Valuing Bioactive Lipids from Green, Red and Brown Macroalgae from Aquaculture, to Foster Functionality and Biotechnological Applications

Marine edible macroalgae have functional proprieties that might improve human health and wellbeing. Lipids represent a minor fraction of macroalgae, yet with major interest as main carriers of omega 3 polyunsaturated fatty acids and intrinsic bioactive properties. In this study, we used lipid extracts from the green macroalgae Ulva rigida and Codium tomentosum; the red Gracilaria gracilis, Palmaria palmata and Porphyra dioica; and the brown Fucus vesiculosus, produced in a land-based integrated multitrophic aquaculture (IMTA) system. We determined the lipid quality indices based on their fatty acid profiles and their bioactivities as putative antioxidant, anti-inflammatory and antiproliferative agents. The results reveal to be species-specific, namely U. rigida displayed the lowest atherogenicity and thrombogenicity indices. Palmaria palmata and F. vesiculosus lipid extracts displayed the lowest inhibitory concentration in the free radical scavenging antioxidant assays. Ulva rigida, C. tomentosum, P. palmata and P. dioica inhibited COX-2 activity by up to 80%, while P. dioica and P. palmata extracts showed the highest cytotoxic potential in the MDA-MB-231 breast cancer cells. This work enhances the valorization of macroalgae as functional foods and promising ingredients for sustainable and healthy diets and fosters new applications of high-valued algal biomass, in a species-specific context.


Introduction
Marine organisms are untapped sources of bioactive molecules with unique properties and multiple potential applications in nutraceutical, cosmeceutical, pharmaceutical and functional food products [1]. Marine macroalgae are popular aquatic resources that have been used for centuries by and functional value of the macroalgae's biomass [19][20][21]23,26]. Understanding how these differences can be reflected in their bioactive potential is paramount to advance the state of the art of macroalgae's potential. The present study aimed to evaluate the biological activities and quality indices of the lipid extracts of a set of edible macroalgae cultivated under the same IMTA conditions: the green macroalgae Ulva rigida C. Agardh (1823) and Codium tomentosum Stackhouse (1797) (1997); and the brown Fucus vesiculosus Linnaeus (1753). Lipid extracts of each macroalga were used to characterize their FA profile and evaluate their antioxidant (ABTS and DPPH assays) and anti-inflammatory (COX-2 assay) activity and effect on breast cancer cells proliferation (cell viability assay).

Fatty Acid Profiles and Lipid Quality Indices
Fatty acid profiles present in the lipid extracts of green U. rigida and C. tomentosum; red G. gracilis, P. palmata and P. dioica; and brown F. vesiculosus are summarized in Table 1. The identified FA corresponded to the FA esterified to glycerolipids and were derivatized by transesterification reactions and analyzed as fatty acid methyl esters (FAME). The palmitic acid (16:0) was identified in all macroalgae with an abundance in the order of 20%, with the exception of F. vesiculosus (11.9 ± 0.5%). Some of the FA that were present in higher proportions were 20:4n-6 in G. gracilis (with a relative abundance of 35.4 ± 1.5%) and eicosapentaenoic acid (20:5n-3, EPA) in P. palmata (with a relative abundance of 51.9 ± 6.5%). It is also noteworthy the presence of omega-3 FA in U. rigida, namely 18:4n-3 and α-linolenic acid (18:3n-3, ALA) with relative abundance of 24.4 ± 0.4% and 10.9 ± 0.4%, respectively, and also the FA 16:4n-3 and 22:5n-3 with relative abundance of 19 ± 0.6% and 4.1 ± 0.1%, respectively.
The atherogenicity index (AI) and thrombogenicity index (TI) were calculated from the FA profiles of U. rigida, C. tomentosum, Gracilaria sp., P. palmata, P. dioica and F. vesiculosus and are summarized in Table 2.
The highest percent of inhibition of COX-2 activity was recorded for the lipid extracts of P. palmata with 89.5 ± 0.9%, followed by U. rigida that promoted a COX-2 inhibition of 87.9 ± 0.1%. The lipid extracts of P. dioica and C. tomentosum induced 83.6 ± 8.1% and 82.3 ± 2.2% of inhibition, respectively. Only the lipid extracts of two macroalgae achieved less than 50% of inhibition, those of F. vesiculosus with 34.6 ± 7.1% and G. gracilis that showed no inhibition potential of COX-2 in this assay.
respectively. Significant differences between macroalgae were recorded in IC20 in DPPH • assay but not in its TE (Table 3).

Anti-Inflammatory Activity
The anti-inflammatory activity of U. rigida, C. tomentosum, G. gracilis, P. palmara, P. dioica and F. vesiculosus lipid extracts was determined through cyclooxygenase (COX-2) activity assay ( Figure 1). The highest percent of inhibition of COX-2 activity was recorded for the lipid extracts of P. palmata with 89.5 ± 0.9%, followed by U. rigida that promoted a COX-2 inhibition of 87.9 ± 0.1%. The lipid extracts of P. dioica and C. tomentosum induced 83.6 ± 8.1% and 82.3 ± 2.2% of inhibition, respectively. Only the lipid extracts of two macroalgae achieved less than 50% of inhibition, those of F. vesiculosus with 34.6 ± 7.1% and G. gracilis that showed no inhibition potential of COX-2 in this assay.

Effect on Breast Cancer Cell Proliferation
The antiproliferative activity of U. rigida, C. tomentosum, G. gracilis, P. palmata, P. dioica and F. vesiculosus lipid extracts was evaluated measuring the cell proliferation of a highly aggressive MDA-MB-231 breast cancer cell line ( Figure 2).

Effect on Breast Cancer Cell Proliferation
The antiproliferative activity of U. rigida, C. tomentosum, G. gracilis, P. palmata, P. dioica and F. vesiculosus lipid extracts was evaluated measuring the cell proliferation of a highly aggressive MDA-MB-231 breast cancer cell line ( Figure 2).
The macroalgae P. dioica and P. palmata showed the lowest IC50s at 35.5 ± 10.5 and 40.4 ± 19.2 µg/mL, respectively, followed by the macroalgae F. vesiculosus with 52.5 ± 10.9 µg/mL and C. tomentosum with 66.4 ± 12 µg/mL. The two macroalgae that required higher concentrations of lipid extract to inhibit 50% of cancer cells growth were G. gracilis and U. rigida, with IC50s of 74.7 ± 19.1 and 82.7 ± 19.1 µg/mL, respectively (Table 4). There were no significant differences in IC50 between the macroalgae species being compared.  ANOVA test revealed significant differences in MDA-MB-231 cell viability between lipid extract concentrations for all macroalgae: U. rigida (p = 0.011), C. tomentosum (p < 0.001), G. gracilis (p < 0.01), P. palmata (p < 0.01), P. dioica (p < 0.001) and F. vesiculosus (p < 0.001). The antiproliferative effects of the different lipid extract concentrations from the same alga were compared using a post hoc analysis (Tukey's HSD). The results are present in Figure 2. The lipid extract concentration of each macroalga needed to inhibit 50% of cell proliferation (IC50) was calculated (Table 4).  (Table 4). There were no significant differences in IC50 between the macroalgae species being compared

Discussion
The global increase in consumption of seafood products including macroalgae arises from consumers awareness that these products are an important source of nutrients including proteins,

Discussion
The global increase in consumption of seafood products including macroalgae arises from consumers awareness that these products are an important source of nutrients including proteins, polysaccharides, vitamins and lipids [34][35][36][37]. In macroalgae, lipids are primary metabolites and accounted for approximately 0.2-8% dry weight (DW), while carbohydrates accounted for 12-71% DW and proteins for approximates 4.3-47% DW [19,38]. However, despite the lower abundance of lipids compared with the other primary metabolites, lipids from macroalgae are a natural source of PUFA, particularly omega-3 FA, being an interesting alternative to fish, especially macroalgae produced under sustainable aquaculture practices, because of its cultivation under controlled conditions [23,39]. In addition, the polar lipids of macroalgae have been reported to display several bioactivities [25,40,41], although this research field remains rather untapped [42].
The set of macroalgae covered in this study, originating from three different phyla (Chlorophyta, Rhodophyta and Ochrophyta) showed a FA profile consistent with previous studies [20,21,23,25,26,41]. In fact, FA are usually characteristic of each macroalga species, however the abundance of several FA species can change in relative amounts depending on season and/or growth conditions [19,23]. Although some of the studied species belong to the same phylum, there are clear species-specific FA profiles (Table 1). Some macroalgae extracts have high PUFA content. Higher abundance of EPA was found in P. palmata (51.9 ± 6.5%) and P. dioica (20.5 ± 2.3%) lipid extracts, and greater abundance of ALA was found in C. tomentosum (14 ± 0.6%) and U. rigida (10.9 ± 0.4%). This illustrates how variable the nutritional value of macroalgae lipids can be, along with their functional properties.
The composition of FA on a diet has implications in risk factor of coronary artery diseases. There are two measures to evaluate the quality and health benefits of ingested lipids, namely atherogenicity index (AI) and thrombogenicity index (TI) from FA profile, and both are used as cardiovascular risk predictors. Atherogenicity index measures the portion of pro-atherogenic and anti-atherogenic FA, evaluating the ratio between FA that favor the adhesion of lipids to cells of the immunological and circulatory systems, and FA that inhibit the formation of lipid plaques [43][44][45]. On the other hand, TI defines the relationship between the pro-thrombogenic (saturated) and the anti-thrombogenic FA showing the tendency of clots formation in blood vessels [43][44][45]. Higher values of AI and TI (>1.0) are detrimental to human health [46,47]. Macroalgae species studied herein had AI and TI less than or equal to 1.1 (Table 2). Among the set of studied macroalgae, U. rigida displayed the highest content of unsaturated FA compared to saturated FA and also the higher proportion of omega-3 FA compared to omega-6 FA, thus supporting the lowest AI and TI. The values reported for these indices are in line with those retrieved from previous studies addressing macroalgae and marine fish, as well as other added value species (e.g., the ragworm Hediste diversicolor, used as feed for marine fish and shrimp broodstock) [48,49].
The growing demand for natural compounds with antioxidant activity is transversal to food, pharmaceutical and cosmetical industries [50][51][52]. Antioxidant activities from natural sources are usually associated with their profile and content in phenolic compounds [52]. While phenolic compounds are not commonly abundant in macroalgae, because they are secondary metabolites produced under stress conditions, other compounds such as lipids, particularly PUFA, have already been reported as potent antioxidants but have been less thoroughly studied [53]. Antioxidant activity usualy evaluates the capacity of redox molecules in foods and biological systems to scavenge free radicals [54,55]. In ABTS •+ assay, all lipid extracts evaluated showed an IC50 lower than 100 µg/mL. Lower IC50 indicates higher radical scavenging activity and antioxidant potential. The lipid extract with the highest IC50 was that from G. gracilis (86.4 ± 3.4 µg/mL). The results gathered for the red macroalga G. gracilis show a better antioxidant performance in the ABTS •+ assay than the ethanolic (0.689 ± 0.045 mg/mL) and methanolic extracts (1.090 ± 0.073 mg/mL) of Gracilaria manilaensis, which exhibited high phenolic and flavonoid content [56]. The results of the DPPH • assay is not comparable with other studies because studied lipid extract only achieved 20% of inhibition and we cannot extrapolate for IC50 values. The oxidative stress is multifactorial, involving species with different reactivity and solubility. Therefore, it is crucial to evaluate antioxidant properties by using different assays with distinct mechanisms to quench, scavenge and reduce both aqueous and lipophilic radicals. This approach will allow gaining a more in-depth knowledge on antioxidant compounds present in the samples being screened. Lipid extracts from the red P. palmata and the brown macroalgae F. vesiculosus achieved the lowest IC50 and IC20, respectively. Extracts from both species displayed a high content in EPA, which may be related to their potent antioxidant action [53].
The search of compounds with anti-inflammatory activity is yet another area of research which deserves further exploration, as inflammatory diseases have emerged as a prominent health issue [57,58]. Diet plays a relevant role in modulating inflammatory state, highlighting food as a potential therapeutic tool in disorders with an inflammatory basis [57]. Fatty acids composition of diets can control inflammation through biosynthesis of lipid mediators, which derive primarily from membrane phospholipids and reflect dietary FA intake [59]. The ratio between omega-6 and omega-3 PUFA is recognized to play a key role in inflammation [60,61]. While several diseases, such as atherosclerosis, sepsis, mastitis and cancer, involve derivatives of omega-6 PUFA, an increased consumption of omega-3 from marine source was shown to lower the risks of cardiovascular diseases and cancer [62][63][64][65][66][67]. Therefore, the use of lipid extracts rich in omega-3 PUFA from macroalgae may hold great potential to be used as ingredients in the formulation of new products for food and feed enrichment, supplements and nutraceuticals.
In the anti-inflammatory process, the cyclooxygenase (COX) enzyme, which includes the constructive (COX-1) and inducible (COX-2) isoforms, are involved in the synthesis of prostaglandins, thromboxanes and prostacyclins [68][69][70]. The COX-2 isoform is selectively induced by proinflammatory cytokines during inflammation. This enzyme catalyzes prostaglandin biosynthesis from arachidonic acid (20:4n-6, AA), and its inhibition translates in subsequent inhibition of prostaglandins, instigating an anti-inflammatory effect [70]. The lipid extracts of U. rigida, C. tomentosum, P. palmata and P. dioica achieved over 80% COX-2 inhibition without significant differences being recorded between them. The macroalga F. vesiculosus reached lower COX-2 inhibition and the lipid extract of G. gracilis did not show any activity. The content of AA, which is the FA with the greatest relative abundance in G. gracilis and the second most abundant in F. vesiculosus, seems to be the best explanation for the poor results obtained in the lipid extracts of these two macroalgae. The COX-2 enzyme catalyzes the conversion of AA into endoperoxides, which initiates the biosynthetic pathway to the prostaglandins [71]. As the lipid extracts of G. gracilis and F. vesiculosus contain higher AA content, and considering that the AA is the substrate of COX-2 enzyme, the poorer performance of the lipid extracts from these two macroalgae could somehow be anticipated. To our knowledge there are few publications that evaluated COX-2 inhibition by macroalgae lipid extracts, and the ones published used cell lines assays, but not in vitro COX-2 assays. Previous studies evaluated the COX-2 expression, evidencing a reduction of the enzyme expression in cell lines [72][73][74][75], but polar lipid extracts from terrestrial plants and microalgae showed modulation capacity of COX-2 using in vitro assays [75,76]. The ethanolic extract of the small herbaceous plant Polygonum minus (Huds) achieved 25% of COX-2 inhibition at concentration of 100 µg/mL [76] which is in the same order of magnitude we obtained in the present study (about 80% inhibition at a concentration of 500 µg/mL). In addition, the ethanolic extract of the microalga Skeletonema sp. [77] achieved an inhibition of 82 ± 2% of COX-2 at concentrations of 1 mg/mL, which highlights the potential of the results for the lipid extracts of the macroalgae being reported in this is study.
Some metabolites from macroalgae such as lipids, sterols, terpenoids, pigments and phenolic compounds were tested as chemopreventive and anti-cancer agents [78,79]. The evaluation of antiproliferative activity of lipid extracts from U. rigida, C. tomentosum, G. gracilis, P. palmata, P. dioica and F. vesiculosus showed that all of these macroalgae have an inhibitory capacity in the range of 10-100 µg/mL. The lowest IC50 was obtained by P. dioica and P. palmata lipid extracts. A similar study using a lipid extract of Gracilaria sp. [25] reported an IC50 of 12.2 µg/mL and 12.9 µg/mL for T-47D and 5637 cancer cells, respectively. Another study that used methanolic extract of P. palmata [80], rich in polyphenolic compounds, achieved an IC50 of 2.30 mg/mL in HeLa cell line, which is higher than the concentration reported in our study. The lipid extracts of P. dioica and P. palmata that showed higher inhibition are those that have a higher content of EPA. It was previously demonstrated that EPA and docosahexaenoic acid (22:6n-3, DHA) decrease the proliferation and increase the apoptosis of human breast cancer cells by inhibition of signaling through the Akt/NFκB cell survival pathway [81]. Since Akt requires translocation to the plasma membrane for activation, MDA-MB-231 cell membrane enrichment of omega-3 FA after exposure to omega-3 PUFA might affect the activation of signaling molecules such as Akt that are recruited to the membrane for activation [81]. In addition, omega-3 PUFA reduced DNA binding activity of both p50-and p65-containing NFκB as a result of decreased Akt phosphorylation [81][82][83][84].
The bioactivities of lipid extracts herein demonstrated are not exclusive of FA. The FA are esterified in polar lipids, which have been associated with intrinsic bioactive proprieties, as mentioned for the inhibition of DNA polymerase α/β by sulfolipids and galactolipids as antitumoral agents [85][86][87][88]. The sulfonic acid linkage and the number of ethylenic double bonds in unsaturated FA was reported to be relevant structural features in their action [85][86][87]. Likewise, other polar lipids such as phospholipids from marine origin have anti-inflammatory properties, since phospholipids are better delivers of PUFA than triglycerides [89][90][91].
Several studies have shown that macroalgal lipid profiles are highly prone to change under contrasting biotic and abiotic conditions [23,39,92]. The set of macroalgae present in this study were produced under the same IMTA system conditions. The production of macroalgae in this kind of system allows obtaining more biochemically stable, safer and more sustainable biomass. Moreover, this culture framework makes it possible to fine-tune abiotic conditions that may favor certain lipid profiles, thus allowing to customize the lipid extracts of farmed macroalgae for distinct applications.
The present study unveils the functionality of macroalgal lipid extracts towards diverse species-specific bioactivities, in line with the unique lipid signature of the macroalgae addressed (green U. rigida and C. tomentosum; red G. gracilis, P. palmata and P. dioica; and brown F. vesiculosus) [20][21][22][23]25,26,41]. Even though the experimental protocols employed were specific for lipid extraction, one cannot ignore the putative role that the residual presence of other molecules, such as pigments and phenolic compounds, may have played in our findings. Potential synergistic effect between these residual molecules and lipids should also be investigated in future studies aiming to add value to sustainably farmed macroalgae biomass.

Macroalgae Biomass
Samples of fresh biomass of U. rigida, C. tomentosum, G. gracilis, P. dioica, P. palmata and F. vesiculosus were provided by a local aquaculture producer-ALGAplus (Aveiro, Portugal, 40 • 36 43 N, 8 • 40 43 W). Macroalgae were produced in an open-flow outdoor tank system integrated with finfish production on earthen ponds. All macroalgae were harvested in May 2018, cleaned to remove the epiphytes and washed using sterilized seawater. Macroalgae were then dried at 25 • C in an air tunnel until 10-12% total moisture was reached. Five (n = 5) 250 mg aliquots were obtained from bulk production of each macroalga species and were used for total lipid extraction.

Lipid Extraction
Macroalgal biomass was grounded using a mortar and pestle with liquid nitrogen. Lipid extracts were prepared according to a modified Bligh and Dyer protocol [25]. Briefly, 250 mg of macroalgae biomass (five replicates) were mixed with 2.5 mL of MeOH and 1.25 mL of CH 2 Cl 2 in a glass PYREX tube and homogenized by vortexing for 2 min, followed by an incubation period of 2 h and 30 min on ice on a rocking platform shaker (Stuart equipment, Bibby Scientific, Stone, UK). The mixture was centrifuged (Selecta JP Mixtasel, Abrera, Barcelona, Spain) for 10 min at 2000 rpm and the organic phase collected. The biomass residue was re-extracted twice with 2.5 mL of MeOH and 1.25 mL of CH 2 Cl 2 . To wash the lipid extract and induce phase separation, 2.25 mL of Milli-Q water and 1.25 mL of CH 2 Cl 2 were added to the final organic phase, followed by a new centrifugation for 10 min at 2000 rpm. The organic lower phase was collected and transferred to amber vials that were dried under nitrogen stream, weighed and stored at −20 • C. Lipid content was estimated as a dry weight percentage.

Fatty Acid Analysis
Gas chromatography mass spectrometry (GC-MS) was used to determine FA profile of each macroalgae species. FA methyl esters (FAMEs) were prepared using a methanolic solution of potassium hydroxide (2.0 M) [93]. A sample volume of 2 µL of hexane solution containing FAMEs was analyzed by GC-MS on a GC system (Agilent Technologies 6890 N Network, Santa Clara, CA, USA) equipped with a DB-FFAP column with the following specifications: 30 m long, 0.32 mm internal diameter and 0.25 µm 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 range m/z of 50 to 550 in a one second cycle in full scan mode acquisition. The oven temperature was programmed from an initial temperature of 80 • C for 3 min, a linear increase to 160 • C at 25 • C/min, followed by linear increase at 2 • C min −1 to 210 • C, then at 30 • C min −1 to 250 • C, and standing at 250 • C for 10 min. The injector and detector temperatures were 220 and 280 • C, respectively. Helium was used as the carrier gas at a flow rate of 1.4 mL/min. The antioxidant scavenging activity against the 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS •+ ) was evaluated using a previously described method [94,95] 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, K2S2O8 (2.45 mmol/L in water). 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 wells microplate 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 the evaluation of the radical scavenging potential, a volume of 150 µL of each lipid extract from each macroalgae species (12.5 (only for P. palmata), 25, 50, 100 and 250 µg/mL in ethanol) or 150 µL of Trolox standard solution (4,8,16,28,40 and 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 antioxidant compounds present in the lipid extracts was monitored by measuring the decrease in absorbance during the reaction, thereby quantifying radical scavenging activity, which is accompanied by a radical color change. All measurements were performed in triplicate. The percent of ABTS •+ remaining after reaction with antioxidant compounds and percent of inhibition was calculated as follows: % ABTS •+ remaining = (Abs samples after 120 min/Abs sample at the beginning of reaction) × 100 (3) Inhibition% = ((Abs ABTS •+ − (Abs samples − Abs control))/Abs ABTS •+ ) × 100 (4) The concentration of samples able to reduce 50% of ABTS radical after 120 min (IC50) was calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity is expressed as TE (µmol Trolox/g of sample), according to:

2,2-Diphenyl-1-Picrylhydrazyl Radical Assay-DPPH Radical Scavenging Activity
The antioxidant scavenging activity against the 2,2-diphenyl-1-picrylhydrazy radical (DPPH • ) was evaluated using a previously described method [94,95] 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 measurement at 517 nm every 5 min. For evaluation of the radical scavenging potential, a volume of 150 µL of each lipid extract from each macroalgae species (25,50,100, 250 µg/mL in ethanol) or 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 measurement at 517 nm every 5 min. Control lipid extracts were also assayed by replacing 150 µL of DPPH • diluted solution by 150 µL of ethanol. Radical reduction by antioxidant compounds present in the lipid extracts was monitored by measuring the decrease in absorbance during the reaction, thus quantifying radical scavenging activity, which is accompanied by a radical color change. All measurements were performed in triplicate. The percent of DPPH radical remaining after reaction with antioxidant compounds and percent of inhibition was calculated as followed: % DPPH • remaining = (Abs samples after 120 min/Abs sample at the beginning of reaction) × 100 (6) Inhibition% = ((Abs DPPH • − (Abs samples − Abs control))/Abs DPPH • ) × 100 (7) The concentration of samples capable of reducing 20% of DPPH radical after 120 min (IC20) was calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity is expressed as TE (µmol Trolox/g of sample), according to: TE = IC20 Trolox (µmol/L) × 1000/IC20 of samples (µg/mL) (8)

In Vitro Cyclooxygenase Inhibition Assay
Lipid extracts were dissolved in 100% DMSO at 500 µg/mL. The inhibition potential against COX-2 was carried out by enzyme immunoassay (EIA) using in vitro enzymatic assay kit (catalog No. 701230, Cayman Chemical Company, Ann Arbor, MI, USA), carried out according to the instructions provided by the manufacturer (https://www.caymanchem.com/pdfs/701230.pdf). The COX-2 inhibitor screening assay measures the amount of prostaglandin F 2α generated from AA in the cyclooxygenase reaction, which was determined by spectrophotometry at 412 nm using a Multiskan GO 1.00.38 (Thermo Scientific, Hudson, NH, USA) controlled by the SkanIT software version 3.2 (Thermo Scientific). The results were expressed as percentage of inhibited COX-2.

Cell Viability Assay on MDA-MB-231 Breast Cell Line
The antiproliferative activity of macroalgal lipid extracts was evaluated in MDA-MB-231 human breast cancer cell line using crystal violet colorimetric assay to determine the viability of cultured cells. Tumor cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM-F12, Invitrogen Life Technologies, Paisley, UK) with 10% fetal bovine serum (FBS; Gold, PAA) and 50 µg/mL penicillin/streptomycin (PEST) (Invitrogen) in a humidified incubator at 37 • C under an atmosphere of 5% CO 2 . Ten thousand cells per well were plated on 96-well plates and allowed to attach for 24 h; 100 µL of cell suspension (1-2 × 10 4 cell/well in complete medium) were used. Following this step, 200 µL of the treatment solution in a range of 5-100 µg/mL were applied to the culture. The lipid extract was dissolved in ethanol and diluted to a final concentration of 0.1% ethanol in phenol-red free DMEM-F12 medium supplemented with 2% charcoal treated FBS (DCC), 2 mM glutamine and 50 µg/mL PEST. The same concentration of ethanol was used in untreated controls. The treatment medium was changed after 48 h incubation and cells allowed to grow for additional 24 h. At the end of incubation, the growth medium was removed, cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.5% crystal violet for 20 min [96]. Thereafter, wells were washed and air dried for 24 h, and then methanol was added to solubilize the pigment and absorbance was measured at 570 nm. Experiments were carried out in quadruplicates and three independent experiments were performed.

Statistical Analysis
Kruskal-Wallis tests followed by Dunn's post-hoc comparisons were performed to identify significant differences between macroalga species in AI, TI, IC50 and TE from ABTS •+ antioxidant assay, IC20 and TE from DPPH • antioxidant assay and IC50 of MDA-MB-231 cells assay. p-values were corrected for multiple testing using Benjamin-Hochberg method (q values). Differences with q-value < 0.05 were considered statistically significant. Differences in the percentage of COX-2 inhibition between different macroalgal lipid extracts, the antiproliferative activity (MDA-MB-231 cell viability) of lipid extracts at different concentrations and IC50 of MDA-MB-231 cells from different macroalgal lipid extracts were analyzed using one-way ANOVA. Post hoc Tukey's HSD test was used to investigate significant differences between lipid extracts concentrations from the same algae in the antiproliferative activity test. Assumptions of normality and homogeneity of variance were verified prior to analysis through Shapiro-Wilks and Levene's tests, respectively. Statistical analyses were performed using R 3.6.0 [97] in RStudio version 1.1.442 [98]. Experimental data from FA analysis are shown as a mean ± standard deviation of 5 replicates per macroalga species (n = 5), while, for bioactivities, data are shown as a mean ± standard deviation of 3 replicates (n = 3).

Conclusions
This study demonstrates the functionality of lipid extracts from macroalgae sustainably produced in aquaculture. The set of green (U. rigida and C. tomentosum), red (G. gracilis, P. dioica and P. palmata) and brown (F. vesiculosus) macroalgae distributed in three phyla (Chlorophyta, Rhodophyta and Ochrophyta) showed species-specific results for the different bioactivity tests. The lipid quality indices of U. rigida achieved the lower AI and TI. For antioxidant potential (ABTS •+ and DPPH • assays), the lower IC and higher TE were obtained for P. palmata and F. vesiculosus, respectively. The macroalgae U. rigida, C. tomentosum, P. palmata and P. dioica showed the most potent anti-inflammatory potential. The macroalgae F. vesiculosus and G. gracilis showed low and no inhibition, respectively, possibly due to their content in AA, which represents an assay limitation. The antiproliferative activity in breast cancer cells reached the lowest IC50 for the red P. dioica and P. palmata lipid extracts. The lipid content already reported in previous studies along with their PUFA compositions is likely the main cause for the different bioactivities; however, the presence of other lipid-soluble molecules and the synergistic effect between them should not be ignored. This work contributes to the species-specific valorization of macroalgae in line with ONU 2030 sustainable development goals (SDG). Its contribution fulfills the gap on the need of sustainable health diets and new therapeutic strategies for disease prevention of non-communicable diseases (SDG 1); promoting well-being for all at all ages (SDG 3); and boosting the consumption of algae and algae-based products from aquaculture as an alternative of fish consumption, contributing to restoring fish stocks and sustainable use of marine resources (SDG 14).

Conflicts of Interest:
The authors declare no conflict of interest.