DGDG) glycolipids lipid species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate dividing the normalized peak area of each lipid species to the sum of normalized peak areas for all identified in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after the number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with the lipid species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) dividing the normalized peak area of each lipid species to the sum of normalized peak areas for all higher RA was DGDG (38:9) for both seaweeds. indicate the number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The the lipid species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) species with higher RA was DGDG (38:9) for both seaweeds. indicate the number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA was DGDG (38:9) for both seaweeds. Betaine lipids identified in B. bifurcata and S. muticum were assigned to the diacylglyceroltrimethylhomoserine (DGTS) and diacylglyceroltrimethyl-β-alanine (DGTA) classes, Betaine lipids identified in B. bifurcata and S. muticum were assigned to the which were identified in the LC–MS spectra in positive ion mode, as [M + H] ions. The DGTA class diacylglyceroltrimethylhomoserine (DGTS) and diacylglyceroltrimethyl-β-alanine (DGTA) classes, is a structural isomer of DGTS and both were discriminated on the basis of having different retention which were identified in the LC–MS spectra in positive ion mode, as [M + H] ions. The DGTA class is a structural isomer of DGTS and both were discriminated on the basis of having different retention Antioxidants 2020, 9, x FOR PEER REVIEW 11 of 20 times [39]: DGTA molecular species eluted at RT 9 min while DGTS molecular species are eluted at RT 6 min. Overall, five lipid species of DGTS (Figure 4a, Table S1) and 26 lipid species of DGTA (Figure 4c and Table S1) were found in B. bifurcata, and 13 lipid species of DGTS (Figure 4b, Table S2) and 37 lipid species of DGTA (Figure 4d, Table S1) were present in S. muticum. The predominant DGTS species in B. bifurcata was detected at m/z 738.6, corresponding to DGTS (34:1) (Figure 4a and Table S1) while in S. muticum the predominant DGTS was detected at m/z 736.6, corresponding to DGTS (34:2), (Figure 4b, Table S2). DGTA assigned as DGTA (36:4) at m/z 760.6 had similar predominance for both seaweed species (Figure 4c,d, and Tables S1 and S2). The structural features of betaine lipids were confirmed through the identification of the typical product ions and fragment Antioxidants 2020, 9, 642 11 of 20 pathways observed in the LC–MS/MS spectra (Figure S6). Figure 4. Relative abundance (RA) of betaine lipid species identified as diacylglyceroltrimethy Figure 4. Relative abundance (RA) of betaine lipid species identified as lhomoserine (DGTS) (a) and diacylglyceroltrimethyl- -alanine (DGTA) (c) in B. bifurcata, and DGTS diacylglyceroltrimethylhomoserine (DGTS) (a) and diacylglyceroltrimethyl-β-alanine (DGTA) (c) in (b) and DGTA (d) in S. muticum. The results are expressed as relative percentage after dividing the B. bifurcata, and DGTS (b) and DGTA (d) in S. muticum. The results are expressed as relative normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid species percentage after dividing the normalized peak area of each lipid species to the sum of normalized within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number of peak areas for all the lipid species within the class obtained after LC–MS analysis. Numbers in carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA were parentheses (C:N) indicate the number of carbon atoms (C) and double bounds (N) in the fatty acid DGTS (34:1) and DGTA (36:4) for B. bifurcata and DGTS (34:2) and DGTA (36:4) for S. muticum. side chains. The species with higher RA were DGTS (34:1) and DGTA (36:4) for B. bifurcata and DGTS (34:2) and DGTA (36:4) for S. muticum. Phospholipid classes identified in B. bifurcata and S. muticum included phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). Sargassum Phospholipid classes identified in B. bifurcata and S. muticum included phosphatidylcholine (PC), muticum also contained lysoPC, lysoPE, and lysoPG species, while no lyso-forms of PL were identified phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). in B. bifurcata (Table 2). Overall, 10 lipid species of PG (Figure 5a, Table S1), seven PI (Figure 6a, Table Sargassum muticum also contained lysoPC, lysoPE, and lysoPG species, while no lyso-forms of PL S1), three PC (Figure 7a, Table S1), and 24 PE (Figure 8a, Table S1) were identified in B. bifurcata. On the were identified in B. bifurcata (Table 2). Overall, 10 lipid species of PG (Figure 5a, Table S1), seven PI other hand, in S. muticum, 15 species of PG (Figure 5b, Table S2), one LPG (Table S2), eight PI (Figure 6b, (Figure 6a, Table S1), three PC (Figure 7a, Table S1), and 24 PE (Figure 8a, Table S1) were identified Table S2), 10 PC (Figure 7b, Table S2), three LPC (Table S2), 34 of PE (Figure 8b, Table S2), and two of in B. bifurcata. On the other hand, in S. muticum, 15 species of PG (Figure 5b, Table S2), one LPG (Table lysophosphatidylethanolamine (LPE) (Table S2) were identified. The predominant PG of B. bifurcata S2), eight PI (Figure 6b, Table S2), 10 PC (Figure 7b, Table S2), three LPC (Table S2), 34 of PE (Figure was assigned as PG (34:1) at m/z 747.5 (Figure 5a and Table S1) while the predominant PG of S. muticum 8b, Table S2), and two of lysophosphatidylethanolamine (LPE) (Table S2) were identified. The was assigned as PG (34:4) at m/z 741.5 (Figure 5b and Table S2). PG and lysophosphatidylglycerol predominant PG of B. bifurcata was assigned as PG (34:1) at m/z 747.5 (Figure 5a and Table S1) while (LPG) classes were identified in negative LC MS spectra as [M H] ions and respective typical the predominant PG of S. muticum was assigned as PG (34:4) at m/z 741.5 (Figure 5b and Table S2). MS/MS spectra are represented in Figure S7a,b. The predominant PI of B. bifurcata was assigned as PI PG and lysophosphatidylglycerol (LPG) classes were identified in negative LC−MS spectra as [M − (38:8) at m/z 877.5 (Figure 6a and Table S1) while the predominant PI of S. muticum was assigned as H] ions and respective typical MS/MS spectra are represented in Figure S7a,b. The predominant PI PI (34:2) at m/z 833.5 (Figure 6b and Table S2). The PI class was identified in negative LC–MS spectra of B. bifurcata was assigned as PI (38:8) at m/z 877.5 (Figure 6a and Table S1) while the predominant as [M H] ions, and a characteristic MS/MS spectrum is shown in Figure S5c. LPC, PC, LPE, and PI of S. muticum was assigned as PI (34:2) at m/z 833.5 (Figure 6b and Table S2). The PI class was PE molecular species were identified in positive LC–MS spectra as [M + H] ions. The predominant identified in negative LC–MS spectra as [M − H] ions, and a characteristic MS/MS spectrum is shown PC for B. bifurcata was assigned as PC (30:3) at m/z 700.5 (Figure 7a, Table S1), while the predominant in Figure S5c. LPC, PC, LPE, and PE molecular species were identified in positive LC–MS spectra as PC species for S. muticum were PC (30:3) at m/z 700.5 and PC (36:4) at m/z 782.6 (Figure 7b and Table S2). The predominant PE for both seaweed species was assigned as PE (40:8) at m/z 788.5 (Figure 8a,b, Tables S1 and S2). Figure S8 shows the typical fragmentation pathways observed in the LC-MS/MS spectra of PC, LPC, PE, and LPE. Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 20 Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 20 [M + H] ions. The predominant PC for B. bifurcata was assigned as PC (30:3) at m/z 700.5 (Figure 7a, Table S + 1), while the predominant PC species for S. muticum were PC (30:3) at m/z 700.5 and PC (36:4) [M + H] ions. The predominant PC for B. bifurcata was assigned as PC (30:3) at m/z 700.5 (Figure 7a, at m/z 782.6 (Figure 7b and Table S2). The predominant PE for both seaweed species was assigned as Table S1), while the predominant PC species for S. muticum were PC (30:3) at m/z 700.5 and PC (36:4) PE (40:8) at m/z 788.5 (Figure 8a,b, Tables S1 and S2). Figure S8 shows the typical fragmentation at m/z 782.6 (Figure 7b and Table S2). The predominant PE for both seaweed species was assigned as pathways observed in the LC-MS/MS spectra of PC, LPC, PE, and LPE. PE (40:8) at m/z 788.5 (Figure 8a,b, Tables S1 and S2). Figure S8 shows the typical fragmentation pathways observed in the LC-MS/MS spectra of PC, LPC, PE, and LPE. Antioxidants 2020, 9, 642 12 of 20 Figure 5. Relative abundance (RA) of lipid species of the phosphatidylglycerol (PG) class identified Figure in B. bifurcat 5. Relative a (a) a abundance nd S. muticu (RA) m (b). The re of lipid species sults are of the expressed phosphatidylglycer as relative percentage afte ol (PG) class identified r dividing in Figure 5. Relative abundance (RA) of lipid species of the phosphatidylglycerol (PG) class identified B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing the the normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid species species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the the normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number of number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA were higher RA were PG (34:1) for B. bifurcata and PG (34:4) for S. muticum. number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with PG (34:1) for B. bifurcata and PG (34:4) for S. muticum. higher RA were PG (34:1) for B. bifurcata and PG (34:4) for S. muticum. Figure 6. Relative abundance (RA) of lipid species of the phosphatidylinositol (PI) class identified in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing the Figure 6. Relative abundance (RA) of lipid species of the phosphatidylinositol (PI) class identified in normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid species B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing the Figure 6. Relative abundance (RA) of lipid species of the phosphatidylinositol (PI) class identified in within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number of normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid species B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing the carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA were within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid species PG (38:8) for B. bifurcata and PI (34:2) for S. muticum. of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number were PG (38:8) for B. bifurcata and PI (34:2) for S. muticum. of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA were PG (38:8) for B. bifurcata and PI (34:2) for S. muticum. Antioxidants 2020, 9, x FOR PEER REVIEW 13 of 20 Antioxidants 2020, 9, 642 13 of 20 Antioxidants 2020, 9, x FOR PEER REVIEW 13 of 20 Figure 7. Relative abundance (RA) of lipid species of the phosphatidylcholine (PC) class identified Figure 7. Relative abundance (RA) of lipid species of the phosphatidylcholine (PC) class identified in in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing the Figure 7. Relative abundance (RA) of lipid species of the phosphatidylcholine (PC) class identified the normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid species in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number of the normalized peak area of each lipid species to the sum of normalized peak areas for all the lipid number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA were species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the higher RA were PC (30:3) for B. bifurcata, and PC (30:3) and PC (36:4) for S. muticum. PC (30:3) for B. bifurcata, and PC (30:3) and PC (36:4) for S. muticum. number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The species with higher RA were PC (30:3) for B. bifurcata, and PC (30:3) and PC (36:4) for S. muticum. Figure 8. Relative abundance (RA) of lipid species of the phosphatidylethanolamine (PE) class identified Figure 8. Relative abundance (RA) of lipid species of the phosphatidylethanolamine (PE) class in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after dividing the identified in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after normalized Figure 8. Rela peak tive area abu of n each dance (RA) lipid spe of cies lip toithe d specie sum of s of normalized the phosphatid peak ary eas lethanolam for all theine (P lipidE) species class dividing the normalized peak area of each lipid species to the sum of normalized peak areas for all identified in B. bifurcata (a) and S. muticum (b). The results are expressed as relative percentage after within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) indicate the number of the lipid species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) carbon dividing the atoms n (C) ormalized and double peabounds k area of (N) eain ch l the ipid fatty spe acid cies side to the sum of chains. The nor species malized with peak a higher reas for RA was all indicate the number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The PC (40:8) for both seaweeds. the lipid species within the class obtained after LC–MS analysis. Numbers in parentheses (C:N) species with higher RA was PC (40:8) for both seaweeds. indicate the number of carbon atoms (C) and double bounds (N) in the fatty acid side chains. The We compared the common and contrasting lipid species in the lipidome of these two brown species with higher RA was PC (40:8) for both seaweeds. We compared the common and contrasting lipid species in the lipidome of these two brown seaweeds, members of the Fucales order. We found 124 lipid species that were common (52.5% of total seaweeds, members of the Fucales order. We found 124 lipid species that were common (52.5% of number of lipid species), 19 lipid species found only in the lipidome of B. bifurcata (8.1%), and 93 lipid We compared the common and contrasting lipid species in the lipidome of these two brown total number of lipid species), 19 lipid species found only in the lipidome of B. bifurcata (8.1%), and species identified only in the lipidome of S. muticum (39.4%). The common lipid species included 24 seaweeds, members of the Fucales order. We found 124 lipid species that were common (52.5% of 93 lipid species identified only in the lipidome of S. muticum (39.4%). The common lipid species MGDG, 25 SQDG, 12 DGDG species, three PC, 22 PE, eight PG, and two PI species and five DGTS and total number of lipid species), 19 lipid species found only in the lipidome of B. bifurcata (8.1%), and included 24 MGDG, 25 SQDG, 12 DGDG species, three PC, 22 PE, eight PG, and two PI species and 23 DGTA species (Table S3). In the case of B. bifurcata, there were two MGDG, two DGDG, three SQDG, 93 lipid species identified only in the lipidome of S. muticum (39.4%). The common lipid species five DGTS and 23 DGTA species (Table S3). In the case of B. bifurcata, there were two MGDG, two two PE, two PG, five PI, and three DGTA unique species. Many lipid species were only assigned to S. included 24 MGDG, 25 SQDG, 12 DGDG species, three PC, 22 PE, eight PG, and two PI species and DGDG, three SQDG, two PE, two PG, five PI, and three DGTA unique species. Many lipid species muticum lipidome and included 11 MGMG, five DGDG, seven SQDG, seven PC, 12 PE, seven PG, six five DGTS and 23 DGTA species (Table S3). In the case of B. bifurcata, there were two MGDG, two were only assigned to S. muticum lipidome and included 11 MGMG, five DGDG, seven SQDG, seven PI, and betaine lipids (eight DGTS, and 14 DGTA). Moreover, 10 MGMG, three LPC, two LPE, and DGDG, three SQDG, two PE, two PG, five PI, and three DGTA unique species. Many lipid species PC, 12 PE, seven PG, six PI, and betaine lipids (eight DGTS, and 14 DGTA). Moreover, 10 MGMG, one LPG were only found in S. muticum lipidome and not in B. bifurcata, since these classes were not were only assigned to S. muticum lipidome and included 11 MGMG, five DGDG, seven SQDG, seven three LPC, two LPE, and one LPG were only found in S. muticum lipidome and not in B. bifurcata, detected in the latter. PC, 12 PE, seven PG, six PI, and betaine lipids (eight DGTS, and 14 DGTA). Moreover, 10 MGMG, since these classes were not detected in the latter. three LPC, two LPE, and one LPG were only found in S. muticum lipidome and not in B. bifurcata, 3.2. Antioxidant Activity since these classes were not detected in the latter. 3.2. Antioxidant Activity The antioxidant activity of B. bifurcata and S. muticum lipid extracts was evaluated The antioxidant activity of B. bifurcata and S. muticum lipid extracts was evaluated by 2,2´-azino- 3.2. Antioxidant Activity by 2,2´-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation assay (ABTS ) and ●+ bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation assay (ABTS ) and 2,2-diphenyl-1- 2,2-diphenyl-1-picrylhydrazyl radical assay (DPPH ) that measured the free radical scavenging The antioxidant activity of B. bifurcata and S. muticum lipid extracts was evaluated by 2,2´-azino- ●+ bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation assay (ABTS ) and 2,2-diphenyl-1- Antioxidants 2020, 9, 642 14 of 20 Antioxidants 2020, 9, x FOR PEER REVIEW 14 of 20 picrylhydrazyl radical assay (DPPH ) that measured the free radical scavenging capacity of the lipid capacity of the lipid extracts. The percentage of radical inhibition in the presence of lipid extracts extracts. The percentage of radical inhibition in the presence of lipid extracts was calculated after 120 was calculated after 120 min. Seven varying concentrations of lipid extracts ranging from 5 to 250 g −1 min. Seven varying concentrations of lipid extracts ranging from 5 to 250 μg mL of S. muticum and 1 1 mL of S. muticum and four varying concentrations ranging from 25 to 250 g mL of B. bifurcata −1 four varying concentrations ranging from 25 to 250 μg mL of B. bifurcata were tested for ABTS -1 were tested for ABTS antioxidant activity, while concentrations ranging from 25 to 250 g mL of B. -1 antioxidant activity, while concentrations ranging from 25 to 250 μg mL of B. bifurcata and S. bifurcata and S. muticum were tested for DPPH antioxidant activity. A dose-dependent increase in the muticum were tested for DPPH antioxidant activity. A dose-dependent increase in the scavenging -1 scavenging capacity was observed for all concentrations tested (Figure 9). The 250 g mL extracts -1 capacity was observed for all concentrations tested (Figure 9). The 250 μg mL extracts showed the showed the best antioxidant activity, where among them, the S. muticum extract showed the highest best antioxidant activity, where among them, the S. muticum extract showed the highest antioxidant antioxidant potential both in ABTS (98.4% 0.19%) and DPPH (62.59%1.29%). potential both in ABTS (98.4% ± 0.19%) and DPPH (62.59% ±1.29%). a) b) Bifurcaria bifurcata Sargassum muticum 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 25 50 100 250 5 10152550 100 250 −1 −1 Concentration, μg mL Concentration, μg mL Figure 9. Free radical-scavenging activity (%) on 2,2´-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid Figure 9. Free radical-scavenging activity (%) on 2,2´-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS) (a) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assays (b) radicals of B. radical cation (ABTS) (a) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assays (b) radicals of B. bifurcata (a) and S. muticum lipid extracts. Each value is expressed as mean standard deviation (n = 3). bifurcata (a) and S. muticum lipid extracts. Each value is expressed as mean ± standard deviation (n = For B. bifurcata, 50% of inhibition (IC ) of ABTS radical was attained at a concentration of 3). 50 -1 1 134.19 2.12 g mL (IC ), representing a TE of 100.33 1.57 mol g , while for DPPH , only 25% of ●+ inhibition For B. bifurcata (IC ) was , 50% o attained f inh at ibition (IC a concentration 50) of ABT of S 155.00 radi cal w 3.96 as at g t mL ained at repr a concen esenting tra atTE ion of of 74.89 134.19 -1 −1 ● ± 2. 1.931 2 mol μg mL g (T (IC able 50), representi 3). Sargassum ng a muticum TE of 1lipid 00.33 ± 1.57 extractsμ showed mol g , wh antioxidant ile for DPPH activity , only 25% against both of + + −1 inhibition DPPH and (IC ABTS 25) was at radicals tained at (T able a conce 3). n For tratABTS ion of 15 radical 5.00 ± 3 scavenging .96 μg mL activity represe,n IC ting a was TE o attained f 74.89 at ± 1 1 −1 1. 23.42 93 μ mol g 0.79 (Tabl g mL e 3 , r ). epr Saesenting rgassum m a u TE ticof um 648.35 lipid extra 21.43 cts showed mol g a and ntioxida for DPPH nt acti assay vity agai the IC nst both was 1 1 ● ●+ ●+ DPPH attained and ABTS at a concentration radicalsof (T188.43 able 3) . F2.55 or A Bg TS mL radic repr al sc esenting avenging a TE act of iv124.19 ity, IC50 wa 14.39 s attai mol ned gat −1 −1 23 (Table .42 ± 0. 3).79 μg mL , representing a TE of 648.35 ± 21.43 μmol g and for DPPH assay the IC50 was −1 −1 attained at a concentration of 188.43 ± 2.55 μg mL representing a TE of 124.19 ± 14.39 μmol g (Table Table 3. Antioxidant activity of B. bifurcata and S. muticum total lipid extracts expressed as inhibitory 3). concentration (IC) values (g mL ) for ABTS and DPPH assays, and Trolox Equivalents (TE) (mol g ) for each assay. Table 3. Antioxidant activity of B. bifurcata and S. muticum total lipid extracts expressed as inhibitory −1 concentration (IC) values (μg mL ) for ABTS and DPPH assays, and Trolox Equivalents (TE) (μmol ABTS Assay DPPH Assay −1 g ) for each assay. Species IC TE IC * TE 1 1 1 1 (g mL ) (mol g ) (g mL ) (mol g ) ABTS Assay DPPH Assay B. bifurcata 134.19 2.12 100.33 1.57 155.00 3.96 74.89 1.93 Species IC50 TE IC* TE S. muticum 23.42 0.79 648.35 21.43 188.43 20.55 124.19 14.39 −1 −1 −1 −1 (μg mL ) (μmol g ) (μg mL ) (μmol g ) * IC for B. bifurcata and IC for S. muticum. 25 50 B. bifurcata 134.19 ± 2.12 100.33 ± 1.57 155.00 ± 3.96 74.89 ± 1.93 S. muticum 23.42 ± 0.79 648.35 ± 21.43 188.43 ± 20.55 124.19 ± 14.39 4. Discussion Seaweeds are currently r *I ecognized C25 for B. bifas urca pr taomising and IC50 fo sour r S. muticum ces of di . erent bioactive metabolites, making them attractive for prospection eorts in the search of new functional ingredients and 4. Discussion pharmacophores/drugs [21]. In this work we studied the lipid composition and antioxidant activity of S. muticum, an invasive species of eastern European shores. The invasive S. muticum species may Seaweeds are currently recognized as promising sources of different bioactive metabolites, represent an especially attractive prospective resource because of its abundance and wide availability, making them attractive for prospection efforts in the search of new functional ingredients and pharmacophores/drugs [21]. In this work we studied the lipid composition and antioxidant activity % ABTS inhibition % DPPH inhibition Antioxidants 2020, 9, 642 15 of 20 and also because of the prospect of removing its biomass from the invaded habitats. In order to study the promise and pertinence of using S. muticum as an industry resource, we compared its composition and antioxidant potential to those of a native species of the Iberian Atlantic coast inhabiting the same geographic niche for some decades, in B. bifurcata. Commercially viable solutions such as the use of lipids from brown seaweeds as source of functional lipids for food and cosmetic, pharmaceutical products could encourage their harvesting, which became particularly relevant in the case of invasive species. To our knowledge, the present study represents the first detailed characterization of the lipidomic signature of these two brown seaweeds, B. bifurcata and S. muticum, from the Portuguese coast. The lipid content obtained for B. bifurcata was 7.29% 0.51% DW, while for S. muticum it was 2.84% 0.16% DW. These values were similar with the ones reported in the literature for both seaweeds, B. bifurcata [7,25] and S. muticum [27,33,48]. Both seaweeds showed the presence of SFA in their FA profile, namely 16:0, MUFA and PUFA, namely 18:1 n-9, 18:4 n-3, 20:4 n-6, and 20:5 n-3, respectively, in accordance with the available literature for the FA composition of B. bifurcata [7,24,25] and S. muticum [26,27]. Both seaweeds attract specific interest as edible seaweeds. The low n-6/n-3 ratio found in both algae guarantees appealing nutritional and health characteristics, since omega-3 FA are proposed to be beneficial for the prevention of cardiovascular diseases and other chronic diseases [21,49], while also being associated with antimicrobial, antiviral, anti-inflammatory, and antitumoral properties [29,50,51]. Sargassum muticum displayed a higher PUFA content and n-3 FA, unveiling a profitable avenue for further research in the nutraceutical/dietary supplements scope. Polar lipids identified in the two seaweeds include GL, phospholipids, and betaine lipids. Sargassum muticum presented a much more varied and complex polar lipidome (217 vs. 143 dierent lipid species detected) making it a much more attractive target if searching for bioactive lipids. That happens in the case of GL, with S. muticum presenting 62 dierent lipid species from the class, while B. bifurcata only presented 40. GL from algae have especially been assigned bioactivities such as antioxidant, anti-inflammatory, antiviral, antibacterial, and antitumoral activities [46,52]. Besides glycolipids, phospholipids also represented a major component of the lipidome of B. bifurcata and S. muticum. Phospholipids are major components of cell membranes and can be important signaling molecules in living organisms, as well as in seaweeds [53]. In addition, considering seaweeds as foods or as raw materials for other biotechnological applications, the PL rich in PUFA can be potentially used as ingredients for supplements or for food fortification in n-3 FA, since there is evidence that PLs are better at delivering PUFA than the triglycerides from diet [42,46]. Betaines are also important components of brown seaweeds, although far less studied [39,40]. It was suggested that DGTS species have the same biological function as PC due to their similar zwitterionic structure, with both lipid classes being able to interchange their roles within the cell [54]. Some species of Phaeophyta can have DGTS and/or DGTA, the latter formed by conversion of DGTS into DGTA [55]. The Fucales order, to which B. bifurcata and S. muticum belong, are known to accumulate high amounts of DGTA/DGTS, while displaying reportedly low amounts of PC [39,56]. However, the bioactivities and nutritional value of the betaine lipids are yet to be unraveled. The biological potential of polar lipids present in both lipidomes enhance the commercial viability to turn these seaweeds into components with value for dierent biotechnological applications and industries, as well as explored for compounds with bioactivities, representing viable options in the prevention and management of modern lifestyle diseases [57]. In this work, we explored the antioxidant activity of lipid extracts from both seaweeds, fostering their valorization. Total lipid extracts from B. bifurcata and S. muticum showed antioxidant activity potential in the g mL range. The values of IC for ABTS free radical scavenging assays obtained for the B. bifurcata lipid extract (Table 3), are comparable to ones reported by Santos et al. in dichloromethane extract [24] (ABTS , IC 116.25 2.54 g mL ). Otherwise, slightly higher DPPH radical scavenging activity has been reported in methanol extract [7] and dichloromethane extracts [58], revealing the impact of extraction solvents selection in antioxidant activities, with dierent solvent polarities altering their ecacy to Antioxidants 2020, 9, 642 16 of 20 extract specific antioxidant components [31]. Therefore, our study contributes for the valorization B. bifurcata as a promising source of bioactive compounds and functional ingredients. B. bifurcata may be produced in integrated multi-trophic aquaculture setups, allowing taking advantage of lipid compounds of interest and, otherwise, promoting the aquaculture sector [24]. Sargassum muticum lipid extracts showed higher antioxidant activity than the ones from B. bifurcata in both ABTS and DPPH radical scavenging assays. The IC values presented by S. muticum for ABTS assay (Table 3) are also lower than those presented in the literature for S. serratifolium ethyl acetate (IC , 34.6 0.47 g mL ), or methanol and ethanol extracts, mentioned as eective antioxidants [59]. These results concur to the realization of the great antioxidant potential of S. muticum extracts. The results of the DPPH assay in this survey also unveil a higher antioxidant capacity than that demonstrated for S. muticum chloroform/methanol extracts (1/1, v/v) meaning the extraction procedure used in our study is ecient in the capture of lipid compounds with antioxidant properties [33]. The reported findings also demonstrated that polar lipids can be relevant active lipid players in the antioxidant activity survey of the total lipid extract [33]. Overall, the results from antioxidant assays are more promising for S. muticum than for the native B. bifurcata, supporting the pertinence of using the biomass of this invasive species for production of bioactive liquid extracts, for further bioactivity prospection. Interestingly, from a structural point of view, a distinct lipid profile between both seaweeds highlighted by lipid species rich in PUFA and also the higher PL and GL content obtained in S. muticum extract (PL and GL content, 0.25 0.03 g/100 g DW and 1.31 0.04 g/100 g DW, respectively), may justify the greatest antioxidant performance in this seaweed. The degree of inhibition of ROS was found to be correlated to the amounts of PUFA, the n 3 and n 6 PUFA ratios in the extracts, and with the GL classes MGDG and DGDG, showing greater antioxidant activities than SGDG [60]. Generally, PUFA and n-3 FA have been suggested to hold increased antioxidant potential with regard to other FA [61], and lipid extracts from S. muticum are significantly more abundant in these than those of B. bifurcata, what might concur to the increased antioxidant activity in extracts from the invasive species. Moreover, it was reported that MGDG containing 20:5, 18:3, and 18:4 [42,53–55] and DGDG rich in 20:5 [56,61], which are all FA with increased amounts in S. muticum, present biological activity. Synergic eects of the whole lipid compounds contributing to the eective antioxidant outcome cannot be excluded. Sargassum muticum lipid extracts are dierentiated from those of B. bifurcata by the specific presence of a huge number of galactolipids, phospholipids, and betaine lipids found in the lipidome of this invasive seaweed, some not found in B. bifurcata, which could contribute to its higher antioxidant potential. Oxidative stress is thought to play a pivotal role in a significant range of diseases, including cardiovascular and neurodegenerative diseases, diabetes, autoimmune processes, and cancer [62]. Moreover, inflammation is being positively correlated with chronic metabolic and inflammatory diseases associated with modern lifestyle [63], and a close relationship between oxidative stress and inflammation is established [64]. All this makes the screening for antioxidant properties a good place to start in terms of bioactivity, while also justifying a constant search for novel compounds with antioxidant and anti-inflammatory properties. This study has justified the prospect of deeper research of the scavenging activity in lipid extract of seaweeds. These lipid extracts can be eventually used as a natural antioxidant in the food industry as well as dietary supplements with antioxidant upside or be used in the cosmetic industry. 5. Conclusions This work meaningfully contributed to the valorization of local natural resources in the brown seaweeds B. bifurcata and S. muticum, by providing a thorough characterization of their lipidome and highlighting their unique lipidomics features. Sargassum muticum, an invasive seaweed of the Portuguese coast, revealed a wide diversity in its polar lipid profile, displaying 13 dierent classes with 217 lipid species. Either way, either this high lipid variability is correlated to the adaptation to environmental and geographic changes experienced by S. muticum while intruding dierent settlements, or it contributes to the invasive potential of this species. In its turn, B. bifurcata, a native seaweed Antioxidants 2020, 9, 642 17 of 20 restricted to the environmental conditions particular to a given natural area, revealed a more narrow polar lipid profile, totaling nine dierent classes with 143 lipid species. Both seaweeds of the Portuguese coast present an interesting PUFA content with interesting nutritional value, namely a high n-3 FA content. These features highlight further possible uses in the nutraceutical/supplement food scope. The lipid extracts of the studied seaweeds presented lipid molecular species that have been specifically assigned biological activities, and displayed promising antioxidant properties, especially those of S. muticum. This may open new perspectives for the use of these species of algae for the chemical exploration of novel and more eective natural antioxidant compounds representing innovative functional ingredients in foods or as resources for the pharmaceutical and cosmetic industries. Generally, S. muticum presented a more varied lipid profile and more enticing results in terms of FA composition and antioxidant activity, which may concur for its further exploitation as a natural resource. The prospect of using an invasive species such as S. muticum for further nutritional purposes and as a source of polar lipids with antioxidants activity seems especially attractive, given the great biomass availability and the fact that its use would mean its gradual removal from the invaded habitats. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/7/642/s1; Figure S1. LC MS/MS spectra of SQDG (34:1), Figure S2. LC MS/MS spectra of [M + NH4]+ ions of MGDG (36:2), Figure S3. LC MS/MS spectrum of [M + NH ] ion of DGDG (32:2), Figure S4. LC MS/MS spectra of [M + H] ion at m/z 736.6 of DGTS (34:2), Figure S5. LC MS/MS spectra of [M H] ions of PG (34:4) at m/z 741.5, Figure S6. LC-MS/MS spectra of the [M + H] ion of LPE (20:4) at m/z 502.3, Table S1. Molecular species identified by HILIC-MS in B. bifurcate, Table S2. Molecular species identified by HILIC-MS and MS/MS in S. muticum, Table S3 List of common and unique lipid species in the lipidome of B. bifurcata and S. muticum. Author Contributions: Conceptualization, F.S.; J.P.M.; E.d.C.; M.R.D.; Methodology and formal analysis, F.S., J.P.M., D.D., T.M., E.d.C.; Investigation, and writing—original draft preparation, F.S., J.P.M., E.d.C., MRD.; Data curation, T.M., E.d.C., M.R.D.; Statistic, software and validation, D.L.; E.d.C.; M.R.D. Resources, T.M.; D.L.; E.d.C., M.R.D.; Coordination and supervision, J.P.M.; M.R.D.; Review and editing, all authors. All authors have read and agreed to the published version of the manuscript. Funding: This work was financially supported by project OMICS 4ALGAE: Lipidomic tools for chemical phenotyping, traceability and valorisation of seaweeds from aquaculture as a sustainable source of high added-value compounds (POCI-01-0145-FEDER-030962), funded by Centro2020, through FEDER and PT2020, as well as by project SmartBioR (Centro-01-0145-FEDER-000018), co-funded by the Centro 2020 program, Portugal 2020, European Union, through the European Regional Development Fund. Acknowledgments: The authors are also grateful to FCT/MCTES (Portugal) for the financial support to CESAM (UIDB/50017/2020+UIDP/50017/2020), QOPNA (FCT UID/QUI/00062/2019), LAQV/REQUIMTE (UIDB/50006/2020) and RNEM (LISBOA-01-0145-FEDER-402-022125), through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. João P. Monteiro and Elisabete da Costa (BPD/UI51/5042/2018) acknowledge the scholarship within the framework of Project GENIALG. Tânia Melo thanks the research contract under the project OMICS 4ALGAE (POCI-01-0145-FEDER-030962). Diana Lopes (SFRH/BD/119027/2016) is grateful to FCT (Fundação para a Ciência e Tecnologia), Programa Operacional do Capital Humano (POCH) and European Union through European Social Fund (FSE) for her grant. Conflicts of Interest: The authors declare no conflict of interest. References 1. Nie, C.; Zepeda, L. Lifestyle segmentation of US food shoppers to examine organic and local food consumption. Appetite 2011, 57, 28–37. [CrossRef] [PubMed] 2. Anand, N.; Rachel, D.; Thangaraju, N.; Anantharaman, P. Potential of marine algae (seaweeds) as source of medicinally important compounds. Plant Genet. Resour. Characterisation Util. 2016, 14, 303–313. [CrossRef] 3. Cardozo, K.H.M.; Guaratini, T.; Barros, M.P.; Falcão, V.R.; Tonon, A.P.; Lopes, N.P.; Campos, S.; Torres, M.A.; Souza, A.O.; Colepicolo, P.; et al. Metabolites from algae with economical impact. Comp. Biochem. Physiol. C. Toxicol. 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In Oxidative Stress and Biomaterials; Academic Press: Cambridge, MA, USA, 2016; pp. 35–58. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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