Lipid Composition, Fatty Acids and Sterols in the Seaweeds Ulva armoricana, and Solieria chordalis from Brittany (France): An Analysis from Nutritional, Chemotaxonomic, and Antiproliferative Activity Perspectives

Lipids from the proliferative macroalgae Ulva armoricana (Chlorophyta) and Solieria chordalis (Rhodophyta) from Brittany, France, were investigated. The total content of lipids was 2.6% and 3.0% dry weight for U. armoricana and S. chordalis, respectively. The main fractions of S. chordalis were neutral lipids (37%) and glycolipids (38%), whereas U. armoricana contained mostly neutral lipids (55%). Polyunsaturated fatty acids (PUFA) represented 29% and 15% of the total lipids in U. armoricana and S. chordalis, respectively. In both studied algae, the phospholipids were composed of PUFA for 18%. In addition, PUFA were shown to represent 9% and 4.5% of glycolipids in U. armoricana and S. chordalis, respectively. The essential PUFA were 16:4n-3, 18:4n-3, 18:2n-3, 18:2n-6, and 22:6n-3 in U. armoricana, and 20:4n-6 and 20:5n-3 in S. chordalis. It is important to notice that six 2-hydroxy-, three 3-hydroxy-, and two monounsaturated hydroxy fatty acids were also identified and may provide a chemotaxonomic basis for algae. These seaweeds contained interesting compounds such as squalene, α-tocopherol, cholest-4-en-3-one and phytosterols. The antiproliferative effect was evaluated in vitro on human non-small-cell bronchopulmonary carcinoma line (NSCLC-N6) with an IC50 of 23 μg/mL for monogalactosyldiacylglycerols isolated from S. chordalis and 24 μg/mL for digalactosyldiacylglycerols from U. armoricana. These results confirm the potentialities of valorization of these two species in the fields of health, nutrition and chemotaxonomy.


Introduction
Seaweeds are known for their richness in nutritionally beneficial components and contain bioactive compounds such as proteins, carbohydrates, antioxidants, minerals, dietary fibers, vitamins and polyunsaturated fatty acids (PUFAs) [1].
Furthermore, studies dealing with fatty acids (FA) of seaweeds are also interesting for the usefulness of FA as potential chemotaxonomic biomarkers. Their lipid profiles could assist the assignment of algal taxonomic position and provide signature profiles for use in organic geochemistry and food studies [4]. Such biochemical analysis may also be useful to study the abundance and ecology of these species in the marine environments.
An attractive approach to find sources of marine lipids, could be the exploitation of coastal proliferative seaweeds species especially if they contain interesting substances. This is the case for Solieria chordalis (Rhodophyta) and for Ulva armoricana (Chlorophyta), which are proliferative species found in Brittany (North West of France).
Thus, the present study explores the lipid composition of S. chordalis for the first time and of U. armoricana for extending the knowledge on its lipid composition where little is known about FA composition of individual lipid classes. The aim is the determination of the potential value of lipid composition (total lipid, glycolipid-GL, phospholipids-PL, sterol and unsaponifiable fraction) of these proliferative species, which may constitute a nutritional database for chemotaxonomy and anti-proliferative activity perspectives.
So, for these two species, the present study was designed to clarify the content of lipid and glycolipid (GL) classes, and the FA composition from total lipids, phospholipids (PL), GL and compounds of unsaponifiable fraction. In addition, the GL fractions were evaluated for their anti-proliferative activity against human cancer cell lines (non-small cell lung cancer-NSCLC-N6). This present study particularly focuses on lung cancer and to the best of our knowledge for the first time for these species, lung cancer being the leading cause of cancer-related mortality in both men and women in the world.

Lipid Contents and Lipid Classes Distribution
The specimens of Chlorophyta (Ulva armoricana) and Rhodophyta (Solieria chordalis) used in the present investigation were based on their wide distribution and abundance in Brittany (North West of France). The total lipid (TL) contents and the lipid class distribution are reported in Table 1. The TL content of U. armoricana and S. chordalis was established at 2.62% ± 0.04% dry weight (dw) and 2.96% ± 0.04% (dw), respectively. These contents are in agreement with the literature [5,6]. Table 2 shows the TL contents previously found in various U. armoricana ranging from 0.2% to 7.9% dw and for Solieria sp. whose levels vary from 0.4% to 2.8%. The values determined in this study are higher than those reported for different species of the same genus, as shown in Table 2. Nevertheless, it is interesting to note that brown seaweeds generally contain higher TL levels [5]. Furthermore, the TL contents of U. armoricana and S. chordalis are lower compared to earth vegetables, such as soy or sunflower [6]. Sea of South China -0.3% la [19] dr, dry weight; fw, fresh weight; la, lyophilized algae.
These variations could be related to taxonomic entity, seasonality of sampling, location and macroalgae growth conditions [22], in addition to extraction processing and solvent polarity [23]. Sánchez-Machado et al. reported that, as the temperature increased, the lipid level decreased and remained almost stable until the end of the growing season [24]. In comparison to Grateloupia turuturu (Halymeniaceae), the lipid contents are lower, G. turuturu TL content being 3%-4% dw [25]. Each of the TL extracts was fractionated into three fractions corresponding to lipid classes (Table 1): neutral lipids (NL-storage lipids), glycolipids (GL) and phospholipids (PL-structural lipids). For S. chordalis, the major lipid classes corresponded to NL and GL, accounting for 38% each, followed by PL (24%). In the case of U. armoricana, the main lipid class was NL with 56%, followed by GL (29%) and by PL (15%).

Neutral Lipid and Sterol Composition
U. armoricana and S. chordalis were examined for their hydrocarbons and sterols present in unsaponifiable fractions. The analyses were carried out by gas chromatography coupled to mass spectrometry (GC-MS) and observed compositions are given in Table 3. Hydrocarbons represented 7% of the unsaponifiable fraction for U. armoricana, among which squalene corresponded to 2.7% (Table 3). It is interesting to note that a rich squalene diet enhances anti-tumor activity of some chemotherapeutic agents by increasing immune system efficiency and by lowering blood cholesterol content [26]. Moreover, there is some evidence that squalene reduces colon cancer and skin cancer; this activity likely being related to its antioxidant status [27]. Therefore, squalene may be beneficial in preventive therapy and integrative medicine. Additionally, α-tocopherol represented 5.5% of the unsaponifiable fraction from U. armoricana (Table 3), and is an important natural antioxydant [28].
Moreover, it also contained 19% phytol (Table 3). This particular compound is usually used as a precursor for the industrial synthesis of vitamins E and K [29,30].
The major constituent of the unsaponifiable fraction from U. armoricana was cholesterol (35%), which is one of the main sterols present in seaweeds. Its content in green and brown algae varies from 2% to 76% of total sterol. In red algae, its content is lower; however, it is still among the major components of the sterol fraction [31]. Some green algae, such as Ulva and Chaetomorpha, contain cholesterol or 28-isofucosterol as their principal sterol [32].
Cholest-4-en-3-one was also detected as a minor component for U. armoricana (0.8%, Table 3). This compound is a key intermediate in steroid chemistry, which is known as a cholesterol derivative occurring in both plant and animal tissues [33]. It may result from the biosynthesis or the autoxidation of cholesterol. Cholest-4-en-3-one, known as an intestinal catabolite of cholesterol, has an anti-obesity effect on animals [34].
Finally, the unsaponifiable fraction of U. armoricana also contained other phytosterols. They corresponded to campesterol, brassicasterol, isofucosterol, which are known to exhibit cholesterol-lowering effects by decreasing intestinal cholesterol absorption [35]. Furthermore, the activity of phytosterols on cardiovascular diseases and their potent anti-inflammatory properties have been reported [35]. For Humans, all phytosterols come from dietary sources, as Humans cannot de novo synthesize phytosterols.
Aknin et al. have investigated sterol composition of three Chlorophyceae orders and showed that sterol composition offers distinguishing features for the chemotaxonomic classification of these algae [36]. Isofucosterol is typical for Ulotrichales and particularly the Ulvaceae family. Nobuo et al. have also found that the sterol content in marine Chlorophyta is similar to higher plants [37].
Regarding S. chordalis, hydrocarbons represented 10.2% of the unsaponifiable fraction, among which squalene corresponded to 4.5%. Furthermore, α-tocopherol and phytol were also detected and represented 3.1% and 34% of the unsaponifiable fraction, respectively. The main component of the unsaponifiable fraction was cholesterol (43%). Other minor sterols were detected such as cholest-4-en-3-one, but no isofucosterol was detected (Table 3).

Fatty Acid Composition of Total Lipids
All fatty acids (FAs) of the TL were converted into the fatty acid methyl esters (FAMEs) by transmethylation with methanolic hydrogen chloride. The FAs were identified as FAME by comparing their equivalent chain lengths (ECLs) values with those previously described or by using commercial mixtures. The ECLs of the FAMEs were determined by expressing their elution positions relative to those of known straight-chain saturated FAMEs.
These results are in agreement with the literature concerning seaweeds. Red seaweed species contain significant quantities of PUFA, up to 20 carbons, with four or five double bonds. Their two major PUFAs are AA and EPA. In addition, a high C20/C18 PUFA ratio is observed with high C18:1 content [20]. Green seaweeds are characterized by C16 and C18 PUFA with a high C18/C20 PUFA ratio [14].
Furthermore, marine algae also contain the n-3 PUFAs. The n-3 long-chain PUFAs are abundant in most red algae, and this is similar for the n-6 long-chain PUFAs and 18:4n-3 [43].
Although U. armoricana and S. chordalis displayed high amounts of SFA, the contents of PUFAs ranged from 14.8% to 29.2%. It confirms that seaweeds contain significantly higher levels of PUFAs than land vegetables [6]. Interestingly, marine algae are rich in PUFA of the ω3 and ω6 series, which are considered essential FA. The World Health Organization currently recommends that the n-6/n-3 ratio should not exceed 10 in a diet [45]. Therefore, U. armoricana and S. chordalis may be used for the reduction of the n-6/n-3 ratio, as in TL, the n-3 PUFA of U. armoricana (23.9%) is higher than the n-6 PUFA (3.7%) and the n-3 PUFA of S. chordalis (5%) is lower than the n-6 PUFA (9.8%). The n-6/n-3 ratio was established at 0.1 for U. armoricana and 1.9 for S. chordalis.
In addition, the benefits of PUFAs in human health are well documented, including cardiovascular effects [46]. Particularly, the n-3 PUFA may be beneficial for the prevention of several types of cancer, and exhibits various biological activities such as decrease of blood pressure and improvement of heart and liver function in body fat in animal trials [47]. Generally, the marine n-3 PUFAs exert anti-arteriosclerosis, anti-hypertensive, anti-inflammatory, immune-regulatory, antioxidant and anti-thrombotic effects, and antiarrhythmic responses [48]. In addition, they are precursors of the eicosanoids biosynthesis, which are bioregulators in many cellular processes [39]. The impact of n-3 PUFAs on brain function and mental health has also been recently examined, showing that they are able to improve the mitochondrial function [49]. Both AA and EPA are precursors of prostaglandins, thromboxane and other eicosanoids, which influence inflammation processes and immune reactions [47]. Linoleic acid, linolenic acid and arachidonic acid serve important functions in skin growth and protection. Finally, 20:4n-6, 20:5n-3, and 20:3n-6 lipids have valuable biological activities such as heart and mental health, arthritis, cancer and lung disease [13].

Fatty Acid Composition of Phospholipids
The PL FAs of U. armoricana and S. chordalis were identified by GC-MS analyses as FAMEs and N-acyl pyrrolidides (NAPs) as previously described for FAs of the TL. For many FAs, GC-MS data of the NAP derivatives allowed us to confirm their structures and to determine the location of double bonds, branching and hydroxyl groups [50].
All the spectra of the 2-hydroxy FAME exhibited molecular ions and other diagnostic fragment ions such as the ions at m/z 90 arising from the McLafferty rearrangement (instead of the usual m/z 74 for FAME) and m/z 103, the ion [M-MeOH] + , and a relatively intense [M-COOMe] + ion [25,28,[50][51][52][53]. The mass spectra of their NAP derivatives showed the molecular ion peak and prominent peaks at m/z 98 and 100, and a base peak at m/z 129 (McLafferty, 113 + 16). 3-Hydroxy FA was identified since the mass spectra of methyl esters and NAP derivatives showed base peaks at m/z 103 and 142, respectively [28,52].
The 2-and 3-hydroxy FA are only minor constituents of PL FA but they are ubiquitous in nature. They have been reported in marine sponge lipids, which is a marine organism that has bacterial symbiosis [50,51], and in the red algae Schizymenia dubyi (Gigartinales) [52] and G. turuturu (Halymeniales) [53]. Matsumoto et al. reported that 3-and 2-hydroxy FA in microalgae may be used to classify algal species [54]. Bacteria are also recognized as an important source of the hydroxy FA in the natural environment [55], and the 2-hydroxy FA are known to occur in sulfate-reducing bacteria [56]. Several short-chains 3-hydroxy FA were reported as antifungal substances [57], demonstrating that 2-and 3-hydroxy FA are able to influence the membrane properties. For instance, at lower temperatures, some bacteria modify their membranous fatty acid composition by increasing the amount of 2-and 3-hydroxy FA for maintaining the functional homeoviscous state of their membrane [19]. Moreover, there are several examples of close associations between bacteria and algae [58,59].
In addition, it is the first time that monounsaturated 3-hydroxy C17 was identified in U. armoricana. Until now, the 3-hydroxy short-chain acids were known as typical bacterial FA. Thus, this hydroxy FA identified in U. armoricana could be of symbiotic origin.
Furthermore, unidentified aldehyde dimethylacetals (DMA), were detected at trace levels in the FA of PL of U. armoricana. Their mass spectra displaying the characteristic fragment ion m/z 75 ([(CH3O)2-CH] + ) as the base peak [51]. DMA revealed the presence of particular PL named plasmalogens known for various biological properties probably including protection against oxidation [60]. Such compounds have been reported from sponges and mollusks [51], and, very recently, in our previous study performed on G. turuturu [25].
In addition, variations in the FA composition can be attributed to environmental conditions, habitat, light, salinity, pollution, species and genetic status, location and seasonality, geography of development of the seaweed and to the method used for extracting oil [43]. Some recent investigations have demonstrated that FA profiles were specific to taxonomic groups [20,43,61,62]. Al-Hasan et al. have reported variations in macroalgae FA concentrations, but not in the composition pattern when the temperature varied [63]. According to Hotimchenko, light conditions influence the FA lipid contents and ratios [64].
In red seaweed, phosphatidylcholine and phosphatidylglycerol have been reported to be the major polar phospholipids, besides minor phosphatidylethanolamine, diphosphatidylglycerol and unidentified compounds [65].

Antiproliferative Activity against Human Non-Small Cell Lung Cancer
This present study particularly focuses on the valorization of the major glycolipids, such as MGDG and DGDG. These lipids are reported to exhibit diverse biological functions, particularly antitumor activity, especially those from marine organisms.
The effect of MGDG and DGDG from U. armoricana and S. chordalis was evaluated for their capacity to inhibit in vitro the growth of human tumor cell Lines: NSCLC-N6 cell lines derived from a human non-small-cell bronchopulmonary carcinoma (moderately differentiated, rarely keratinized, classified as T2N0M0).
Silva et al. showed that Ulva rigida and the red macroalga Gelidium microdon Kützing (Gelidiales), collected from the Azorean archipelago, exhibited in vitro growth inhibitory effect on human tumor cell lines: NCI-H460 (non-small cell lung cancer) [67]. The crude methanol extracts (after removal of chlorophylls) of both macroalgae were found moderately active against cell lines (IC50 = 42 µg/mL for U. rigida and IC50 = 65 µg/mL for G. microdon).

Proposed Algal Glycolipid Structures of Bioactive Constituents
To further identify the active constituents of U. armoricana and S. chordalis GL fraction, they were profiled by liquid chromatography coupled to high resolution mass spectrometry (HR-MS) [68]. The combination of the HR-MS data with the FA composition obtained by GC-MS of the active fraction was used to identify compounds responsible of the observed antitumor activity.
From the U. armoricana, the activity of the DGDG fraction was related to an ion m/z = 887.573 Da corresponding to the molecular formula of C47H82O15. This corresponded to a DGDG containing two Fas, 14:0 and 18:3n-3 (the major PUFA in DGDG). In the case of S. chordalis MGDG fraction, the antitumor activity was related to an ion m/z = 708.399 Da corresponding to the molecular formula of C39H62O10, the two FAs branched on the MGDG being 14:0 and 16:1n-5. In both cases the S1 and S2 position on the glycerol as well as the sugar moiety was undetermined.

Samples
Ulva armoricana (Ulvales, Chlorophyceae) was collected on the beach in Plestin-les-Grèves (48°39′28″ N, 3°37′47″ W), English Channel (Brittany, France), on 18 June 2012. Solieria chordalis (Rhodophyta, Gigartinales, Solieriaceae) was collected in October 2013 from the littoral zone of the Saint Gildas de Rhuys (47°30′0″ N, 2°49′60″ W, Atlantic coast, France). The algae were stored, and then thoroughly cleaned to remove epiphytes, sediment, organic debris, and macrofauna. Samples were successively rinsed with distilled water. The seaweeds were ground to pieces of about 3 mm with a hammer mill. The crushed seaweeds were frozen immediately at −25 °C and were thawed at the time of lipid analysis. The moisture content (%, MC) of seaweeds was determined by drying 2.00 g of samples in a thermo regulated incubator at 105 °C until constant weight and water content was determined gravimetrically. MC (%) = (mi − mod/mi) × 100; with mi = initial mass of wet seaweed specimen; mod = oven dry mass of seaweed specimen.

Lipid Extraction, Lipid Classes, Fatty Acid and Sterol
Total lipids were extracted from fresh algae crushed (1 kg), with a mixture of chloroform/methanol (1:1, v/v) over 2 days at room temperature under agitation for 5 h. The extract was filtered using a Büchner funnel and washed with distilled water. The lipid content was determined by the gravimetric method and as a percentage of the algae dry weight.
The proportion of lipid relative to the dry mass corresponds to the weight ratio of the total lipid extract and biomass. The percentage (%) of lipid was calculated according to the following equation: % Fat = total lipid/(total lipid + dry mass without lipid) × 100. Mass of dry S. chordalis = 71.8 g; Mass of lipid content of S. chordalis = 2.2 g; Mass of dry U. armoricana = 88.6 g; Mass of lipid content of U. armoricana = 2.4 g.
One part of lipids (1 g) was fractionated into NL (dichloromethane), GL (acetone) and PL (methanol) by normal phase on flash column chromatography (SI-Std, 25 G, 50 μm, 22 bar, 20 mL/mn, IR-50SI/25G, Puri Flash ® Interchim, Montluçon, France). Fractions were evaporated to dryness and the percentage was determined as percentage of 1 g of lipids. Another part of lipids (50 mg) was saponified with 2 M ethanolic potassium hydroxide. A part of the unsaponifiable matter was acetylated using acetic anhydride and pyridine giving a mixture containing sterol acetates. The aqueous phase containing potassium salts of FA was acidified by 2 M HCl (pH = 4-5) and FA were extracted by dichloromethane. FAMEs were prepared by transmethylation (1 h at 80 °C with 6% methanolic hydrogen chloride). A part of these FAMEs was heated at 85 °C in a mixture of pyrrolidine and acetic acid for 1 h in order to obtain the N-acyl pyrrolidides (NAP). The FAMEs of GL were obtained by acidic methanolysis by heating 10 mg of GL with methanol/water/hydrochloric acid (29:4:3, v/v/v, 5 mL) at 80 °C for 18 h. The reaction mixture was extracted with water/hexane (3:9, v/v, 12 mL), the organic layer containing the FAME mixture was dried on anhydrous sodium sulfate, filtered and weighed after solvent evaporation. A part (1/3) of the FAME was preserved; the other was transformed into NAP, as described above.
Total FAs derivatives, PL FAs derivatives and GL FAs derivatives (FAME and NAP), sterols (as free forms and acetates), NL, were analyzed by gas chromatography coupled with mass spectrometry (GC-MS).

Thin Layer Chromatography
Thin layer chromatography (TLC) was carried out for the whole of sterols and glycolipids, in order to visualize the families of products. Thin layer chromatography was carried out on a plate of size 20 cm × 20 cm, consisted of an analytical polyester support and of a silica gel (60F254, 60 Å, 15 µm) of 0.25 mm thickness.
The sterols and unsaponifiable fractions composition were studied by TLC in addition to the analysis by GC-MS, using hexane, diethyl ether and acetic acid as eluent (85:15:0.1, v/v/v, double elution), with a standard cholesterol and cholesterol acetate, phytol, β carotene. Plates were visualized by spraying with sulfuric vanillin followed by heating in an oven.

Gas Chromatography-Mass Spectrometry Analyses of Fatty Acid and Sterol Derivatives
FAMEs, NAPs, sterols (as free forms and acetates), and neutral lipids were analyzed by GC-MS. The samples were analyzed using a Hewlett Packard 6890 series GC system coupled with a MS HP 6890 series, equipped with silica capillary column SLB™-5ms (60 m × 0.25 mm × 0.25 µm), the carrier gas was helium (1 mL·min −1 ). The analyses were carried out in electron impact (70 eV). Detector was set at 280 °C, and the injector at 250 °C. The samples were injected in splitless mode. Helium was used as the carrier gas under a constant flow rate (1 mL/min). Three different temperature gradients were used for FAME analysis, as follows: temperature was held at 170 °C for 4 min and programmed to 300 °C at 3 °C·min −1 ; for NAP analysis, 200 °C for 4 min then 3 °C·min −1 up to 310 °C and held for 20 min; and for sterols analysis, 200 °C then 3 °C·min −1 to 310 °C and held for 25 min. The solvent delay was 7 min for FAME and NAP analyses and 8 min for sterols.
The identification of FAs, sterols and unsaponifiable fractions is carried by identification their mass spectra. Thus, the FAs were identified as FAMEs by comparing their ECL values with those previously described or using commercial mixtures. The ECLs of the FAMEs were determined by expressing their elution positions relative to those of known straight-chain saturated FAME. The FAMEs were converted to N-acyl pyrrolidides in order to locate double bonds and branching.

Cellular Studies (NSCLC-N6)
The antiproliferative activity of glycolipids of U. armoricana and S. chordalis were evaluated on the NSCLC-N6 cell line [69] derived from a human non-small-cell bronchopulmonary carcinoma, moderately differentiated, rarely keratinized, classified as T2N0M0. The cell lines were cultured in RPMI 1640 medium with 5% fetal calf serum, to which were added 100 IU penicillin·mL −1 , 100 mg streptomycin·mL −1 and 2 mM glutamine, at 37 °C in an air/carbon dioxide atmosphere (95:5, v/v).
Cytotoxicity was determined by continuous drug exposure. Experiments were performed in 96 wells microtiter plates (105 cells·mL −1 for NSCLC-N6). Cell growth was estimated by a colorimetric assay based on the conservation of tetrazolium dye (MTT) to a blue formazan product by live mitochondria [70]. Eight repeats were performed for each concentration. Control growth was estimated from 8 determinations. Optical density at 570 nm corresponding to solubilized formazan was read for each well on a Titertek Multiskan MKII. Experiments were performed at least in triplicate, 4 wells per glycolipid concentration being used. IC50 values were calculated from the dose-response curves.

Liquid Chromatography-Mass Spectrometry Analysis of Glycolipids
Active GL fractions were analyzed by high performance liquid chromatography coupled with an electrospray ionization ion trap time-of-flight multistage mass spectrometer (LCMS-IT-TOF) analyses to determine molecular formula. A Shimadzu LCMS-IT-TOF instrument composed of two LC-20ADxr pumps, a SIL-20ACxr autosampler, a CTO-20AC column oven, a SPD-M20A DAD detector, a CBM-20A system controller, an ESI ion source, and an IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) was used. Chromatographic conditions were adapted from Knittelfelder et al. [68]. LC-MS profiles were recorded with a 150 × 2.1 mm, 2.6 µm Kinetex C18 column (Phenomenex) in gradient mode at a flow rate of 0.4 mL·min −1 at the temperature of 60 °C. A solvent system composed of (A) water/MeOH 1:1 and (B) iso-propanol ; both eluant (A and B) being modified with 0.1% v/v formic acid and 10 mM of ammonium formate. The gradient consisted of 5 min at 25% B, followed by an increase to 45% B from 5 min to 13 min, followed by an increase to 75.27% B from 13 min to 45 min. The column was then washed with 100% iso-propanol for 7 min and re-equilibrated to 25% B for 7 min.

Statistical Analysis
All measurements were made in triplicate for each alga (n = 3), except for GL analyses, which were carried out on only one sample. All data are reported as mean ± standard deviation (SD). The statistical analysis was carried out on SPSS v20 (IBM, Chicago, IL, USA) using one-way analysis of variance (ANOVA).

Conclusions
The macroalgae U. armoricana and S. chordalis revealed low lipid contents. However, they exhibited high amounts of nutritionally essential n-6 and n-3 PUFAs, including EPA, AA, DHA, 16:4n-3, 18:4n-3, 18:3n-3, 18:2n-3, and 18:2n-6, but at lower levels than other edible red seaweeds such as Chondrus crispus or Gracilaria verrucosa. Therefore, U. armoricana and S. chordalis may be potential sources of n-3 and n-6 lipids. The health benefiting n-6/n-3 ratio in macroalgae allows their use in the formulation of functional foods and nutraceuticals. In this study, U. armoricana and S. chordalis can be considered as a source of dietary PUFAs, since they showed n-6/n-3 ratios ranging from 0.1 for U. armoricana to 1 for S. chordalis. Moreover, some FAs were identified for the first time in seaweeds as minor components such as 3-hydroxyoctadecanoic and the 2-hydroxy acid and two monounsaturated hydroxy acids. Hence, FAs compositions may provide a chemotaxonomic basis for macro-algae. These seaweeds contained interesting compounds such as phytol (precursor for the industrial synthesis of vitamins E and K), α-tocopherol (vitamin E) and squalene. Phytosterols were identified, namely brassicasterol, chondrillasterol, fucosterol and isofucosterol. The sterol composition showed also the presence of cholest-4-en-3-one. It would be of interest to isolate and identify the most important ones in terms of biological activities. Interestingly, glycolipids (MGDG, DGDG and SQDG) from U. armoricana and S. chordalis showed promising anti-proliferative activities on cancer cell lines.