Antioxidant, Immunomodulatory and Potential Anticancer Capacity of Polysaccharides (Glucans) from Euglena gracilis G.A. Klebs

The present study was carried out to determine the bioactivity of polysaccharides extracted from Euglena gracilis (EgPs). These were characterized by FT-IR and GC-MS. Cytotoxicity analyses (MTT) were performed on healthy human gingival fibroblast cell lines (HGF-1), obtaining an IC50 of 228.66 µg mL−1, and cell lines with anticancer activity for colon cancer (HCT-116), breast cancer (MCF-7), human leukemia (U-937, HL-60) and lung cancer (NCl-H460), showing that EgPs have anticancer activity, mainly in HTC-116 cells (IC50 = 26.1 µg mL−1). The immunological assay determined the immunomodulatory capacity of polysaccharides for the production of proinflammatory cytokines IL-6 and TNF-α in murine macrophages (RAW 264.7) and TNF-α in human monocytes (THP-1). It was observed that the EgPs had a stimulating capacity in the synthesis of these interleukins. The antioxidant capacity of polysaccharides and their biomass were analyzed using the ABTS method (18.30 ± 0.14% and (5.40 ± 0.56%, respectively, and the DPPH method for biomass (17.79 ± 0.57%). We quantitatively profiled HGF-1 proteins by liquid chromatography–tandem mass spectrometry analysis, coupled with 2-plex tandem mass tag labelling, in normal cells. In total, 1346 proteins were identified and quantified with high confidence, of which five were considered to be overexpressed. The data is available through ProteomeXchange, under identifier PXD029076.


Introduction
Microalgae are unicellular organisms classified as prokaryotes, and eukaryotes can grow autotrophic, heterotrophic, and some of the mixotrophic, modes. They are generally highly efficient at CO 2 fixation and utilization of solar energy to produce biomass, with an efficiency up to four times higher than that of plants. The importance of microalgae lies in their role as primary producers in the food chain, in which they are the initial producers of organic matter. These phytoplankton organisms have developed different strategies for survival. They are present in all environments with water, such as lakes, seas, and rivers, but can be found approach by means of liquid chromatography high-resolution mass spectrometry analysis (LC/MS), coupled with 2-plex tandem mass tag labelling (TMT 2-plex). This research facilitated an in-depth study of EgPs and the discovery of metabolites of interest as a source of bioinspiration. In the present study of E. gracilis elemental analysis was performed on the algal biomass and the extracted polysaccharides. Biomass showed a high percentage of carbon (44.59%), followed by nitrogen (10.54%) and hydrogen (5.57%). The sulphur content was practically null (0.03%).
Concerning the elemental analysis of the EgPs, the highest content was C, with 1.24%, followed by N and H, with 0.24 and 0.20%, respectively. The S content was also analyzed, obtaining a higher percentage of Sin polysaccharides than in biomass with 0.16% (Table 1). Accordingly, the obtained molar (C/N ratio) value was 4.93 in biomass and 5.27 in polysaccharides.

Protein, Carbohydrates, Lipids, Inorganic Compounds and Moisture
Analysis of E. gracilis biomass is summarized in Table 2, i.e., total proteins, carbohydrates, lipids, moisture, and inorganic compounds. FT-IR spectrum was recorded in the region of 4000-400 cm −1 . Figure 1 shows the FT-IR spectrum of EgPs, which exhibited the characteristic band features for polysaccharide fractions. EgPs showed the broadband positioned at 3351 cm −1 , which was a distinctive sign of the band of axial stretching of OH. The band at 2954 cm −1 and 2919 cm −1 signified the presence of C-H groups (Str of asymmetric (CH 2 ) and Str of aliphatic (C-H)). The signals around 1640 cm −1 and 1405 cm −1 were attributed to asymmetric and symmetric stretching of a carboxylate anion group (C=O). The stretching at 1226 cm −1 identified the S=O vibration and showed the presence of the sulphate group. The intense absorption band at around 1068 cm −1 was attributed to the coupling of C-O-C, C-C stretching mode, and C-O-H bending mode in compounds like sugar. The FT-IR spectrum of EgPs exhibited the signal at 908 cm −1 which was attributed to typical β-configurations, indicating the presence of β-glucans. Additionally, the signal at 882 cm −1 represented the presence of β-pyranoses. This result confirmed the presence of sulphate groups in EgPs.
the presence of C-H groups (Str of asymmetric (CH2) and Str of aliphatic (C-H)). The signals around 1640 cm −1 and 1405 cm −1 were attributed to asymmetric and symmetric stretching of a carboxylate anion group (C = O). The stretching at 1226 cm −1 identified the S = O vibration and showed the presence of the sulphate group. The intense absorption band at around 1068 cm −1 was attributed to the coupling of C-O-C, C-C stretching mode, and C-O-H bending mode in compounds like sugar. The FT-IR spectrum of EgPs exhibited the signal at 908 cm −1 which was attributed to typical β-configurations, indicating the presence of β-glucans. Additionally, the signal at 882 cm −1 represented the presence of βpyranoses. This result confirmed the presence of sulphate groups in EgPs.

Gas Chromatography-Mass Spectrometry (GC-MS)
The major peak in the GC-MS spectrum of polysaccharides corresponded to the monosaccharide glucose, with a retention time of 29.73 min ( Figure 2; Table 3). The other peaks belonged to ribose and mannose. Other monosaccharides with lower relative abundances were identified as galactose and fucose.

Gas Chromatography-Mass Spectrometry (GC-MS)
The major peak in the GC-MS spectrum of polysaccharides corresponded to the monosaccharide glucose, with a retention time of 29.73 min ( Figure 2; Table 3). The other peaks belonged to ribose and mannose. Other monosaccharides with lower relative abundances were identified as galactose and fucose.   Table 3. The results showed a maximum value of antioxidant activity for the polysaccharides and the biomass of E. gracilis of 5.40 ± 0.26% and 18.30 ± 0.14%, respectively, at the concentration of 500 µ g mL −1 (Figure 3).  Table 3. The results showed a maximum value of antioxidant activity for the polysaccharides and the biomass of E. gracilis of 5.40 ± 0.26% and 18.30 ± 0.14%, respectively, at the concentration of 500 µg mL −1 (Figure 3).  Figure 3. Scavenging effects % of the sample on ABTS radical. The data are the mean of 3 replicate measurements ± standard error. Similar letters indicate no significant differences (Tukey, p < 0.05) between the different concentrations.

Antioxidant Activity (DPPH Method)
The antioxidant activity assay, using the DPPH method, for the biomass from E. gracilis showed the highest antioxidant activity at the concentration of 200 µ g mL −1 with a value of 17.79 ± 0.57%, although at the studied concentrations, there was not much variation between the data obtained from this method. (Figure 4).

Figure 3.
Scavenging effects % of the sample on ABTS radical. The data are the mean of 3 replicate measurements ± standard error. Similar letters indicate no significant differences (Tukey, p < 0.05) between the different concentrations.

Antioxidant Activity (DPPH Method)
The antioxidant activity assay, using the DPPH method, for the biomass from E. gracilis showed the highest antioxidant activity at the concentration of 200 µg mL −1 with a value of 17.79 ± 0.57%, although at the studied concentrations, there was not much variation between the data obtained from this method. (Figure 4).   The HTC-116, MCF-7, U-937, HL-60, and NCl-H460 cells were treated with different concentrations of EgPs to assay the potential effects of this compound on anticancer activity. The results obtained with the MTT assay showed that the tested polysaccharides inhibited cell proliferation in a dose-dependent manner, so its anticancer ability in these tumor cells was shown.
According to results obtained from the anticancer activity assay of EgPs, it was observed that the highest IC50 value was obtained in MCF-7 (110.53 µ g mL −1 ) ( Figure 5C), followed by NCl-H460 (107.11 µ g mL −1 ) ( Figure 5B). In the case of HTC-116, the IC50 was lower than in the others, being 26.10 µ g mL −1 ( Figure 5A). In the U-937 and HL-60 cells, IC50 of 27.54 µ g mL −1 was obtained ( Figure 5D) and 18.20 µ g mL −1 (Figure 5E) respectively. These values indicated that these polysaccharides have great potential against these cells at a very low level to inhibit cell growth in concentrations of 50%. The HTC-116, MCF-7, U-937, HL-60, and NCl-H460 cells were treated with different concentrations of EgPs to assay the potential effects of this compound on anticancer activity. The results obtained with the MTT assay showed that the tested polysaccharides inhibited cell proliferation in a dose-dependent manner, so its anticancer ability in these tumor cells was shown.
According to results obtained from the anticancer activity assay of EgPs, it was observed that the highest IC 50 value was obtained in MCF-7 (110.53 µg mL −1 ) ( Figure 5C), followed by NCl-H460 (107.11 µg mL −1 ) ( Figure 5B). In the case of HTC-116, the IC 50 was lower than in the others, being 26.10 µg mL −1 ( Figure 5A). In the U-937 and HL-60 cells, IC 50 of 27.54 µg mL −1 was obtained ( Figure 5D) and 18.20 µg mL −1 (Figure 5E) respectively. These values indicated that these polysaccharides have great potential against these cells at a very low level to inhibit cell growth in concentrations of 50%.

Cell Viability of Line HFG-1 more and less Proliferating
This test was carried out to make a comparative study between healthy cells (HGF-1) in a proliferative state, and, therefore, simulating the typical state of tumor cells, and in a healthy state.

Cell Viability of Line HFG-1 more and less Proliferating
This test was carried out to make a comparative study between healthy cells (HGF-1) in a proliferative state, and, therefore, simulating the typical state of tumor cells, and in a healthy state.
These cells were treated with different concentrations of EgPs to test the potential effects of this compound in both conditions (Figures 6 and 7). The results obtained with the MTT assays showed that the tested polysaccharides inhibited cell proliferation in a dosedependent manner, obtaining an IC 50 value one order of magnitude lower in proliferative HGF-1 (similar to cancerous conditions) cells, compared to HGF-1 less proliferative (normal healthy condition) cells. That is, the IC 50 value in more proliferating HGF-1 cells with EgPs was 147.86 µg mL −1 (Figure 6), while the IC 50 value of less proliferating HGF-1 cells was 228.66 µg mL −1 (Figure 7). dose-dependent manner, obtaining an IC50 value one order of magnitude lower in proliferative HGF-1 (similar to cancerous conditions) cells, compared to HGF-1 less proliferative (normal healthy condition) cells. That is, the IC50 value in more proliferating HGF-1 cells with EgPs was 147.86 µ g mL −1 (Figure 6), while the IC50 value of less proliferating HGF-1 cells was 228.66 µ g mL −1 (Figure 7). . % Survival of cell line HGF-1 (human gingival fibroblasts), very proliferative, exposed to different concentrations of EgPs. Similar letters (a to g) indicate that there were no significant differences (Tukey, p < 0.05) between the different concentrations.   This trial evaluated the production of proinflammatory cytokines, TNF-α and IL-6. For this purpose, the RAW 264.7 mouse macrophage cell line was used with the following polysaccharide concentrations: 1 μg mL −1 , 5 μg mL −1 , 25 μg mL −1 , 50 μg mL −1 , 75 μg mL −1, and 100 μg mL −1 . For the positive control, E. coli lipopolysaccharide (LPS) was used at a concentration of 0.5 μg mL −1 .

Log 10 Concentration Polysaccharides (µg mL
The maximum concentration of Interleukin-6 obtained within the concentrations studied was 3056.60 pg mL −1 when a concentration of polysaccharides of 100 μg mL −1 (Figure 8) was applied in the case of TNF-α, and was 564.05 pg mL −1 (Figure 9). Both figures show that for IL-6 and TNF-α, the synthesis and accumulation by RAW 264.7 cells increased as a higher concentration of EgPs was applied. Specifically, IL-6 synthesis increased exponentially from 108 to 3057 pg mL −1 as it passed a polysaccharide concentra- Figure 7. % Survival of cell line HGF-1 (human gingival fibroblasts), low proliferative, exposed to different concentrations of EgPs. Similar letters (a to g) indicate that there are no significant differences (Tukey, p < 0.05) between the different concentrations.
The maximum concentration of Interleukin-6 obtained within the concentrations studied was 3056.60 pg mL −1 when a concentration of polysaccharides of 100 µg mL −1 Pharmaceuticals 2022, 15, 1379 9 of 24 ( Figure 8) was applied in the case of TNF-α, and was 564.05 pg mL −1 (Figure 9). Both figures show that for IL-6 and TNF-α, the synthesis and accumulation by RAW 264.7 cells increased as a higher concentration of EgPs was applied. Specifically, IL-6 synthesis increased exponentially from 108 to 3057 pg mL −1 as it passed a polysaccharide concentration of 5 to 100 µg mL −1 . Similarly, TNF-α production increased exponentially from 87 to 564 pg mL −1 when the polysaccharide concentration increased from 5 to 100 µg mL −1 . However, in none of the cases did the cytokine levels become saturated.  Figure 9. Concentration of Tumor Necrosis Factor-α by RAW 264.7 macrophages exposed to different concentrations of EgPs. Data points represent the average of three samples ± SD.

Determination of TNF-α with Human THP-1 Cell Line
Human THP-1 were exposed to 10 ng mL −1 phorbol-12-myristate-13-acetate (PMA) for 48 h to differentiate them into macrophages and to ensure that samples with detectable quantities of TNF- were obtained. After that, THP-1-derived macrophages were exposed to different concentrations of EgPs for 24 h. As shown in Figure 10, cells exposed to crescent concentrations of EgPs increased TNF- production from a concentration of 15.6 µ g mL −1 , reaching its maximum effect at 250 µ g mL −1 . Figure 9. Concentration of Tumor Necrosis Factor-α by RAW 264.7 macrophages exposed to different concentrations of EgPs. Data points represent the average of three samples ± SD.

Determination of TNF-α with Human THP-1 Cell Line
Human THP-1 were exposed to 10 ng mL −1 phorbol-12-myristate-13-acetate (PMA) for 48 h to differentiate them into macrophages and to ensure that samples with detectable quantities of TNF-α were obtained. After that, THP-1-derived macrophages were exposed to different concentrations of EgPs for 24 h. As shown in Figure 10, cells exposed to crescent concentrations of EgPs increased TNF-α production from a concentration of 15.6 µg mL −1 , reaching its maximum effect at 250 µg mL −1 .

Proteomic Analysis in HGF-1 Cells
We quantified the proteomic changes between treated and control HGF-1 cell groups utilizing TMT isobaric labelling of the protein extracts derived from culture cells, followed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Data analysis identified a total of 1346 proteins with high confidence (<1% false discovery rate and more than two different peptides identified per protein). In total, five proteins were upregulated (Log2 fold-change > 0.3, p-value < 0.05) as compared with the controls. Significantly changed proteins are listed in Table 4 and illustrated in Figure 11.

Proteomic Analysis in HGF-1 Cells
We quantified the proteomic changes between treated and control HGF-1 cell groups utilizing TMT isobaric labelling of the protein extracts derived from culture cells, followed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Data analysis identified a total of 1346 proteins with high confidence (<1% false discovery rate and more than two different peptides identified per protein). In total, five proteins were upregulated (Log 2 fold-change > 0.3, p-value < 0.05) as compared with the controls. Significantly changed proteins are listed in Table 4 and illustrated in Figure 11.  * The sum PEP score corresponds to the score calculated based on the posterior error probability (PEP) values of the peptide spectrum matches (PSM). The PEP indicates the probability that an observed PSM was a random event. The Sum PEP score was calculated as the negative logarithms of the PEP values of the connected PSM.

STRING Analysis of Protein Networks
In addition, we carried out a bioinformatics analysis of protein interaction networks for upregulated proteins, which was built using the database and web-tool STRING

STRING Analysis of Protein Networks
In addition, we carried out a bioinformatics analysis of protein interaction networks for upregulated proteins, which was built using the database and web-tool STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) version 11.5 [20]. This analysis summarized the network of predicted associations for TNF-α, IL-6, and the upregulated proteins in HGF-1 cells after treatment with EgPs ( Figure 12), allowing us to delve deeper into the functional pathways where these proteins were involved. The analysis of the networks showed that proteins had more interactions among themselves than what would be expected for a random set of proteins of the same size and degree distribution drawn from the genome (11 vs. 5), as shown in Figure 12C, which meant that the proteins were at least partially biologically connected, as a group. Therefore TNF-α and IL-6 were significantly linked to the overexpressed proteins. Table 5 reports all the significant interactions between the different proteins. (Search Tool for the Retrieval of Interacting Genes/Proteins) version 11.5 [20]. This analysis summarized the network of predicted associations for TNF-α, IL-6, and the upregulated proteins in HGF-1 cells after treatment with EgPs ( Figure 12), allowing us to delve deeper into the functional pathways where these proteins were involved. The analysis of the networks showed that proteins had more interactions among themselves than what would be expected for a random set of proteins of the same size and degree distribution drawn from the genome (11 vs. 5), as shown in Figure 12C, which meant that the proteins were at least partially biologically connected, as a group. Therefore TNF-α and IL-6 were significantly linked to the overexpressed proteins. Table 5 reports all the significant interactions between the different proteins.

Discussion
The total content of C and N in the biomass of E. gracilis was higher than that obtained in other microalgae, such as Tetraselmis suecica [21] or Porphyridium purpureum [22], as was the content of H, with respect to P. purpureum. However, the S content was lower than that of P. purpureum. Regarding the polysaccharides, it was observed that the C content of the EgPs was lower than that obtained in the polysaccharides of T. suecica [21] and P. purpureum [22]. In the case of total Nitrogen, it was lower than that of T. suecica, and higher than in P. purpureum. The H content was lower in EgPs than in the polysaccharides of P. purpureum. When comparing the content of S, it was observed that the same percentage was obtained in EgPs and in polysaccharides of P. purpureum.
The study by Metsoviti et al. 2019 [23] compared the growth rates and nutrient contents of five species of microalgae grown in greenhouses, including E. gracilis, the samples of which were taken under different conditions of light intensity and temperature. The data for ash, protein and moisture content were lower than those obtained in our study in all cases, while the percentage of lipids varied according to light intensity and temperature, with the lipid content being higher than that obtained in our study at a higher intensity of light and temperature, and lower in conditions of lower intensity of light and temperature. If we compare our results with different strains of Tetraselmis and with Pyramimonas from the study by Tzovenis et al. 2009 [24], the contents of inorganic compounds, and mainly carbohydrates, were higher than that obtained with E. gracilis. However, the percentages of proteins and lipids were lower than that obtained in E. gracilis. It is known that the concentration of lipids in microalgae is extremely variable, reaching productive peaks of 70-80% for those used in oil production [25].
The bands obtained in the FT-IR of the EgPs coincided with those obtained in polysaccharides from other algae, as determined by different authors. In the case of the band at 3351 cm −1 , this was also found in polysaccharides from Sarcodia ceylonensis, Ulva lactuca, Gracilaria lemaneiformis and Durvillaea antarctica, according to He et al. (2016) [26]. An important reserve polysaccharide in Euglenophytes is the paramylon that presents bands at 2954 cm −1 and 2919 cm −1 corresponding to the CH group, as indicated by Synytsya, A. & Novak, M (2014) [27], and a peak at 908 cm −1 , characteristic of β-glucans [28], and at 882 cm −1 of β-pyranose [26]. The presence of sulphate groups (S=O) and carboxylate anions (C=O) identified in EgPs have also been described by other authors in polysaccharides from algae [29][30][31]. The appearance of intense bands around 1068 cm −1 was attributed to the presence of compounds, such as sugars [32,33]. According to Meng et al. (2014) [34], the FTIR method is a validated spectroscopic method, which determines the existence of polysaccharides, lipids and proteins in algal samples and evaluates the physiology of microalgae. This study indicated that the bands at 1200-950 cm −1 characterized the absorption of C-O-C vibrations of polysaccharides, as was detected in our samples. This characterization method could be used to temporarily observe the extraction dynamics of different algae compounds and compare them with spectra from digital libraries, which would allow the identification of fractions of biotechnological interest and industrial application.
The identification of the derivatized samples was carried out in a GC-MS system, particularly by comparing their retention times with those of the standard monosaccharides Arb, Rib, Fuc, Glc, Gal, Rha, Fru, Mann, Xyl, Gluc-A and Api. The monosaccharide units identified were mainly Glc, although Rib and Mann, and other minor monosaccharides, such as Gal and Fuc, were also detected. There have been very few characterization tests of EgPs conducted. However, those that have been conducted agree with the results obtained in our test. Thus, Barras and Stone, 1965 [35] indicated that Glc, Gal, Mann, Fuc, Xyl and Rha were detected on the cell surface of E. gracilis, as well as Xyl, associated with flagella [36]. Farrah et al., 2019 [37] detected glucose and matinol in the aqueous extracts of E. gracilis in GC-MS, this being a derivative of mannose. The differences between the carbohydrate composition of E. gracilis in our and other studies could be explained by differences in the extraction methods, the type of derivation, and the carbohydrate analysis strategies.
There are studies that show the antioxidant activity of microalgae [38,39]. These contain various compounds that have antioxidant characteristics, such as carotenoids, vitamins C and E, florotannines, ω3 polyunsaturated fatty sulphates, polysaccharides and others [40]. However, the most interesting ones are carotenoids, especially astaxanthin, β-carotene and lutein [41]. According to [42], the antioxidant activity of the biomass, determined by means of the ABTS method in Galdieria sulphuraria, has much higher activity than that obtained at the studied concentrations of E. gracilis and the same happens with Spirulina plantensis [43]. However, the activity of E. gracilis is comparable and, in many cases, superior, to various foods, such as extracts of fruits, vegetables, and drinks, as demonstrated in the study carried out by Pellegrini et al., 2003 [44]. In the case of the biomass test using the DPPH method, the antioxidant activity of S. platensis was higher than that obtained in E. gracilis [45]. The antioxidant activity of EgPs, determined by means of the ABTS test was lower than the polysaccharides of other microalgae, such as Porphyridium cruentum [46] as well as the S. plantensis [47] polysaccharides. It was shown that Tetraselmis suecica polysaccharides have antioxidant activity in both autotrophic and heterotrophic cultures in [21].This indicated that the antioxidant activity was higher or lower according to different factors, such as the monomers that form the polysaccharides [48], the oligomeric components, sulphates and glycoproteins of the polymer or the combination of these factors [49]. Other authors, [50,51] reported similar values of antioxidant activity (35-40 and 66.5 µmol Trolox g −1 DW) in polysaccharides from the fungus Cordyceps sinensis.
Studies show that algae (micro and macroalgae) produce polysaccharides with biological activities: anti-inflammatory, antioxidant, antiviral, anticoagulant, anticancer and immunomodulant [52]. The results obtained in this research showed anticancer activity of EgPs in the cell lines studied (U-937, HL-60, NCl-H460, HTC-116 and MCF-7). Microalgae polysaccharides are interesting candidates for antitumor therapies. Polysaccharides from Tribonema sp. and Phaedactylum tricornum induced apoptosis in the liver cancer cell line (HepG2) [53,54]. The anticancer activity of polysaccharides from S. platensis, on the cell lines MCF-7 and HepG2 (liver hepatocellular carcinoma), were studied by Abd El Baky H. et al. respectively. Polysaccharides of T. suecica also exhibited anticancer activity, mainly in the HL-60 cell line, presenting an IC 50 of 36 µg mL −1 [21].
Polysaccharides from various algae [56,57] and higher plants [58,59] have been reported to increase phagocytic and phagocyte-secreting activities. According to the data obtained, the effect of EgPs on the immune system is due to a strong stimulation of the macrophages in relation to the synthesis of interleukins with a maximum concentration in Il-6 of 3056.60 pg mL −1 and in TNF-α of 564.05 pg mL −1 . As is known, polysaccharides from numerous microalgae have been reported to stimulate the activity of macrophages and phagocytes. For example, according to [60] the polysaccharides extracted from P. cruentum are potent inducers of cytokine secretion IL-6 and TNF-α [22]. De Jesus et al. (2013) [52] indicated that polysaccharides of marine microalgae, such as Porphyridium, Phaeodactylum and Chlorella stigmatophora, showed anti-inflammatory activity and acted as immunomodulatory agents. The direct stimulating effect of Phaeodactylum tricornutum on immune cells was evidenced by positive phagocytic activity assayed in vitro or in vivo. Polysaccharides from other algae, such as Laminaria ochroleuca, Porphyra umbilicalis and Gelidium corneum [61], have also been studied for their immunomodulatory ability, demonstrating the high capacity of these molecules to activate macrophages and for secretion of pro-inflammatory cytokines IL-6 and TNF-α, especially the polysaccharides from Laminaria ochroleuca. Sun [62] showed that EPS (sulphated polysaccharides) showed immunostimulant activity in mice with S180 tumors, by increasing both the spleen index and thymus index, and also the spleen lymphocyte index. It has also been reported that polysaccharides from P. cruentum induced an increase in the production of TNF-α and IL-6 in the mouse macrophage cell line (RAW 264.7) [22]. These results demonstrate the ability of algae polysaccharides to directly stimulate the immune system.
In the present study, we analyzed the proteomic profile of control and EgP-treated HGF-1 cells to understand the molecular mechanisms underlying their biological effects. A comparative proteomics approach allowed us to identify five key proteins that could be involved in their biological activity: alpha-2-HS-glycoprotein (AHSG), myosin regulatory light chain 12B (MYL12B), thymosin beta-10 (TMSB10), Superoxide dismutase-2 (SOD2) and metallothionein-2 (MT2). Four of them have been reported as anti-inflammatory agents: AHSG, MYL12B, SOD2, and MT2. AHSG deficiency is associated with inflammation and links vascular calcification to mortality in patients on dialysis [63]. Although MYL12B has its most important role in regulation of cytokinesis and cell locomotion [64], it was recently reported as a ligand of the CD69 molecule, a type II transmembrane protein that is recognized as a marker of lymphocyte activation [65]. In addition, many studies have shown that there are antioxidant enzymes that have protective effects during inflammation by free radical scavenging, as occurs with SOD. These play an essential role in inflammatory diseases because they catalyze the conversion of superoxide into hydrogen peroxide and oxygen, affecting immune responses [66]. As is the case with SOD, MT2 also acts as an antioxidant. Mammals have four general metallothionein isoforms (MT1, 2, 3, 4). Though the functions of metallothioneins (MTs) have not been fully elucidated, they appear to participate in detoxifying heavy metals [67], storing and transporting zinc, and redox biochemistry [68]. In fact, the redox regulating capacity of MTs is approximately 50 times greater than that of the major antioxidant, glutathione [69]. It has been suggested that induction of MTs in cell therapy may provide protection by serving as antioxidant and anti-inflammatory agents [70]. What is more, cytokines, such as TNF-α and IL-6 modulate MTs and Zn metabolism in non-immunological organs, such as the hepatic tissue [71].
Regarding the antitumor effects that EgPs could have, several observations suggest that TMSB10 plays a significant. and possibly obligatory, role in cellular processes controlling apoptosis, possibly by acting as an actin-mediated tumor suppressor [72].

Biological Material
E. gracilis used for the realization of this study was within the species that we had in our cepari, in this case the coding was Eg 01. Once collected from the cepari, E. gracilis was inoculated into a 1 litre flask for growth. The medium used was 3N-BBM + V (Bold Basal Medium with 3 times Nitrogen and Vitamins, modified) enriched with vitamin B12 (Sigma-Aldrich, St. Louis, MO, USA), B1 (Sigma-Aldrich, St. Louis, MO, USA) and Biotin (Sigma-Aldrich, St. Louis, MO, USA). The culture was kept at a pH 7 in a culture chamber at 25 • C, 100 µmol of photons m −2 s −1 lighting and in constant agitation by air bubbling. Once a steady state was reached, the biomass was inoculated in larger flasks, maintaining the same conditions, to obtain a greater amount of biomass in order to carry out all the subsequent tests.

Total Carbon (C), Hydrogen (H), Nitrogen (N) and Sulphur (S)
To determine the total carbon, nitrogen and sulphur of the biomass and polysaccharide samples, the combustion technique was used, using the LECO TruSpec Micro CHNSO elemental analyser (St. Joseph, MI, USA). The result obtained was given in % with respect to the weight of the sample for each of the elements (C, H, N, S).

Protein Content
Once the samples were analysed with the CNH Perkin-Elmer 2400 Mar Biotechnol (Perkin-Elmer, Waltham, MA, USA) analyzer, the protein content was calculated by multiplying the per cent of total nitrogen by a factor reported by Lourenço 4.99 [73]. To calculate the total protein concentration from the nitrogen content in the microalgae in the early stationary phase, the use of the average overall N-Prot Factor of 4.99 is recommended, as it has not yet been studied for its specific factor.

Determination of Total Carbohydrates
The total carbohydrates were determined according to the method of Dubois [74]. The technique is described by Kochert [75]. Five mg of lyophilized biomass of E. gracilis were taken and 1 mL of H 2 SO 4 (1 M) was added. The mixture was sonicated for 5 min. Then, 4 mL of H 2 SO 4 (1 M) (Sigma Aldrich, St. Louis, MO, USA) were added and the mixture was placed in a water bath (temperature 100 • C) for 1 h. After that, the samples were cooled to ambient temperature. Then, the samples were centrifuged at 4000 rpm at 10 • C for 15 min. After this, 0.25 mL of the samples were taken. This extract was mixed with 0.75 mL of H 2 SO 4 (1 M) and 1 mL of 5% phenol (Sigma Aldrich, St. Louis, MO, USA) and allowed to stand for 40 min at room temperature. Finally, 5 mL of concentrated H 2 SO 4 was slowly added. After cooling to room temperature, the sample was measured using a spectrophotometer at 485 nm. The blank sample was made in the same way, substituting the sulphated extract of the sample with 1 mL of H 2 SO 4 (1 M).

Determination of Lipids
Lipids were determined by the Folch method [76]. For this purpose, 200 mg of lyophilized algae were taken and 5 mL of chloroform (Sigma Aldrich, St. Louis, MO, USA) and methanol mixture (2:1) was added with 0.01% butylhydroxytoluene (BHT) (Sigma Aldrich St. Louis, MO, USA). The solution was homogenized, and 2 mL of 0.88% KCl was added. The mixture was centrifuged for 5 min at 2000 rpm. Two phases appeared. The upper phase was discarded, and the lower was collected and filtered. It was cooled at −20 • C for at least 20 min. The thin layer of salt that formed on the surface was removed. The lipids were weighed and stored in a nitrogen atmosphere.
The percentage (%) of lipids was calculated according to the Equation (1):

Determination of Inorganic Compounds
To determine the inorganic compounds, a weight of 2 g of DW of E. gracilis was placed into a previously weighed crucible. The crucible was placed in a muffle for 24 h at 550 • C. During this process, all organic matter was eliminated. The weight of the crucible with the samples was determined by subtracting the final weight of the crucible from the initial weight. Finally, the percentage of inorganic compounds in the sample was calculated.

Extraction of Polysaccharides
The polysaccharide extraction process was carried out according to Pajares et al. (2012) [77] with some modifications. For this extraction, we first depigmented the biomass, for which 10 g of dry biomass of E. gracilis were taken, added in 400 mL of EtOH (Sigma-Aldrich, St. Louis, MO, USA) at 80% (v/v), stirred at room temperature for 16 h, and then centrifuged (4000 rpm, 10 min). This was done repeatedly until a colourless supernatant was obtained (usually after four times). After the last centrifugation, the pellet was taken and added to 300 mL of dH 2 O, and heated at 100 • C with continuous stirring for 40 min. After this time, it was allowed to cool and centrifuged (4500 rpm, 15 min). The supernatant was saved for further processing. This was repeated up to two times so that the greatest possible quantity of polysaccharides was extracted. The supernatants obtained were mixed in a beaker and the alginates were removed with polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, St. Louis, MO, USA) reagent. To do this, 2 spatula tips were added to the supernatant, it was stirred and left for 5 min, then centrifuged at 3000 rpm for 5 min, eliminating the polyphenols. Once this was done, the entire supernatant was poured back into a beaker and placed on a tray with ice, 2% (w/v) O-N-cetylpyridinium bromide (Cetavlon) (Merk, Darmstadt, Germany) was added according to Abdala   [78] for the selective precipitation of polysaccharides. The polysaccharides were flocculated after this process and were recovered by centrifugation (4500 rpm, 10 min). The precipitated polysaccharide was purified with 4 M NaCl (Sigma-Aldrich, St. Louis, MO, USA) and the solution was stirred at 60 • C for 30 min. The polysaccharide was flocculated again with 96% (v/v) ethanol (Sigma-Aldrich, St. Louis, MO, USA), centrifuged (4500 rpm, 10 min). The pellet was recovered and placed in a dialysis membrane (Dialysis Tubing Cellulose, Sigma Aldrich St. Louis, MO, USA).The membrane with the polysaccharides was placed in a 0.5 M NaCl solution and shaken overnight at 4 • C. After dialysis, the entire content of the membrane was recovered, and washed with 96% EtOH. The mixture was kept at 4 • C until the flocculation of the polysaccharides had occurred. A final centrifugation pulse (3000 rpm 2 min, room temperature) was applied to recover the polysaccharides, which were then frozen at −80 • C and lyophilized (Lyophilizer Cryodos, Telstar, Terrasa, Spain).

Fourier Transform Infrared Spectroscopy (FT-IR)
To perform the Fourier transform infrared characterization, the spectra were collected in a Thermo Nicolet Avatar 360 IR spectrophotometer (Thermo Electron Inc., Waltham, MA, USA) with a resolution of 4 cm −1 , equipped with a DTGS detector and OMNIC software 7.2 (Thermo Nicolet, Waltham, MA, USA). Baseline adjustment was performed using Thermo Nicolet OMNIC (Thermo Nicolet, Waltham, MA, USA) software to flatten the baseline in each spectrum. For this, a self-supporting disc had been previously made, pressed from the mixture of EgPs-KBr (1% p/p), which was introduced in the spectrophotometer for its characterization.

Antioxidant Activity (ABTS Method) in Polysaccharides and Biomass
The ability of EgPs to scavenge the ABTS radicals was evaluated sing an ABTS assay, as reported by Re et al. (1999) [79], with a few modifications. ABTS radical cation was generated by a reaction of 7 mM ABTS (Sigma-Aldrich, St. Louis, MO, USA) with 2.45 mM potassium persulphate (Sigma-Aldrich, St. Louis, MO, USA). This reaction mixture was stored for 16 h at room temperature and used within two days. After incubation, the mixed solution was diluted to a 0.7 absorbance unit at 413 nm with the deionized water. From a weight of 10 mg of the lyophilized samples (biomass and polysaccharides) dissolved in 1 mL of phosphate, buffer dilutions were made so that the final concentrations in the cuvettes were 12.5, 25, 50, 100, and 200 µg mL −1 . Then, 50 µL of these samples were mixed with 940 µL of phosphate buffer and 10 µL of ABTS solution in the cuvettes. Then, the mixture was determined at 413 nm. ABTS radical scavenging capacity was calculated according to the Equation (2): where, A 0 is the absorbance of the ABTS radical in phosphate buffer at time 0; A 1 is the absorbance of the ABTS radical solution mixed with the sample after 8 min. From a stock of Trolox ® 2.5 mM a calibration curve was made with different concentrations of Trolox ® . The serial dilutions were obtained at concentrations of 5, 10, 15, and 20 µM. All determinations were performed in triplicate (n = 3) [80]. The antioxidant capacity was expressed as % antioxidant activity.

Antioxidant Activity (DPPH Method) in Biomass
The antioxidant and radical scavenging activities of biomass extracts were evaluated by the 2,2-diphenyl-1-picrylhydrazil (DPPH) free radical method of Brand-Williams et al., (1995) [81]. From 1 mg of dry weight of the crushed algae, the biomass extract was obtained, and 1 mL of MeOH at 80% was added. Dilutions were made so that the final concentrations in the cuvettes were 200, 100, 50, 25 and 12.5 µg mL −1 . DPPH (Sigma-Aldrich, St. Louis, MO, USA) (1000 µL), with 80% MeOH and 200 µL sample, was added to the cuvettes. The initial absorbance was measured at 517 nm. The samples were incubated for 30 min and the absorbance was recorded again at 517 nm, using 80% MeOH (Sigma-Aldrich, St. Louis, MO, USA) as the target. The absorbance was then transformed into a percentage inhibition against 80% MeOH.
The percentage of the antioxidant activity was calculated according to Equation (3): where, A 0 is the absorbance at time 0 min and A 1 is the absorbance at the end of the reaction (30 min) at 517 nm. A calibration curve was made with different concentrations of Trolox ® , from a stock of Trolox ® 1.268 mM. Dilutions were obtained at concentrations from 0 to 6 µM. All determinations were performed in triplicate (n = 3) [80]. The antioxidant capacity was expressed as % of antioxidant activity.

Lipopolysaccharides (LPS) Contamination Assay
LPS contamination in EgP was evaluated using the Limulus Endosafe ® -PTS amebocyte lysate (LAL) assay kit (Charles River Laboratories, Charleston, SC, USA) following the manufacturer's instructions. For this, 25 µL of the polysaccharide solution were loaded into each of the four channels of the cartridge. The LAL reagent and sample were automatically mixed by the reader in two channels, just like the sample, with the LAL reagent and positive product in the other two channels, as a control. The channels were incubated and combined with the chromogenic substrate. Following this, the optical densities of all four channels were measured and compared to an internal standard curve. The result of the amount of endotoxin in the sample was expressed as endotoxin units (EU) mL −1 . HCT-116, MCF-7, RAW 264.7, and HGF-1 were routinely grown in Dulbecco's Modified Eagle's Medium (DMEM) (Biowest, Nuaillé, France), supplemented with 10% fetal bovine serum (FBS) (Biowest, Nuaillé, France), 2 mM L-glutamine (Biowest, Nuaillé, France), 5 mL penicillin-streptomycin (Biowest, Nuaillé, France) and 2.5 mL amphotericin B (Biowest, Nuaillé, France). The U-937 and HCl-N460 were cultured in RPMI-1640 medium (Biowest, Nuaillé, France), supplemented with 10% FBS, 2 mM L-glutamine, 5 mL penicillin-streptomycin, and 2.5 mL amphotericin B. In the case of HL-60, 5 mL of penicillin-streptomycin and 2.5 mL of amphotericin B and THP-1 were grown in RPMI-1640 medium, supplemented with 20% FBS, with 0.05 mM, 2-mercaptoethanol (Sigma-Aldrich, St. Luis, MO, USA), 10% FBS, 5 mL penicillin-streptomycin, and 2.5 mL amphotericin B. These were incubated at 37 • C in humidified air with 5% CO 2 . Cultured cells were harvested when confluency reached 80%.

Cytotoxicity Assay MTT with Healthy Cell Line (HGF-1)
MTT assays were performed with HGF-1 cells. The same supplements were used at the same concentrations except for FBS. To resemble what occurs in tumor cells, less cells (very proliferative) were taken and the concentration of FBS used was 10%. In the case of comparing healthy cell behaviour, the number of cells taken was ten times more than the amount previously taken (low proliferating), and the FBS concentration was 2%. With this test, we wanted to observe the behaviour of the cells in different amounts against the treatment used in our study.

Determination of Cytokines with RAW 264.7 Cell Line
RAW 264.7 cells were cultured in the presence of different concentrations of polysaccharides (0-100 µg mL −1 ) in 96-well microplates (5 × 10 4 cells well −1 ) in a total volume of 100 µL. Bacterial LPS (50 ng mL −1 ) was used as a positive control for the activation of macrophages and wells with treated and untreated medium, for 24 h. After this, the culture medium was taken from all the wells and tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were quantified, using the ELISA Ready-SET-Go mouse TNF alpha kit and a mouse IL-6 Ready-SET-Go ELISA (Invitrogen, Thermo Fisher Scientific, Madrid, Spain). following the supplier's instructions.

Determination of Cytokines with Human THP-1 Cell Line
Human THP-1 cells were seeded in 96-well microplates (5 × 10 4 cells per well) and derived into macrophages after a 10 ng mL −1 PMA stimulation for 48 h. Then, wells were washed with fresh medium and exposed to different polysaccharide concentrations for a further 24 h. Human TNF-α present in the conditioned media was quantified by using a Human TNF alpha uncoated ELISA kit (Invitrogen, Thermo Fisher Scientific, Spain).

Proteomics Analysis
To investigate the effects of EgPs on protein expression levels in non-tumor cells, proteomics analysis of the HGF-1 cell line was carried out.

Cell Treatment and Protein Extraction
HGF-1 cells were cultured in DMEM without fetal bovine serum and treated with 320 µg mL −1 of EgPs for 24 h, the highest concentration that produced no cytotoxic effect on cells was noted. After treatments, cells were washed with ice-cold phosphate-buffered saline and solubilized in 100 mM triethylammonium bicarbonate buffer (TEAB)-10% SDS containing Pierce universal nuclease (Thermo Scientific, MA). Then, cells were sonicated, centrifuged at 16,000× g for 10 min at 4 • C, and the supernatant was carefully separated.

In-Solution Tryptic Digestion and 2-Plex Tandem Mass Tag (TMT) Labeling
The protein concentrations of samples were determined by the bicinchoninic acid protein assay and normalized to the same concentration (1 ug µL −1 ). For reduction and alkylation, 5 µL de 200 mM Tris (2-carboxyethyl) phosphine was added and incubated at 55 • C for 1 h. Proteins were alkylated with 60 mM iodoacetamide at room temperature for 30 min, protected from light. Samples were subjected to acetone precipitation to purify proteins by incubating with 6 volumes of ice-cold acetone at −20 • C for 4 h. Precipitated proteins were centrifuged at 8000× g for 10 min at 4 • C and the pellet redissolved in 100 µL of 50 mM TEAB (pH 8.5). Proteins were digested by trypsin (Pierce trypsin protease, Thermo Scientific, MA) at a ratio of 1:50 (trypsin:protein, w/w) by incubating overnight at 37 • C.
For quantitative proteomics, each digested protein sample was labelled with an appropriate TMT2plex Isobaric Label Reagent (Thermo Scientific, MA), according to the protocol supplied by the manufacturer. Briefly, the control and treated peptide samples were labelled with reagents TMT-126 and TMT-127, respectively. To quench the TMT reaction, 8 µL of 5% hydroxylamine was added to each sample and incubated for 15 min at room temperature. All the labelled peptides were combined in equal amounts and dried using a speed vacuum system.

Liquid Chromatography High-Resolution Mass Spectrometry
The peptides were dissolved in 0.1% formic acid and analyzed using a Q-Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, MA, USA) connected to an Easy-nLC 1200 UHPLC (Thermo Scientific, MA, USA). Solvent A and solvent B were 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile, respectively. Peptide separation was performed using a trap column (Acclaim PepMap 100, 75 µm × 2 cm, C18, 3 µm, 100 A, Thermo Scientific, MA, USA) at a flow rate of 20 µL min −1 and eluted onto a 50 cm analytical column (PepMap RSLC C18, 2 µm, 100 A, 75 µm x 50 cm, Thermo Scientific) with a linear gradient of 2% to 20% solvent B (0.1% formic acid in 80% acetonitrile) for 240 min, followed by a 30 min gradient from 20% to 35% solvent B and, finally, to 95% solvent B for 15 min before re-equilibration to 2% solvent B at a constant flow rate of 300 nL min −1 . The peptides were positively ionized using a nanospray ion source and mass spectrometry was conducted in top 20 data-dependent mode with the following settings: 2.2 kV ion spray voltage; 320 • C ion capillary temperature, 350-1500 mass scan range for full MS and 350-1500 scan range for MS/MS; MS resolution 120,000 for full MS and 30,000 for data-dependent MS/MS; collision energy 32 with high-energy collisional dissociation mode; MS/MS isolation window 0.7 m/z; and dynamic exclusion 20 s.

Data Analysis
The peptides and proteins were identified and quantified using Proteome Discoverer TM 2.4 (Thermo Scientific, MA, USA) platform. Sequest HT ® was utilized as the search engine [82], and the Swiss-Prot part of UniProt for Homo sapiens was used as the database, accessed on 29 September 2021. The search parameters were set as follows: two missed tryptic cleavage site was allowed; full MS and MS/MS tolerances were 10 ppm and 0.02 Da, respectively; fixed modification of carbamidomethylation on Cys, variable modification of oxidation on Met, and N-terminal acetylation on protein were specified; proteins required a minimum of two peptides. Peptide spectral matches and consecutive protein assignments were validated using the Percolator ® algorithm [83] by imposing a strict cut-off of 1% false discovery rate. To calculate the ratio of TMT-labelled proteins between treated and control cells, abundances were based on precursor intensities. Normalization was performed based on total peptide amount, and samples were scaled on all average (for every protein and peptide the average of all samples is 100).
The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (https://www. string-db.org, accessed on 13 October 2021) was used to perform a protein-protein interaction networks functional enrichment analysis.
Three technical replicates of each sample were carried out.

Statistical Analysis
The statistical analyses were performed with the software Statistica 7.0. Figures were made using SigmaPlot versión 12.0, 2015 (Systat Software Inc., Chicago, IL, USA). All values were expressed as means ± standard deviation (SD). To evaluate the differences of each treatment with polysaccharides, a one-way ANOVA was conducted. Later on, when significant differences were found, a post-hoc Tukey HSD test was conducted to compare the means between each treatment. Differences were considered to be statistically significant when p < 0.05.
In proteomics analysis, the normalized and scaled relative abundance of every protein was expressed as mean ± standard deviation of four biological replicates. Protein ratios were directly calculated from the grouped protein abundances. Abundance ratio p-values were calculated by ANOVA, based on the abundance of individual proteins. Only proteins with ANOVA p < 0.05 and Log 2 fold change > 0.3 were considered as significantly deregulated.

Conclusions
The present study concludes that EgPs have potential anticancer, immunomodulatory and antioxidant capacity, in addition to overexpressing five proteins that interact with each other and with TNF-α and IL-6. As this seaweed is a natural product, and its extracted polysaccharides do not present side effects, at least as far as this study has been able to determine, it should be considered a potential nutraceutical.