FTIR Characterization of Sulfated Polysaccharides Obtained from Macrocystis integrifolia Algae and Verification of Their Antiangiogenic and Immunomodulatory Potency In Vitro and In Vivo

The aim of this study was to evaluate the antiangiogenic and immunomodulatory potential of sulfated polysaccharides from the marine algae Macrocystis integrifolia characterized by FTIR. The cytotoxicity of sulfated polysaccharides was evaluated using the 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. Antiangiogenic activity was evaluated using the chicken chorioallantoic membrane (CAM) assay. Immunomodulatory activity was determined on macrophage functionality and allergic response. The results showed that sulfated polysaccharides significantly decreased angiogenesis in chicken chorioallantoic membranes (p < 0.05). Likewise, they inhibited in vivo chemotaxis and in vitro phagocytosis, the transcription process of genes that code the enzymes cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and nitric oxide synthase-2 (NOS-2) and the nuclear factor kappa-light chain enhancer of activated B cells (NF-κB), showing immunomodulatory properties on the allergic response, as well as an in vivo inhibitory effect in the ovalbumin-induced inflammatory allergy model (OVA) and inhibited lymphocyte proliferation specific to the OVA antigen in immunized mice. Finally, these compounds inhibited the histamine-induced skin reaction in rats, the production of immunoglobulin E (IgE) in mice, and the passive response to skin anaphylaxis in rats. Therefore, the results of this research showed the potential of these compounds to be a promising source for the development of antiangiogenic and immunomodulatory drugs.


Introduction
Cancer affects humanity and is currently considered to be one of the main global public health problems. In 2020, it resulted in 1.4 million deaths, 47% of them aged 69 years or younger. The number of cancer cases in the Region of the Americas was estimated to be 4 million in 2020 and is expected to increase up to 6 million by 2040 [1,2]. However, the lack of specificity of anticancer drugs, their harmful side effects, and the appearance of resistance to chemotherapy, led to the appearance of new treatment strategies, such as antiangiogenic therapy. This was based on the inhibition of tumor vascularization (tumor Likewise, the MIF2 and MIF4 fractions are of very low intensity, which suggests that there is only a low quantity. The absorption of the bands around 1270-1220 cm −1 of the spectra of the fractions MIF1 (1261 cm −1 ), MIF2 (1262 cm −1 ), MIF3 (1261 cm −1 ) and MIF4 (1262 cm −1 ), is related to the asymmetric stretching of the vibration of the sulfate group (S=O), a characteristic component of fucoidans [18,19]. The MIF3 fraction presents a very low intensity, which postulated its existence in a low quantity. Likewise, the MIF4 fraction (581 cm −1 ), presents an additional band of symmetrical stretching vibration of the sulfate group at around 600-570 cm −1 .The bands around the 1076-1030 cm −1 of the fractions MIF1 (1037 cm −1 ), MI F3 (1076 cm −1 , 1050 cm −1 , 1030 cm −1 ), MI F4 (1056 cm −1 , 1032 cm −1 ), correspond to symmetrical stretching vibrations characteristic of the hemiacetal bond of the sugar ring (C-O-C). In the MIF3 fraction, more absorption peaks are observed compared to the other fractions, indicating that it presents more types of sugar.
related to the C-H stretching of the pyranoid rings. The intense bands around 1700 cm −1 , 1600 cm −1 in the spectra of fractions MIF1(1612 cm −1 ), MIF2 (1628 cm −1 ), MIF3 (110 cm −1 ) and MIF4 (1627 cm −1 ) are attributed to the stretching vibration of the carboxyl or acetyl group bonds (C=O), due to the presence of uronic acids, which mainly derived from the residues of guluronic and mannuronic acid (C=O) [17]. Likewise, the MIF2 and MIF4 fractions are of very low intensity, which suggests that there is only a low quantity. The absorption of the bands around 1270-1220 cm −1 of the spectra of the fractions MIF1 (1261 cm −1 ), MIF2 (1262 cm −1 ), MIF3 (1261 cm −1 ) and MIF4 (1262 cm −1 ), is related to the asymmetric stretching of the vibration of the sulfate group (S=O), a characteristic component of fucoidans [18,19]. The MIF3 fraction presents a very low intensity, which postulated its existence in a low quantity. Likewise, the MIF4 fraction (581 cm −1 ), presents an additional band of symmetrical stretching vibration of the sulfate group at around 600-570 cm −1 .The bands around the 1076-1030 cm −1 of the fractions MIF1 (1037 cm −1 ), MI F3 (1076 cm −1 , 1050 cm −1 , 1030 cm −1 ), MI F4 (1056 cm −1 , 1032 cm −1 ), correspond to symmetrical stretching vibrations characteristic of the hemiacetal bond of the sugar ring (C-O-C). In the MIF3 fraction, more absorption peaks are observed compared to the other fractions, indicating that it presents more types of sugar.

Chemical Composition of M. integrifolia Polysaccharide Fractions
The main components of brown algae are polysaccharides, where the sulfated polysaccharides constitute a very common type of structural polysaccharide, based mainly on sulfated L-fucose (Fuc), with less than 10% of the other monosaccharides such as mannose

Evaluation of Antiangiogenic Activity Using Chorioallantoic Membrane (CAM) Assay
In vivo CAM assay is a very effective and popular method to evaluate the antiangiogenic potency of natural products such as plant extracts. In addition, it is an alternative animal model that is adopted to screen different substances comprising cosmetics or natural products such as essential oils, sulphated polysaccharides, or extracts [25]. The probable antiangiogenic effect of the four sulfated polysaccharide fractions MIF1, MIF2, MIF3 and MIF4 was determined using different concentrations for each, namely, 10, 50 and 100 µg/mL, in addition to the normal control. CAM is a vascular chick membrane that is composed of two mesodermal layers: chorionic and allantois epithelium. CAM transverse sections displayed allantoic epithelium, chorionic epithelium showing small blood vessels and mesenchyme revealing medium-size blood vessels [26].
The microvascular density analysis illustrated that the number of vessels/9000 µm 2 on CAM was 10.4 ± 0.15 for control, 4.8 ± 0.2, 7.2 ± 0.25, 8.0 ± 0.15 and 9.2 ± 0.2 for MIF1, MIF2, MIF3 and MI F4 at 100 µg/mL, respectively. At 200 µg/mL, values ranged between 3.6 ± 0.3, 6.8 ± 0.25, 7.2 ± 0.1 and 8.8 ± 0.15, respectively. Furthermore, in MIF1, MIF2, MIF3 and MI F4 revealed 2.5 ± 0.32, 6.2 ± 0.2, 6.9 ± 0.33 and 7.5 ± 0.4 to be the number of vessels/9000 µm 2 on CAM at 400 µg/mL. Thus, from the results illustrated in Figure 2, it was clear that the studied fractions showed antiangiogenic activity in vivo in the chicken chorioallantoic membrane (CAM) model, showing a statistically significant decrease (p < 0.05) in the number of average blood vessels, where the MIF1 fraction showed the most promising activity at all tested doses. This coincides with previous publications verifying the use of the same model that sulfated polysaccharides decrease capillary density in vivo in addition to inhibiting tubulogenesis in rabbit aortic endothelial cells (RAEC), further consolidating the antiangiogenic effect of these compounds [27,28]. Likewise, the MIF1 fraction presents a higher percentage of sulfates (33.2%) and a lower molecular weight (49.2 kDa) compared to the other fractions. Previous studies noted that brown algae fucoidans possess various biological activities, including antiangiogenic activities [29,30], and this could be attributed to the degree of sulfation, which significantly influences the antiangiogenic activity level of fucoidans [31], which, in turn, could significantly suppress the mitogenic and chemotactic actions of vascular endothelial growth factor 165 (VEGF 165 ) by preventing VEGF 165 from binding to its cell surface receptor [32]. Furthermore, various investigations mentioned that fucoidans with MW > 30 kDa and a high degree of sulfation have an antiangiogenic effect [33,34].
in the number of average blood vessels, where the MIF1 fraction showed the most promising activity at all tested doses. This coincides with previous publications verifying the use of the same model that sulfated polysaccharides decrease capillary density in vivo in addition to inhibiting tubulogenesis in rabbit aortic endothelial cells (RAEC), further consolidating the antiangiogenic effect of these compounds [27,28]. Likewise, the MIF1 fraction presents a higher percentage of sulfates (33.2%) and a lower molecular weight (49.2 kDa) compared to the other fractions. Previous studies noted that brown algae fucoidans possess various biological activities, including antiangiogenic activities [29,30], and this could be attributed to the degree of sulfation, which significantly influences the antiangiogenic activity level of fucoidans [31], which, in turn, could significantly suppress the mitogenic and chemotactic actions of vascular endothelial growth factor 165 (VEGF165) by preventing VEGF165 from binding to its cell surface receptor [32]. Furthermore, various investigations mentioned that fucoidans with MW > 30 kDa and a high degree of sulfation have an antiangiogenic effect [33,34]. Data are presented as mean ± SD (n = 3). * indicates statistically significant differences (p < 0.05) between the treatments and the control, determined with the ANOVA statistic. Lowercase letters indicate statistical differences between the concentrations of the same fraction and uppercase letters indicate the statistical differences between fractions (p < 0.05) with the Tukey multirank test.

Determination of Cytotoxicity by MTT Assay
In the present study, the cell viability of fractions of sulfated polysaccharides from Macrocystis integrifolia algae (fractions MIF1, MIF2, MIF3 and MIF4) was evaluated using the MTT method in secondary cultures of chicken embryo fibroblasts (SCEFP) (Figure 3). The MIF1 fraction was observed to have a higher percentage of cell viability than all fractions at tested concentrations ranging from 25 to 1000 μg/mL, which coincides with other studies, where it is observed that sulfated polysaccharide compounds do not present significant toxicity in vitro, further reflecting their relative safety [12]. Data are presented as mean ± SD (n = 3). * indicates statistically significant differences (p < 0.05) between the treatments and the control, determined with the ANOVA statistic. Lowercase letters indicate statistical differences between the concentrations of the same fraction and uppercase letters indicate the statistical differences between fractions (p < 0.05) with the Tukey multirank test.

Determination of Cytotoxicity by MTT Assay
In the present study, the cell viability of fractions of sulfated polysaccharides from Macrocystis integrifolia algae (fractions MIF1, MIF2, MIF3 and MIF4) was evaluated using the MTT method in secondary cultures of chicken embryo fibroblasts (SCEFP) (Figure 3). The MIF1 fraction was observed to have a higher percentage of cell viability than all fractions at tested concentrations ranging from 25 to 1000 µg/mL, which coincides with other studies, where it is observed that sulfated polysaccharide compounds do not present significant toxicity in vitro, further reflecting their relative safety [12].

In vivo Cellular Chemotaxis Assay
Thioglycolate-induced peritonitis (TIP) is a well-established model that was adopted to evaluate the acute chemotactic responses of immune cells to the peritoneal cavity [35,36], reaching an accumulation of 25 × 10 6 cell/mL ( Figure 4A), where more than 80% of the cell population was macrophages ( Figure 4B). As a result of the ip administration of the sulfated polysaccharide fractions at 10, 50 and 100 mg/kg, a significant reduction (p < 0.05) in the total number of cells that migrated to the peritoneum in response to the inflammatory stimulus was evidenced ( Figure 4A). It was clear that MIF1, MIF2, MIF3, and MIF4 reduced the total number of cells in the peritoneal exudate by 66%, 59%, 48% and 44%, respectively, at a dose of 10 mg/kg, compared to the control group that received thioglycolate. Meanwhile, at a dose of 50 mg/kg, they showed a reduction of 76%, 68%, 72%, and 46%, respectively, in the total number of cells in the peritoneal exudate relative to the control group. However, the greatest reduction was observed at a dose of 100 mg/kg, where MIF1, MIF2, MIF3, and MIF4 showed a reduction of 83%, 79%, 68%, 60%, respectively, relative to the control group ( Figure 4A). The reduction in the number of total peritoneal cells corresponded to a reduction in the number of macrophages, since the proportion of these in the total cell population remained constant ( Figure 1B); that is, the reduction was not at the expense of a population of peritoneal cells other than macrophages. The illustrated results showed that the MIF1 fraction showed the greatest inhibition of cellular chemotaxis with statistically significant differences (p < 0.05), evidenced by the reduced number of cells in the peritoneal exudate, as well as the reduced percentage of macrophages in the total cell population. It should be noted that macrophages play an important role in the immune response, so overstimulation could cause tissue failure and even necrosis [37]. Sulfated polysaccharides such as fucoidans were investigated in experimental models, showing immunomodulatory effects [38,39]. Indeed, fucoidan from F. vesiculosus induces macrophage activation [40]. Furthermore, fucoidan of Undaria pinnatifitida, an edible seaweed, may increase inflammation by increasing the production of proinflammatory cytokines [41]. In this sense, the evidence affirms a relationship between the structure and immunomodulatory effects of fucoidan, where low-molecular-weight fucoidan presents more favorable bioactivity in vitro and in vivo studies compared to highmolecular-weight fucoidan [42][43][44].

. In vivo Cellular Chemotaxis Assay
Thioglycolate-induced peritonitis (TIP) is a well-established model that was adopted to evaluate the acute chemotactic responses of immune cells to the peritoneal cavity [35,36], reaching an accumulation of 25 × 10 6 cell/mL ( Figure 4A), where more than 80% of the cell population was macrophages ( Figure 4B). As a result of the ip administration of the sulfated polysaccharide fractions at 10, 50 and 100 mg/kg, a significant reduction (p < 0.05) in the total number of cells that migrated to the peritoneum in response to the inflammatory stimulus was evidenced ( Figure 4A). It was clear that MIF1, MIF2, MIF3, and MIF4 reduced the total number of cells in the peritoneal exudate by 66%, 59%, 48% and 44%, respectively, at a dose of 10 mg/kg, compared to the control group that received thioglycolate. Meanwhile, at a dose of 50 mg/kg, they showed a reduction of 76%, 68%, 72%, and 46%, respectively, in the total number of cells in the peritoneal exudate relative to the control group. However, the greatest reduction was observed at a dose of 100 mg/kg, where MIF1, MIF2, MIF3, and MIF4 showed a reduction of 83%, 79%, 68%, 60%, respectively, relative to the control group ( Figure 4A). The reduction in the number of total peritoneal cells corresponded to a reduction in the number of macrophages, since the proportion of these in the total cell population remained constant ( Figure 1B); that is, the reduction was not at the expense of a population of peritoneal cells other than macrophages. The illustrated results showed that the MIF1 fraction showed the greatest inhibition of cellular chemotaxis with statistically significant differences (p < 0.05), evidenced by the reduced number of cells in the peritoneal exudate, as well as the reduced percentage of macrophages in the total cell population. It should be noted that macrophages play an important role in the immune response, so overstimulation could cause tissue failure and even necrosis [37]. Sulfated polysaccharides such as fucoidans were investigated in experimental models, showing immunomodulatory effects [38,39]. Indeed, fucoidan from F. vesiculosus induces macrophage activation [40]. Furthermore, fucoidan of Undaria pinnatifitida, an edible seaweed, may increase inflammation by increasing the production of pro-inflammatory cytokines [41]. In this sense, the evidence affirms a relationship between the structure and immunomodulatory effects of fucoidan, where low-molecular-weight fucoidan presents more favorable bioactivity in vitro and in vivo studies compared to high-molecular-weight fucoidan [42][43][44]. Values represent the mean ± SD (n = 3). * indicates statistically significant differences (p < 0.05) between the treatments and the control, determined with the ANOVA statistic. Lowercase letters indicate statistical differences between the concentrations of the same fraction and uppercase letters indicate statistical differences between fractions (p < 0.05) with the Tukey multi-rank test.

In Vitro Assay to Determine Phagocytic Activity
Phagocytosis is one of the main functions of macrophages and, to a certain extent, reflects the state of immune function within the body. They can remove cellular debris and pathogenic organisms to maintain body homeostasis. Previous studies have shown that sulphated polysaccharides can effectively control immunity by regulating macrophage phagocytosis [45]. The results illustrated in Figure 5 showed that different fractions of sulfated polysaccharides MIF1, MIF2, MIF3 and MIF4 inhibited phagocytic activity in a dose-dependent manner relative to the control group, which received normal saline. MIF1, MIF2, MIF3 and MIF4 reduced cell phagocytosis by 47.9%, 41.1%, 33.9% and 21.4% respectively at a dose of 10 mg/kg compared to the control group, while, at a dose of 50 mg/kg, they showed reductions of 53.6%,49.6%,42.9% and 39.3%, respectively, in cell phagocytosis with respect to the control group. However, the greatest reduction in cell phagocytosis was observed at a dose of 100 mg/kg, where MIF1, MIF2, MIF3 and MIF4 showed 64.3%, 57.1%, 50.8% and 48.2% reductions, respectively, compared to the control group ( Figure 5). The results presented in Figure 5 also highlighted that the MIF1 fraction showed the most significant potency in the inhibition of cell phagocytosis, which coincides with the work of Tavarsa et al., who showed that sulphated polysaccharides could Values represent the mean ± SD (n = 3). * indicates statistically significant differences (p < 0.05) between the treatments and the control, determined with the ANOVA statistic. Lowercase letters indicate statistical differences between the concentrations of the same fraction and uppercase letters indicate statistical differences between fractions (p < 0.05) with the Tukey multi-rank test.

In Vitro Assay to Determine Phagocytic Activity
Phagocytosis is one of the main functions of macrophages and, to a certain extent, reflects the state of immune function within the body. They can remove cellular debris and pathogenic organisms to maintain body homeostasis. Previous studies have shown that sulphated polysaccharides can effectively control immunity by regulating macrophage phagocytosis [45]. The results illustrated in Figure 5 showed that different fractions of sulfated polysaccharides MIF1, MIF2, MIF3 and MIF4 inhibited phagocytic activity in a dose-dependent manner relative to the control group, which received normal saline. MIF1, MIF2, MIF3 and MIF4 reduced cell phagocytosis by 47.9%, 41.1%, 33.9% and 21.4% respectively at a dose of 10 mg/kg compared to the control group, while, at a dose of 50 mg/kg, they showed reductions of 53.6%,49.6%,42.9% and 39.3%, respectively, in cell phagocytosis with respect to the control group. However, the greatest reduction in cell phagocytosis was observed at a dose of 100 mg/kg, where MIF1, MIF2, MIF3 and MIF4 showed 64.3%, 57.1%, 50.8% and 48.2% reductions, respectively, compared to the control group ( Figure 5). The results presented in Figure 5 also highlighted that the MIF1 fraction showed the most significant potency in the inhibition of cell phagocytosis, which coincides with the work of Tavarsa et al., who showed that sulphated polysaccharides could improve the inflammatory condition of macrophages through the inhibition of phagocytosis [28]. In addition, a study indicated that excess phagocytosis decreased after treatment with Fucus vesiculosus fucoidan, and that this compound could regulate phagocytosis by modulating TNF-α production [46]. improve the inflammatory condition of macrophages through the inhibition of phagocytosis [28]. In addition, a study indicated that excess phagocytosis decreased after treatment with Fucus vesiculosus fucoidan, and that this compound could regulate phagocytosis by modulating TNF-α production [46]. . The * indicates statistically significant differences (p < 0.05) between the treatments and the control, determined with the ANOVA statistic. Lowercase letters indicate statistical differences between the concentrations of the same fraction and uppercase letters indicate statistical differences between fractions (p < 0.05) with the Tukey multi-rank test.

Determination of the Expression of Genes Encoding Enzymes, Cytokines and Immune Response Transcription Factors
Determination of mRNA Levels of NOS-2, COX-1, COX-2, NF-κB and IκB NO production requires nitric oxide synthase (NOS), where various inflammatory stimuli can trigger the expression of inducible NOS (iNOS), in different types of cells, such as macrophages [47]. Here, NO production was evaluated by RT-PCR determination of iNOS mRNA expression after the addition of sulfated polysaccharide fractions MIF1, MIF2, MIF3 and MIF4 at different doses, namely, 10, 50 and 100 μg/mL. The results illustrated in Figure 6 showed that the sulfated polysaccharides of different fractions significantly reduced (p < 0.05) the mRNA expression levels in a dose-dependent manner, which, when produced in excess, can cause diseases such as rheumatoid arthritis, diabetes mellitus, atherosclerosis, as well as septic shock [48,49]. At a dose of 10 μg/mL, MIF1, MIF2, MIF3 and MIF4 reduced iNOS mRNA expression, as evidenced by the reduction in optical density by 83%, 59%, 58%, and 56%, respectively, relative to the control group. Meanwhile, at 50 μg/mL, they showed 88%, 61%, 60% and 57% reductions in iNOS mRNA expression, respectively, compared to the control group, while the highest reduction was observed at 100 μg/mL, showing 92%, 68%, 65% and 63% with respect to the control group, where MIF1 showed the highest inhibitory activity at all tested doses. This is supported by an investigation using fucoidan extracted from Sargassum fusiforme, where the results demonstrated the inhibition of NO production and PGE2 by down-regulating iNOS and COX-2 [50]. Furthermore, a fucose-rich polysaccharide fraction of Chnoospora minima also decreased the production of PGE2, TNF-α, IL−1β, IL-6, NO, iNOS, and COX-2 expression in a dose-dependent manner. This inhibition of pro-inflammatory cytokine production is a crucial mechanism to control inflammation [51]. The * indicates statistically significant differences (p < 0.05) between the treatments and the control, determined with the ANOVA statistic. Lowercase letters indicate statistical differences between the concentrations of the same fraction and uppercase letters indicate statistical differences between fractions (p < 0.05) with the Tukey multi-rank test.

Determination of the Expression of Genes Encoding Enzymes, Cytokines and Immune Response Transcription Factors
Determination of mRNA Levels of NOS-2, COX-1, COX-2, NF-κB and IκB NO production requires nitric oxide synthase (NOS), where various inflammatory stimuli can trigger the expression of inducible NOS (iNOS), in different types of cells, such as macrophages [47]. Here, NO production was evaluated by RT-PCR determination of iNOS mRNA expression after the addition of sulfated polysaccharide fractions MIF1, MIF2, MIF3 and MIF4 at different doses, namely, 10, 50 and 100 µg/mL. The results illustrated in Figure 6 showed that the sulfated polysaccharides of different fractions significantly reduced (p < 0.05) the mRNA expression levels in a dose-dependent manner, which, when produced in excess, can cause diseases such as rheumatoid arthritis, diabetes mellitus, atherosclerosis, as well as septic shock [48,49]. At a dose of 10 µg/mL, MIF1, MIF2, MIF3 and MIF4 reduced iNOS mRNA expression, as evidenced by the reduction in optical density by 83%, 59%, 58%, and 56%, respectively, relative to the control group. Meanwhile, at 50 µg/mL, they showed 88%, 61%, 60% and 57% reductions in iNOS mRNA expression, respectively, compared to the control group, while the highest reduction was observed at 100 µg/mL, showing 92%, 68%, 65% and 63% with respect to the control group, where MIF1 showed the highest inhibitory activity at all tested doses. This is supported by an investigation using fucoidan extracted from Sargassum fusiforme, where the results demonstrated the inhibition of NO production and PGE 2 by down-regulating iNOS and COX-2 [50]. Furthermore, a fucose-rich polysaccharide fraction of Chnoospora minima also decreased the production of PGE 2 , TNF-α, IL−1β, IL-6, NO, iNOS, and COX-2 expression in a dose-dependent manner. This inhibition of pro-inflammatory cytokine production is a crucial mechanism to control inflammation [51]. Similarly, the results represented in Figure 7A, 7B and 7C showed that the sulfated polysaccharides revealed a more selective inhibition of COX−1 and COX-2, since they inhibit the levels of COX-1 and COX-2 mRNA at concentrations of 10, 50 and 100 μg/mL, in a dose-dependent manner. Therefore, at a dose of 100 μg/mL, MIF1, MIF2, MIF3 and MIF4 reduced the expression of COX−1 mRNA, evidenced by reductions in optical density of 66.7%, 65.0%, 63.3% and 60.0%, respectively, relative to the control group, approaching that of dexamethasone, which revealed a reduction of 58.4% at 40 μg/mL. Similarly, they reduced COX-2 mRNA expression by 82.1, 65.2, 64.1, and 70.2%, respectively, relative to the control group, in a similar manner to that of dexamethasone, which revealed a reduction of 85.2% at 40 μg/mL, where MIF1 revealed the highest inhibitory potential at all examined doses. This coincides with in silico studies of fucoidan-type sulfated polysaccharides, which turned out to be potent inhibitors of COX-1 and COX-2, and their effect may be greater than that of alginates [52]. Another study showed that Fucus vesiculosus fucoidan showed the better inhibition of both isoforms of the COX enzyme compared to indomethacin; the inhibition of COX-2 was higher than COX1 [53]. Furthermore, fucoidan fractions with a low molecular weight and high sulfate content showed the downregulation of iNOS and COX-2 [54]. In addition, a commercial fucoidan from Undaria pinnatifida (with high sulfate and fucose content and unknown Mw) decreased the expression of COX-2 [55]. The evidence points to the fact that molecular weight and sulfate content play a significant role in COX inhibition [56], and fucoidan can down-regulate IL−1β-induced COX-2 expression, confirming its anti-inflammatory potential [53]. Similarly, the results represented in Figure 7A−C showed that the sulfated polysaccharides revealed a more selective inhibition of COX-1 and COX-2, since they inhibit the levels of COX-1 and COX-2 mRNA at concentrations of 10, 50 and 100 µg/mL, in a dose-dependent manner. Therefore, at a dose of 100 µg/mL, MIF1, MIF2, MIF3 and MIF4 reduced the expression of COX-1 mRNA, evidenced by reductions in optical density of 66.7%, 65.0%, 63.3% and 60.0%, respectively, relative to the control group, approaching that of dexamethasone, which revealed a reduction of 58.4% at 40 µg/mL. Similarly, they reduced COX-2 mRNA expression by 82.1, 65.2, 64.1, and 70.2%, respectively, relative to the control group, in a similar manner to that of dexamethasone, which revealed a reduction of 85.2% at 40 µg/mL, where MIF1 revealed the highest inhibitory potential at all examined doses. This coincides with in silico studies of fucoidan-type sulfated polysaccharides, which turned out to be potent inhibitors of COX-1 and COX-2, and their effect may be greater than that of alginates [52]. Another study showed that Fucus vesiculosus fucoidan showed the better inhibition of both isoforms of the COX enzyme compared to indomethacin; the inhibition of COX-2 was higher than COX1 [53]. Furthermore, fucoidan fractions with a low molecular weight and high sulfate content showed the downregulation of iNOS and COX-2 [54]. In addition, a commercial fucoidan from Undaria pinnatifida (with high sulfate and fucose content and unknown Mw) decreased the expression of COX-2 [55]. The evidence points to the fact that molecular weight and sulfate content play a significant role in COX inhibition [56], and fucoidan can down-regulate IL−1β-induced COX-2 expression, confirming its anti-inflammatory potential [53]. COX-2 mRNA levels, respectively, quantified by densitometric analysis, in all cases expressed with respect to the value obtained for GADPH on the same gel. The bars show the mean ±SD (n = 3). * indicates statistically significant differences (p < 0.05) between the treatments and the control, determined with the ANOVA statistic. Lowercase letters indicate statistical differences between the concentrations of the same fraction and uppercase letters indicate statistical differences between fractions (p < 0.05) with the Tukey multi-rank test.
Furthermore, the sulfated polysaccharides showed that, at the tested doses of 10, 50 and 100 µg/mL they inhibited the transcription of the genes encoding the NF-κB/IκB complex evaluated by RT-PCR, particularly MIF1. This revealed the highest inhibitory potential in all doses examined with 70%, 81% and 92% at 10, 50 and 100 µg/mL, respectively, with a p < 0.05. Likewise, M1F1 at 100 µg/mL showed a greater reduction compared to dexamatasone (40 µg/mL), which caused a reduction of 90% ( Figure 8A, 8B and 8C). This indicated that sulfated polysaccharides inhibit the transcriptional activation of genes controlled by this transcription factor, most of which are involved in the immunoinflammatory response. It is important to note that the inhibition of NF-κB can be useful in the treatment of acute and chronic inflammatory diseases. Many anti-inflammatory therapies aim to block NF-κB activity; in this sense, an investigation using Fucoidan purified from Fucus vesiculosus exhibited anti-inflammatory activity by suppressing the NF-κB pathway, suggesting a down-regulation of pro-inflammatory mediators [57]. Furthermore, evidence has shown that when Toll-like receptors such as TLR2 and TLR4 are inhibited, there Furthermore, the sulfated polysaccharides showed that, at the tested doses of 10, 50 and 100 µg/mL they inhibited the transcription of the genes encoding the NF-κB/IκB complex evaluated by RT-PCR, particularly MIF1. This revealed the highest inhibitory potential in all doses examined with 70%, 81% and 92% at 10, 50 and 100 µg/mL, respectively, with a p < 0.05. Likewise, M1F1 at 100 µg/mL showed a greater reduction compared to dexamatasone (40 µg/mL), which caused a reduction of 90% ( Figure 8A-C). This indicated that sulfated polysaccharides inhibit the transcriptional activation of genes controlled by this transcription factor, most of which are involved in the immunoinflammatory response. It is important to note that the inhibition of NF-κB can be useful in the treatment of acute and chronic inflammatory diseases. Many anti-inflammatory therapies aim to block NF-κB activity; in this sense, an investigation using Fucoidan purified from Fucus vesiculosus exhibited anti-inflammatory activity by suppressing the NF-κB pathway, suggesting a down-regulation of pro-inflammatory mediators [57]. Furthermore, evidence has shown that when Toll-like receptors such as TLR2 and TLR4 are inhibited, there is a reduction in NF-κB transcriptional activities [58]; notably, the MyD88 gene, which provides instructions for making a protein involved in signaling within immune cells, showed a decrease in pro-inflammatory cytokine secretion and the NF-κB transcriptional activities of NF-κB [59].
Mar. Drugs 2023, 21, 36 11 of 21 is a reduction in NF-κB transcriptional activities [58]; notably, the MyD88 gene, which provides instructions for making a protein involved in signaling within immune cells, showed a decrease in pro-inflammatory cytokine secretion and the NF-κB transcriptional activities of NF-κB [59].  ; (B,C) IκB and NF-κB mRNA levels, respectively, quantified by densitometric analysis, expressed with respect to the parKa GADPH value obtained on the same gel. The bars show the mean ± SD (n = 3). * indicates statistically significant differences (p < 0.05) between the treatments +and the control, determined with the ANOVA statistic. Lowercase letters indicate statistical differences between the concentrations of the same fraction and uppercase letters indicate statistical differences between fractions (p < 0.05) with the Tukey multi-rank test.

Ovalbumin (OVA) Induced Allergic Inflammation in Vivo Model
Ovalbumin is a glycoprotein in nature and constitutes the main protein components of the chicken egg whites. It is complex and large enough to elicit a mild immune reaction and, therefore, is commonly used as a T-cell-dependent antigen to trigger immunization in scientific research [60]. The results illustrated in Table 2 showed that the fractions tested for sulfated polysaccharides elicited a pronounced anti-inflammatory and anti-allergic behavior, as evidenced by the reveal of a significant reduction in plantar edema induced by Ovalbumin (OVA) at a dose of 100 mg/kg. The control group immunized with Ovalbumin (OVA) showed a marked increase in plantar edema, estimated at 95.6%, compared to the normal group that did not receive immunization. However, MIF1, MIF2, MIF3, and MIF4 treatment significantly reduced plantar edema, by 56, 27, 23 and 18%, respectively, compared to the ovalbumin immunized group. It is worth highlighting that MIF1 exerted anti-inflammatory and antiallergic behavior close to that of 5 mg/kg indomethacin, which caused a 57% reduction in ovalbumin-induced plantar edema. This is in accordance with an investigation that found that Saccharina japonica F-fucoidan showed an inhibitory effect on ear edema, finding that this effect depends on galectin-9, a suppressor of T and B cells, which prevents the binding of IgE to the IgE receptor FcεRI, inhibiting mast cell activation, which are regulatory cells of inflammatory and allergic processes [61].

Histamine Skin Reaction
Histamine causes an increase in capillary and post-capillary venular permeability that ultimately leads to the wheal-flare response [62] reflected in the elevation of extravasated Evans blue in the histamine skin reaction assay. The results illustrated in Table 3 showed that tested fractions of sulfated polysaccharides also inhibited histamine release in vivo. Treatment with MIF1, MIF2, MIF3 and MIF4 significantly reduced histamine-induced skin reaction by 66.9%, 57.6%, 44.9% and 16.3%, respectively, with respect to the control group that received histamine solution only. However, promethazine treatment showed a 95.6% reduction in histamine-induced skin reaction (Table 3). In this context, the Porphyra haitanensis sulfated polysaccharide significantly inhibited histamine levels, also finding that IL-4, IL-5 and IL-13 levels decreased when co-cultured with the sulfated polysaccharide [63]. Furthermore, fucoidan from Undaria pinnatifida decreased histamine and IgE levels. In this sense, fucoidan suppresses Th-2 cytokines, such as IL-4, IL-5 and IL-13, as IgE production, and increases IgG2 production to suppress allergic reactions [64]. Table 3. Effect of pretreatment for seven days with fractions of sulfated polysaccharides and promethazine on histamine-induced skin reaction in rats. Additionally, the results illustrated in Table 4 showed that the fractions tested for sulfated polysaccharides caused a significant reduction in the serum IgE levels of OVAimmunized mice, as estimated by the passive cutaneous anaphylaxis test (PCA). Treatment with 50 mg/kg of MIF1, MIF2, MIF3, and MIF4 showed a notable reduction in serum IgE estimated by 54.4, 44.1, 41.0 and 43.0%, respectively, compared to the control group that receives only OVA, while ketotifen, at a dose of 3 mg/kg, showed a reduction of 66.2% in serum IgE with respect to the control group. Thus, it can be concluded that these compounds are capable of inhibiting the allergic inflammatory response, which coincides with a previous investigation [65]. In addition, low-molecular-weight fucoidan, which is rich in fucose and sulfate from Laminaria japonica, decreased allergen-specific IgE, proving that low-molecular-weight fucoidan had a significant effect on cytokine expression by enhancing the production of IL-2, IL-4 and IFN-γ, favoring TH1 differentiation, and decreases IgE, so these compounds may be useful in counteracting the allergic response because they act as adjuvants to enhance the immune-response-specific antigen [66].

Collection and Identification of Biological Material
Five kilograms of Macrocystis integrifolia algae were collected from the coastal waters of San Nicolás Bay, Marcona district, Ica region, Peru. To preserve algae during collection, the samples were immediately packed with ice and transported to the laboratory, where they were washed with distilled water, cut into small pieces and dried in a forced-air-circulation oven at 40 • C. Species identfication was carried out by the Herbarium Truxillense (HUT) with the deposit voucher HUT 25819.

Chemicals
In this investigation, all the chemicals used were of analytical grade and all were obtained from Sigma-Aldrich (St. Louis, MI, USA)).

Extraction of Sulfated Polysaccharides
One kilogram of dry ground seaweed was taken and extracted with distilled water at 55 • C, with continuous stirring for 4 h. The tissue was removed by simple filtration and the filtrate was centrifuged to remove the sediment until clear solutions were obtained. The clarified solution was precipitated with distilled ethanol and the solid was placed in an electric oven at 50 • C. Subsequently, the solid was subjected to fractionation by ion exchange chromatography, using a column (diameter = 2.2 cm, height = 20 cm) packed with 40 g of diethylaminoethylcellulose (DEAE-C), suspended in 1N Tris-HCl with a pH of 8.3. The extract rich in sulfated polysaccharides (1 g·15 mL −1 ) was dissolved in distilled water and incorporated into the column, using 5 volumes of NaCl (250 mL) at a concentration gradient of 0.5 M, 1.0 M and 2.0 M, and about 40 10 mL of eluates were collected. The eluates that showed a higher concentration was pooled and precipitated with three volumes of ethanol per 24 h. The supernatant was recovered by centrifugation (3000 rpm for 10 min), salt-purified by three consecutive washes with distilled water (10 mL), and precipitated again with three volumes of EtOH (30 mL). The supernatant of each fraction was recovered by centrifugation (3000 rpm for 10 min), dried in its entirety in an oven at 45 • C and stored in a vial [67][68][69]. Four fractions were obtained: MIF1 (350 mg), MIF2 (280 mg), MIF3 (400 mg), MIF4 (430 mg).

Fourier Transform Infrared (FTIR) Spectroscopic Analysis
Infrared spectra were obtained using a PerkinElmer TWO spectrophotometer equipped with a total reflectance (FTIR) attenuator. Each spectrum was taken in the spectral range of 400-4000 cm −1 [69]. Spectragryph optical spectroscopy software was used to process the spectra of the four evaluated fractions (MIF1, MIF2, MIF3 and MIF4).

Determination of the Chemical Composition of M. integrifolia Polysaccharide Fractions
The chemical composition of M. integrifolia polysaccharide fractions was investigated, where the composition of neutral sugar was determined by an HPLC assay after acid hydrolysis meanwhile the composition of uronic acid was determined by the carbazole method and calculated as the equivalent glucuronic acid [70]. Furthermore, the sulphate content of each fraction was determined by turbidimetric assay after acid hydrolysis [71], whereas the respective molecular weights of each polysaccharide fraction were determined using gel permeation chromatography [72].

Evaluation of Antiangiogenic Activity Using Chorioallantoic Membrane (CAM) Assay
Sixty fertilized chicken eggs were used, obtained from the "Mochic" farm, which were washed, disinfected and incubated at 37 • C in a humidified atmosphere. On day 3, the upper surface of the egg was perforated, and 2 mL of albumin was extracted. This was then closed with cellophane paper, and a second 2.5 × 2 cm window was opened, exposing the underlying blood vessels. Bubbles generated by albumin suction were removed. Finally, the window was sealed with transparent tape and the recipients were returned to the incubator for one week. On day 10, a 5 mm diameter sterilized methylcellulose disc (MilliporeTM, Bedford, MA, USA) was placed on the surface of the CAM, in which the following solutions were placed: control group (n = 12): double-distilled water; the first group (n = 12): double distilled water and 100 µM of sulfated polysaccharide fraction (MIF1) 100 µM; the second group (n = 12): double-distilled water and 100 µM of sulfated polysaccharide fraction (MIF2); the third group (n = 12): double-distilled water and 100 µM of sulfated polysaccharide fraction (MIF3); the fourth group 4 (n = 12): double-distilled water and 100 µM of sulfated polysaccharide fraction (MIF4). On day 13, the MACs were removed and fixed in 10% formalin for 24 h, then subsequently washed with water and stained with hematoxylin-eosin (H-E). The microvascular density was determined by counting all blood vessels per 9000 µm 2 (30 µm × 300 µm) in an optical microscope, with a magnification of 400× and an eyepiece located 600 µm from the filter. Counts were carried out in each of the work groups, obtaining sample values of the number of blood capillaries [73,74].

Determination of Cytotoxicity by MTT
Tetrazoyl-salt-reduction-based colorimetric technology (MTT) was used, where the secondary culture of chick embryo fibroblasts (CSFEP) was transferred to 96-well boxes (6000 cells per well) and incubated at 37 • C and 5% CO 2 until a confluence of 80-90% was obtained. The medium was removed from the plates and 100 µL of polysaccharides per well were added at different concentrations: 1, 25, 50, 100, 200, 400, 600, 800 and 1000 µg/mL (three wells for each concentration). Three wells were left for control without sulfated polysaccharides; the assay was performed in triplicate. Plates were incubated for 48 h at 37 • C and 5% CO 2 . 0.025 g of the MTT reagent was prepared in 5 mL of PBS 1×, to obtain a concentration of 5 mg/mL of MTT solution. At the end of the incubation, the D-MEM/F12 medium was removed from the plates with CSFEP treated with sulfated polysaccharides, and 22 µL of the MTT solution was added to each well and incubated at 37 • C until a change in color was observed. A total of 100 µL of dimethylsulfoxide (DMSO) was added to solubilize the formazan salts in all wells, and incubation was performed for 8 min; then, the absorbance measurement was taken at 570 nm in the ELISA reader [73,74].

Isolation of Peritoneal Macrophages for in Vitro Assays
Rats were sacrificed by cervical dislocation in a laminar flow chamber. A total of 30 mL of peritoneal fluid was removed. The cell suspension was washed by centrifugation at 400× g for 10 min at 4 • C and re-suspended in DSBH (Hank's balanced salt solution). The cell concentration was adjusted to 106 cells/mL. The proportion of macrophages in the total population of peritoneal cells was determined by α-naphthyl staining [75,76].

In Vivo Cellular Chemotaxis Assay
The experimental groups containing eight Wistar rats were arranged. To induce a chemotactic response towards the peritoneum of rats, 1 mL of 3% thioglycolate was injected intraperitoneally and 0.5 mL of sulfated polysaccharides was administered at doses of 10, 50 and 100 mg/kg in NaCl saline solution at 0.9%, corresponding to each experimental group. The control group received intraperitoneal saline solution. Five days later, the peritoneal exudate was collected, and the total number of cells was counted on a hemocytometer, determining the proportion of macrophages by staining with α-naphthyl [75,76].

In Vitro Assay to Determine Phagocytic Activity
The phagocytic activity of macrophages was evaluated using the fluorimetric method based on the use of yeast (Kluyveromyces lactis), and marked with fluorescein isothiocyanate (ITCF) [75,76].

In Vitro Assay for the Determination of Nitric Oxide Production
Nitric oxide production by peritoneal macrophages was determined by the Griess reaction by measuring the total amount of nitrites in the culture supernatant [75,76].
3.9. Determination of the Expression of Genes Encoding Enzymes, Cytokines and Immune Response Transcription Factors 3.9.1. Collection and Activation of Murine Peritoneal Macrophages Murine peritoneal macrophages were obtained from Balb/c mice and injected with 0.5 mL of 3% thioglycolate; then, after three days, the peritoneal exudate was obtained. The proportion of macrophages in peritoneal exudate was estimated by determining the nonspecific esterase activity [76,77] This was carried out using the in vivo model of allergic inflammation in seven experimental groups of eight Balb/c mice each, where six groups were immunized with Ovalbumin (OVA) in 0.5 mL of physiological saline. The remaining group only received injections of physiological saline solution. Three weeks after the last immunization, the groups were treated with saturated polysaccharides at doses of 50 mg/kg and 5 mg/kg of indomethacin, while the control group received distilled water. At the end of treatment, the allergic response was carried out using the ovalbumin-induced plantar edema model (OVA), and the proliferative response of splenocytes to OVA was determined [76,77].

Histamine Skin Reaction
Six experimental groups of eight Wistar rats were arranged, who received treatment for 7 days with the administration of sulfated polysaccharides at doses of 50 mg/kg or promethazine at 25 mg/kg. Ten hours after the last administration, the rats were depilated in the dorsal region, drawing six quadrants of 1 × 1. In four of the quadrants, 20 mg/kg of Evans blue and 100 µL of saline solution with 5 µg of histamine were administered. In the remaining two, 100 µL of saline solution was administered. Thirty minutes later, the animals were sacrificed; the skin quadrants were cut and placed in test tubes with 2 mL of formamide and incubated at 37 • C for 48 h, determining the optical density of the transferred dye at 620 nm on a spectrophotometer [78,79].

Determination of Specific IgE in OVA-Immunized Mice
Six experimental groups of eight Balb/c mice were organized and immunized with OVA according to the method described by Park et al. [80]. Then, for 21 days, mice were treated once daily with sulfated polysaccharides at concentrations of 50 mg/kg, or ketotifen (3 mg/kg). The control group only received distilled water. After 21 days, blood was extracted from mice, placed in test tubes, and centrifuged at 400× g for 10 min; serum was extracted and stored at −70 • C for later use. Subsequently, to estimate the levels of a specific IgE, the passive cutaneous anaphylaxis test (PCA) was performed in Wistar rats using the antiserum obtained in mice immunized with OVA and treated with sulfated polysaccharides or ketotifen.

Statistical Analysis
Data were presented as mean ± standard deviation (SD). The ANOVA test was performed to determine the differences between groups. In addition, a Tukey multirank test was used to indicate differences between the concentrations of the same fraction, as well as the differences between fractions. All statistical analyses were performed using SPSS v.25.0 (IBM Corp., Armonk, NY, USA), and Graphpad Prism 6.0 ® (GraphPad Software, San Diego, CA, USA) 4. Conclusions From the current study, it was concluded that M. integrifolia algae are a rich source of sulfated polysaccharides, as revealed by Fourier transform infrared (FTIR) spectroscopic analysis. All tested fractions of sulfated polysaccharides, particularly MIF1, showed notable antiangiogenic activity using the chorioallantoic membranes (CAM) assay. They also showed potent immunomodulatory potential, as evidenced by the inhibition of chemotaxis, phagocytosis, and mRNA expression of NOS-2, COX-1, COX-2, NF-κB and IκB. Additionally, they showed antiallergic potential in ovalbumin (OVA)-induced allergic inflammation in an in vivo model, with concomitant reductions in histamine-induced skin reaction and specific IgE in OVA-immunized mice. The biological activities of sulfated polysaccharides are intimately dependent on the chemical structure and molecular weight, as well as the extraction process; thus, low-molecular-weight sulfated polysaccharides showed better pharmacological activities than high-molecular-weight ones. In addition, the content of the fucose and sulfate groups could influence immunomodulatory activity. In the same way, antiangiogenic activity is related to sulfated polysaccharides with MW > 30 kDa; however, a low molecular weight promotes angiogenesis. Therefore, sulfated polysaccharides and their chemical characteristics may explain the observed activity. This means that M. integrifolia sulfated polysaccharides could serve as promising, naturally occurring, antiangiogenic and immunomodulatory drug entities that could be incorporated into pharmaceutical dosage forms to combat various immune-related disorders. However, further preclinical studies should be conducted to consolidate the obtained results.