The Potential Effect of Polysaccharides Extracted from Red Alga Gelidium spinosum against Intestinal Epithelial Cell Apoptosis

Gut injury is a severe and unpredictable illness related to the increased cell death of intestinal epithelial cells (IECs). Excessive IEC apoptotic cell death during the pathophysiological state entails chronic inflammatory diseases. This investigation was undertaken to assess the cytoprotective action and underlying mechanisms of polysaccharides from Tunisian red alga, Gelidium spinosum (PSGS), on H2O2-induced toxicity in IEC-6 cells. The cell viability test was initially carried out to screen out convenient concentrations of H2O2 and PSGS. Subsequently, cells were exposed to 40 µM H2O2 over 4 h in the presence or absence of PSGS. Findings revealed that H2O2 caused oxidative stress manifested by over 70% cell mortality, disturbed the antioxidant defense, and increased the apoptotic rate in IEC-6 cells (32% than normal cells). Pretreatment of PSGS restored cell viability, especially when used at 150 µg/mL and normal cell morphology in H2O2-callenged cells. PSGS also equally sustained superoxide dismutase and catalase activities and hindered the apoptosis induced by H2O2. This protection mechanism of PSGS may be associated with its structural composition. The ultraviolet visible spectrum, Fourier-transformed infrared (FT-IR), X-ray diffraction (XRD), and high-performance liquid chromatography (HPLC) demonstrated that PSGS is mainly sulfated polysaccharides. Eventually, this research work provides a deeper insight into the protective functions and enhances the investment of natural resources in handling intestinal diseases.


Introduction
The blooming growth in the world population along with changes in the level of lifestyle and eating habits have been crucial in the advent of multiple diseases constituting a severe threat to human life [1]. Inflammatory bowel diseases (IBDs) are chronic idiopathic diseases marked by relapsing gastrointestinal tract inflammation [2]. Bloody diarrhea, abdominal pain, hepatosis, and eye and skin lesion are the main clinical symptoms of these diseases. IBDs involve two forms, ulcerative colitis (UC) and Crohn's disease (CD) [3]. CD represents the more profound, transmural, inflammatory condition in patches throughout the small intestine and the colon. Meanwhile, UC is marked by mucosal inflammation confined to the colon [4].
IBDs constitute a global and significant public health challenge that is increasing in newly industrialized countries, especially those in Africa, Asia, and South America [2]. They On the other side, the uronic acid content of PSGS (14.30%) was similar to that obtained by polysaccharides extracted from Sargassum vulgare (brown alga) [39]. The marine origin, seasonal periods, conditions, and extraction method are determining factors for the variations of all these contents.
The ash contents were estimated at a percentage of 2.64 ± 0.41%. As reported by Rioux et al. [40], the proportion of minerals can be the result of the association between polysaccharides and cations or the inorganic salt in the water absorbed by seaweed. Concerning sulfate esters, the chemical analyses demonstrated amounts of 17.30%, similar to those found in polysaccharides of red seaweed Gelidium pacificum [41]. Figure 1 illustrates the UV spectra of PSGS. The data observed in Figure 1 display two peaks. The first corresponds to a broad absorption around 204 nm, confirming that PSGS is specified as polysaccharides. Jose et al. [42] indicated a significantly prominent absorbance peak at 205-215 nm using sulfated polysaccharide from brown seaweed Padina tetrastromatica. The second slight absorption peaks at 260-280 nm indicate the presence of proteins [43].

UV and FT-IR Spectroscopy Analysis
As it is well known, FT-IR spectroscopy stands as a handy tool to identify the structural features of polymer blends, such as distinct organic groups in the polysaccharide. The infrared spectroscopy results in Figure 2 revealed that PSGS had typical polysaccharide absorption peaks in the region between 400 and 4000 cm −1 . The broad absorptions around 3273 cm −1 and 2928 cm −1 were attributed to O-H stretching vibration and C-H stretching vibration of the -CH-groups, respectively [44], indicating that the sample was a polysaccharide compound. The asymmetric and symmetric vibration of the carboxylate groups appeared at around 1600 cm −1 and 1415 cm −1 , respectively, demonstrating that PSGS was an acidic polysaccharide [45], implying in turn the presence of uronic acids, which was Pharmaceuticals 2023, 16, 444 4 of 17 confirmed by monosaccharide composition. Both hydroxyl and carboxyl groups played an intrinsic role in the biological activities of polysaccharides. Previous studies [46] unveiled that extracellular polysaccharide containing carboxyl and hydroxyl groups can enhance their antitumor and antioxidant activities. This polysaccharide has an absorption peak at 1538 cm −1 , revealing its content of proteins [47], which was proven by UV analysis. The absorption band at 1227 cm −1 was attributed to the S=O stretching vibration [48]. The absorption peak at 1026 cm −1 was assigned for the presence of glycosidic linkage stretch vibration of C-O bond in guluronic units [32]. Combined with an absorption band at 853 cm −1 , originating from the C-O-S stretching vibration [49], the three bands suggest the existence of sulfate in the PSGS, which was corroborated by chemical analyses. Positive specific rotation and the characteristic absorption at 853 cm −1 indicated the α-configuration of the sugar units [50]. As it is well known, FT-IR spectroscopy stands as a handy tool to identify the structural features of polymer blends, such as distinct organic groups in the polysaccharide. The infrared spectroscopy results in Figure 2 revealed that PSGS had typical polysaccharide absorption peaks in the region between 400 and 4000 cm 1. The broad absorptions around 3273 cm −1 and 2928 cm −1 were attributed to O-H stretching vibration and C-H stretching vibration of the -CH-groups, respectively [44], indicating that the sample was a polysaccharide compound. The asymmetric and symmetric vibration of the carboxylate groups appeared at around 1600 cm −1 and 1415 cm −1 , respectively, demonstrating that PSGS was an acidic polysaccharide [45], implying in turn the presence of uronic acids, which was confirmed by monosaccharide composition. Both hydroxyl and carboxyl groups played an intrinsic role in the biological activities of polysaccharides. Previous studies [46] unveiled that extracellular polysaccharide containing carboxyl and hydroxyl groups can enhance their antitumor and antioxidant activities. This polysaccharide has an absorption peak at 1538 cm −1 , revealing its content of proteins [47], which was proven by UV analysis. The absorption band at 1227 cm −1 was attributed to the S=O stretching vibration [48]. The absorption peak at 1026 cm −1 was assigned for the presence of glycosidic linkage stretch vibration of C-O bond in guluronic units [32]. Combined with an absorption band at 853 cm −1 , originating from the C-O-S stretching vibration [49], the three bands suggest the existence of sulfate in the PSGS, which was corroborated by chemical analyses. Positive specific rotation and the characteristic absorption at 853 cm −1 indicated the α-configuration of the sugar units [50]. Numerous polysaccharides correspond to bioactive products, and their biological activities are closely related to their structural characteristics, such as monosaccharide composition, glycosidic bonds, and crystalline structure. Figure 3 illustrates the X-ray diffractogram of PSGS ranging between 0° and 100°. Data demonstrated a major crystalline re-

X-ray Diffractometry (XRD) Analysis
Numerous polysaccharides correspond to bioactive products, and their biological activities are closely related to their structural characteristics, such as monosaccharide composition, glycosidic bonds, and crystalline structure. Figure 3 illustrates the X-ray diffractogram of PSGS ranging between 0 • and 100 • . Data demonstrated a major crystalline reflection at 29 • , and PSGS tends to be a semi-crystalline polymer. Crystalline and semicrystalline structures of materials were directly influenced by various physical properties, including tensile strength, flexibility, solubility, swelling, viscosity, or opaqueness of the bulk polymer [51].

Monosaccharide Composition Analysis
The biological activities of polysaccharides are strongly affected by their monos charide composition. Previous data indicate that algae represent a rich source of mo saccharides [52,53]. In the current work, PSGS monosaccharide composition was inve gated through HPLC-FID analysis. The latter displayed a heterogeneous behavior, wh arabinose, glucuronic acid, and galactose constitute the major monosaccharide units retention times of 5.57; 14.53, and 13.65 min, respectively, according to the elution time monosaccharide standards ( Figure 4). Previous data regarding polysaccharides extrac from green macroalga Chaetomorpha linum also revealed heterogeneous compositions monosaccharides [54]. Referring to the literature, extraction protocol, temperature, a solvent used for precipitation affect the nature of molecules and extraction yield [55].

Monosaccharide Composition Analysis
The biological activities of polysaccharides are strongly affected by their monosaccharide composition. Previous data indicate that algae represent a rich source of monosaccharides [52,53]. In the current work, PSGS monosaccharide composition was investigated through HPLC-FID analysis. The latter displayed a heterogeneous behavior, where arabinose, glucuronic acid, and galactose constitute the major monosaccharide units at retention times of 5.57; 14.53, and 13.65 min, respectively, according to the elution time of monosaccharide standards ( Figure 4). Previous data regarding polysaccharides extracted from green macroalga Chaetomorpha linum also revealed heterogeneous compositions of monosaccharides [54]. Referring to the literature, extraction protocol, temperature, and solvent used for precipitation affect the nature of molecules and extraction yield [55]. gated through HPLC-FID analysis. The latter displayed a heterogeneous behavior, where arabinose, glucuronic acid, and galactose constitute the major monosaccharide units at retention times of 5.57; 14.53, and 13.65 min, respectively, according to the elution time of monosaccharide standards ( Figure 4). Previous data regarding polysaccharides extracted from green macroalga Chaetomorpha linum also revealed heterogeneous compositions of monosaccharides [54]. Referring to the literature, extraction protocol, temperature, and solvent used for precipitation affect the nature of molecules and extraction yield [55].
As displayed in Figure 5A, cell viability increased with increasing PSGS concentrations. Our findings agree with those reported in the study of Qiu et al. [56], which indicated that natural polysaccharides from red seaweed Porphyra haitanensis are nontoxic to IEC-6 cells and promote at the same time cell proliferation. Previous studies emphasized that antioxidant capacities and beneficial effects of natural compounds might be inversed and become lethal for the cells under such conditions as high concentrations [57]. At 200 µg/mL, the cell survival rate reached 15% compared to the control group (p < 0.05), which might damage cells, therefore indicating a dose-dependent relationship between viability rate and concentrations of PSGS.
In our experimental model of cellular oxidative damage, we stimulated IEC-6 cells with different concentrations of H 2 O 2 for 4, 24, and 48 h exposure duration. Exposure concentration significantly altered the cell viability of IEC-6 cells in a dose/time-dependent manner ( Figure 5B). Besides, cell growth was remarkably inhibited, departing from 40 µg/mL by 70% and decreased dramatically to reach 90 % to 80 and 100 µM H 2 O 2 . Based on these results, IEC-6 cells were treated with 40 µM H 2 O 2 for 4 h as an oxidative damage model in the present study. Bettaib et al. [58] selected 40 µM H 2 O 2 for 4 h as a stress condition in IEC-6 cells to assess the cytoprotective effect of phenolic compounds.
The viability of IEC-6 cells exposed to PSGS and H 2 O 2 was investigated through MTT analysis. Our results ( Figure 5C) demonstrated a significant difference between the control group and the model (H 2 O 2 ) group, suggesting that the survival rate of IEC-6 cells significantly decreased after 4 h of H 2 O 2 stimulation. However, pre-incubation of 20; 50; 70; 100, and 150 µg/mL PSGS, for 24 h, significantly increased the survival rate of IEC-6 cells. PSGS were collectively beneficial in protecting IEC-6 cells against H 2 O 2 -induced injury. Meanwhile, it was inferred that the sulfate group is highly related to free radicals, including superoxide and hydroxyl scavenging effects. This can further explain why PSGS showed significant improvement in the viability of IEC-6 cells. Thus, sulfate polysaccharides display an outstanding protective ability to handle the adverse effects of H 2 O 2 , which is consistent with a previous report [59].

Effect of PSGS and H 2 O 2 on the Morphological Aspect
To further explore the protective effects of PSGS on IEC-6 cells, morphological changes were examined under an inverted microscope. As exhibited in Figure 6B, observing IEC-6 cells under the inverted photonic microscope demonstrated impressive changes in cell shape. H 2 O 2 crosses the membrane and interferes with cell attachment to initiate cellular damage, such as cell shape changes and mitochondrial dysfunction, leading to metabolic alterations [58,60]. In addition, a high number of dead cells in response to H 2 O 2 toxicity was observed. Referring to previous studies [61], the time of incubation as well as the dose of H 2 O 2 tightly influence the survival rates of IEC-6 cells. However, cells co-treated with both H 2 O 2 and PSGS showed a morphology close to that of control cells, suggesting the protective effect of PSGS against the toxicity induced by H 2 O 2 .
As displayed in Figure 5A, cell viability increased with increasing PSGS concentrations. Our findings agree with those reported in the study of Qiu et al. [56], which indicated that natural polysaccharides from red seaweed Porphyra haitanensis are nontoxic to IEC-6 cells and promote at the same time cell proliferation. Previous studies emphasized that antioxidant capacities and beneficial effects of natural compounds might be inversed and become lethal for the cells under such conditions as high concentrations [57]. At 200 μg/mL, the cell survival rate reached 15% compared to the control group (p < 0.05), which might damage cells, therefore indicating a dose-dependent relationship between viability rate and concentrations of PSGS. In our experimental model of cellular oxidative damage, we stimulated IEC-6 cells with different concentrations of H2O2 for 4, 24, and 48 h exposure duration. Exposure concentration significantly altered the cell viability of IEC-6 cells in a dose/time-dependent manner ( Figure 5B). Besides, cell growth was remarkably inhibited, departing from 40 μg/mL by 70% and decreased dramatically to reach 90 % to 80 and 100 μM H2O2. Based on these results, IEC-6 cells were treated with 40 μM H2O2 for 4 h as an oxidative damage

PSGS Supported Enzymatic Defense against H2O2 Toxicity
The organism balances pro-and antioxidant systems in response to oxidative stress conditions. As an important index for detecting oxidative cell damage, SOD corresponds to an antioxidant enzyme catalyzing superoxide anions' dismutation to H2O2 and O2 [22]. CAT is a common antioxidant enzyme that utilizes oxygen and catalyzes the degradation or reduction of H2O2 to water and molecular oxygen. Consequently, it completes the detoxification process initiated by SOD [62]. Compared to the control group, the activity of SOD in IEC-6 cells increased after treatment by 40 μM of H2O2 for 4 h ( Figure 7A). It is to be noted that, the SOD activity in PSGS-treated cells gradually decreased according to the PSGS concentrations. The result indicates the dose-dependent relationship between SOD activity and polysaccharides concentrations. Overproduction of ROS, including superoxide, singlet O2−, and hydroxyl radical, was the chief cause of oxidative damage and cell apoptosis [63]. Based on the mechanism stated above, several native sulfated polysaccharides isolated from red algae were also found to increase the SOD activity [64] remarkably.

PSGS Supported Enzymatic Defense against H 2 O 2 Toxicity
The organism balances pro-and antioxidant systems in response to oxidative stress conditions. As an important index for detecting oxidative cell damage, SOD corresponds to an antioxidant enzyme catalyzing superoxide anions' dismutation to H 2 O 2 and O 2 [22]. CAT is a common antioxidant enzyme that utilizes oxygen and catalyzes the degradation or reduction of H 2 O 2 to water and molecular oxygen. Consequently, it completes the detoxification process initiated by SOD [62]. Compared to the control group, the activity of SOD in IEC-6 cells increased after treatment by 40 µM of H 2 O 2 for 4 h ( Figure 7A). It is to be noted that, the SOD activity in PSGS-treated cells gradually decreased according to the PSGS concentrations. The result indicates the dose-dependent relationship between SOD activity and polysaccharides concentrations. Overproduction of ROS, including superoxide, singlet O 2 −, and hydroxyl radical, was the chief cause of oxidative damage and cell apoptosis [63]. Based on the mechanism stated above, several native sulfated polysaccharides isolated from red algae were also found to increase the SOD activity [64] remarkably. Error bars represent standard deviations of three replications. Significant differences between the treated groups and the normal group were mentioned as follows: *** p < 0.001.
However, the binding effect of CAT activity was achieved at 150 μg/mL of PSGS (Figure 7B). Therefore, the protective capacity of these promising polymers against H2O2-induced oxidative stress might reside under their antioxidant actions through enhancing endogenous antioxidant enzyme activities. Evidence indicated that polysaccharides containing uronic acid have significant antioxidant activity owing to carboxyl groups, which behaved as important electron or hydrogen donors in the antioxidant activity [65].

Figure 7. (A) Effects of PSGS on levels of SOD in H 2 O 2 -injured IEC-6 cells, (B) Effects of PSGS on levels of CAT in H 2 O 2 -injured IEC-6 cells. NG (normal group), HG (model group treated with H 2 O 2 ).
Error bars represent standard deviations of three replications. Significant differences between the treated groups and the normal group were mentioned as follows: *** p < 0.001. However, the binding effect of CAT activity was achieved at 150 µg/mL of PSGS ( Figure 7B). Therefore, the protective capacity of these promising polymers against H 2 O 2induced oxidative stress might reside under their antioxidant actions through enhancing endogenous antioxidant enzyme activities. Evidence indicated that polysaccharides containing uronic acid have significant antioxidant activity owing to carboxyl groups, which behaved as important electron or hydrogen donors in the antioxidant activity [65].

Effect of PSGS on H 2 O 2 Induced Apoptosis in IEC-6 Cells
Apoptosis is a fundamental and crucial biological phenomenon that plays an intrinsic role in clearing abnormal cells [66]. Thus, IECs renewal is necessary for maintaining tissue homeostasis. Still, excessive IEC cell death disrupts intestinal barrier integrity and permits the invasion of luminal antigens into the lamina propria, a hallmark of intestinal inflammation [67]. H 2 O 2 is a membrane-permeable ROS generator that is widely used to induce oxidative damage and apoptosis in cells [47]. Sound evidence revealed that oxidative stress causes programmed cell death [68]. As far as our study is concerned, IEC-6 cells were stained with Oxazole Yellow, and apoptotic cells were computed using fluorescence microscopy. As plotted in Figure 8, cells treated with H 2 O 2 alone exhibited a higher apoptosis rate of 32% than normal cells, indicating that H 2 O 2 induced apoptosis. Zhuang et al. [69] indicated that 40 ng/mL H 2 O 2 induces apoptosis in Chondrocytes. As expected, the cell apoptosis rate decreased after pretreatment with PSGS, suggesting that the PSGS can effectively mediate oxidative damage and protect IEC-6 cells against H 2 O 2 -induced apoptosis. Different concentrations of polysaccharides displayed more potent anti-apoptotic effects. Thus, due to their marked effect on cell viability, the pretreatment with 150 µg/mL of polysaccharides displayed a lower percentage of apoptosis (13.2 ± 0.64). Previous studies revealed that oxidative stress and apoptosis are detected in many diseases and are caused by an imbalance between free radical generation and antioxidant defense capacity [70]. Therefore, the results suggest that PSGS can protect endothelial cells from apoptosis. At this stage of analysis, it is noteworthy that polysaccharides from Gelidium spinosum might be considered as promising molecules in terms of injury recovery and degenerative diseases referring to their anti-apoptotic capacities. For a deeper and better understanding of the effect of polysaccharides on apoptotic cell death, Ma et al. [68] highlighted that sulfated polysaccharides can effectively mediate oxidative damage and significantly protect PC12 cells against H 2 O 2 -induced apoptosis.

Effect of PSGS on H2O2 Induced Apoptosis in IEC-6 Cells
Apoptosis is a fundamental and crucial biological phenomenon that plays an intrinsic role in clearing abnormal cells [66]. Thus, IECs renewal is necessary for maintaining tissue homeostasis. Still, excessive IEC cell death disrupts intestinal barrier integrity and permits the invasion of luminal antigens into the lamina propria, a hallmark of intestinal inflammation [67]. H2O2 is a membrane-permeable ROS generator that is widely used to induce oxidative damage and apoptosis in cells [47]. Sound evidence revealed that oxidative stress causes programmed cell death [68]. As far as our study is concerned, IEC-6 cells were stained with Oxazole Yellow, and apoptotic cells were computed using fluorescence microscopy. As plotted in Figure 8, cells treated with H2O2 alone exhibited a higher apoptosis rate of 32% than normal cells, indicating that H2O2 induced apoptosis. Zhuang et al. [69] indicated that 40 ng/mL H2O2 induces apoptosis in Chondrocytes. As expected, the cell apoptosis rate decreased after pretreatment with PSGS, suggesting that the PSGS can effectively mediate oxidative damage and protect IEC-6 cells against H2O2-induced apoptosis. Different concentrations of polysaccharides displayed more potent anti-apoptotic effects. Thus, due to their marked effect on cell viability, the pretreatment with 150 μg/mL of polysaccharides displayed a lower percentage of apoptosis (13.2 ± 0.64). Previous studies revealed that oxidative stress and apoptosis are detected in many diseases and are caused by an imbalance between free radical generation and antioxidant defense capacity [70]. Therefore, the results suggest that PSGS can protect endothelial cells from apoptosis. At this stage of analysis, it is noteworthy that polysaccharides from Gelidium spinosum might be considered as promising molecules in terms of injury recovery and degenerative diseases referring to their anti-apoptotic capacities. For a deeper and better understanding of the effect of polysaccharides on apoptotic cell death, Ma et al. [68] highlighted that sulfated polysaccharides can effectively mediate oxidative damage and significantly protect PC12 cells against H2O2-induced apoptosis.

Seaweed Collection and Processing
The red alga Gelidium spinosum was collected in March 2021 from the coastal area of Sidi Jmour, Djerba, Tunisia. Google maps coordinates are (33 • 51 22.4 N 10 • 44 33.4 E). Gelidium spinosum was authenticated by a specialist in ecology, Professor "Asma Hamza", accredited with the World Register of Marine Species (WoRMS) under the following identifier "145594" (Figure 9).

Seaweed Collection and Processing
The red alga Gelidium spinosum was collected in March 2021 from the coastal area of Sidi Jmour, Djerba, Tunisia. Google maps coordinates are (33°51′22.4″ N 10°44′33.4″ E). Gelidium spinosum was authenticated by a specialist in ecology, Professor "Asma Hamza", accredited with the World Register of Marine Species (WoRMS) under the following identifier "145594" (Figure 9) Collected seaweed was washed thoroughly to remove surface impurities, salt, and sand particles, and epiphytes. The water was drained off, and the seaweed sample was dried in the dark. The dried seaweed was powdered in the grinder and preserved in a limp sterile for further studies.

Polysaccharides Extraction
The polysaccharide extraction procedure was performed according to the method reported by Gong et al. [71], using hot water for extraction and ethanol as a precipitating agent. Notably, the seaweed flour (50 g) was dispersed in distilled water, stirred at 90 °C for 4 h, and filtered. The filtrate was centrifuged at 3600× g for 10 min. After centrifugation and concentration, the ethanol was incorporated (V/3V) to a concentration of 95% for alcohol precipitation at 4 °C for 24 h. The crude polysaccharide (PSGS) was obtained after centrifugation using a refrigerated centrifuge and lyophilization. The yield was expressed in terms of the ratio of the dry weight of the polysaccharide extracted (g) against the dry Collected seaweed was washed thoroughly to remove surface impurities, salt, and sand particles, and epiphytes. The water was drained off, and the seaweed sample was dried in the dark. The dried seaweed was powdered in the grinder and preserved in a limp sterile for further studies.

Polysaccharides Extraction
The polysaccharide extraction procedure was performed according to the method reported by Gong et al. [71], using hot water for extraction and ethanol as a precipitating agent. Notably, the seaweed flour (50 g) was dispersed in distilled water, stirred at 90 • C for 4 h, and filtered. The filtrate was centrifuged at 3600× g for 10 min. After centrifugation and concentration, the ethanol was incorporated (V/3V) to a concentration of 95% for alcohol precipitation at 4 • C for 24 h. The crude polysaccharide (PSGS) was obtained after centrifugation using a refrigerated centrifuge and lyophilization. The yield was expressed in terms of the ratio of the dry weight of the polysaccharide extracted (g) against the dry weight Gelidium spinosum (g) in percentage. The dried PSGS was stored at −20 • C for further studies.

Chemical Characterization of PSGS
Determination of Total Carbohydrate, Protein, Uronic Acid, Sulfate, and Ash Content As reported by Huang et al., carbohydrate content was quantified using the phenol sulfate acid method [72]. Basically, 0.1 mg/mL standard glucose solution was prepared; 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL were pipetted in a test tube. Next, 1 mL of distilled water and 1 mL of 3% phenol were added. Afterward, 4 mL of concentrated sulfuric acid was inserted gradually. The absorbance was measured at 490 nm after reaction for 30 min at room temperature.
Soluble proteins in PSGS were quantified by colorimetric assay [73]. The content of the uronic acid was assessed through the use of the Carbazole-sulfate method [74]. Notably, the galacturonic acid solution was configured similarly and served as a standard. In an ice bath, 6 mL of superior pure sulfuric acid were added, shaking while adding. Subsequently, 0.2 mL of 0.1% carbazole-ethanol (25 mg carbazole dissolved in 25 mL ethanol) was added, and the reaction was carried out for 2 h. The absorbance was measured at 530 nm.
Sulfate content was determined according to the gelatin-barium method [75], using 1 mg/mL of potassium sulfate (K 2 SO 4 ) as standard.
The amount of ash contents in PSGS was measured according to the method reported by Seedevi et al. [76]. In brief, 0.5 g of the dried polysaccharides taken in a porcelain crucible was burnt at 550 • C for 8 h in a muffle furnace. The weight of the residue, which represents the ash content, was recorded and the results are given as percentage of the dry weight of polysaccharides.

Ultraviolet and Fourier Transform Infrared (FT-IR) Spectroscopic Analysis
The ultraviolet spectrum of PSGS was recorded using an UV-vis spectrophotometer (JENWAY/7315, Staffordshire, UK) in the 200-800 nm range.
The infrared spectrum of PSGS was determined on a Nicolet FT-IR spectrometer. The spectrum was acquired at a resolution of 4 cm −1 , and the measurement range was 4000-400 cm −1 at room temperature. OPUS data collection software program was next used to analyze the data (Bruker, Ettlingen, Germany) [43].

X-ray Diffractometry (XRD) Analysis
An X-ray diffractogram of PSGS was recorded using an X-ray diffractometer (D8 advance, Bruker, Bremen, Germany). The data were obtained in the 2θ ranges 5-80 • with a step size of 0.05 • and a counting time of 5 s/step.

Monosaccharide Composition Analysis
HPLC-FID recorded the Monosaccharide composition of PSGS according to the method described by Xie et al. [77]. A five-milligram sample was dissolved within 3 mL of 2 mol/L TFA and hydrolyzed at 110 • C for 3 h. Subsequently, TFA was removed by washing with methanol.
Hence, 50 mg of sodium tetrahydruroborate were added to reduce the hydrolyzed product. Then, pyridine and acetic acid anhydride were added at 40 • C for 2 h to acetylation. The acetylated sample was filtered and analyzed by HPLC-FID.
3.6. Cytoprotective Activity of PSGS on IEC-6 Cells 3.6.1. Measurement of IEC-6 Cells Viability Cell viability was determined using an MTT assay. IEC-6 cells were seeded into a 96-well plate at 2 × 10 4 cells/well. After incubation in 5% of CO 2 at 37 • C for 24 h, different concentrations of PSGS ranging from 20-200 µg/mL were inserted into the well for coculturing during 24 h. 150 µL of 0.05% MTT solution was added for 2 h. Then, 100 µL of DMSO was added to dissolve the produced formazan prior to incubation with IEC-6 cells for 30 min. This procedure was followed by measuring the absorbance at 540 nm using a microplate reader.
Different concentrations (10-100 µM) of H 2 O 2 were used to stimulate IEC-6 cells for 4, 24, and 48 h in 96-well plates to estimate a suitable level for the cell injury model.
The cytoprotective effect of PSGS was then evaluated. IEC-6 cells were incubated with different concentrations of PSGS for 24 h and then stimulated with 40 µM H 2 O 2 for 4 h. The obtained results are indicative of the mean of three independent experiments. Cell viability is suggestive of the absorbance of treated groups relative to that of the control group.

Cell Morphology Observation
After each treatment, as depicted above, cell morphology was examined with an inverted microscope (Olympus Optical, Rungis, France).

Determination of Antioxidant Enzymes Activity
IEC-6 cells were placed in a Petri dish at 2 × 10 4 cells/well. Then, they were exposed to 40 µM H 2 O 2 for 4 h, both with and without PSGS at different concentrations (20; 50; 70; 100; 150 µg/mL). After treatment, cells were rinsed with ice-cold PBS, scraped, and sonicated at 4 • C. After homogenization and centrifugation for 10 min at 4 • C, protein concentration was determined in the supernatant according to the method of Bradford [78]. Finally, samples were stored at −80 • C for subsequent analysis.
3.7.1. Superoxide DISMUTASE Activity SOD activity was measured using a commercially available kit (SOD activity Elabscience, Houston, TX, USA). The principle of the method relies on the ability of SOD to neutralize superoxide ions created by the xanthine/xanthine oxidase system and subsequently inhibit the reduction of WST-1 (water-soluble tetrazolium salt) to WST-1 formazan. In this respect, IEC-6 cells obtained 24 h after oxidative stress induction were washed with ice-cold 1× PBS and lysed, as described in the kit protocol. The supernatant of each sample was collected, and the total SOD activity was assayed spectrophotometrically at 450 nm. SOD concentration, expressed in units per milligram of protein, was specified using the SOD standard curve.

Catalase Activity
CAT activity was estimated using a commercially available kit (CAT activity Elabscience, Houston, TX, USA). The principle of the method rests on the ability of CAT to decompose hydrogen peroxide. Ammonium molybdate can stop this reaction, and the residual H 2 O 2 reacts with ammonium molybdate to generate a yellow complex. The production of the yellow complex which can calculate CAT activity at 405 nm and CAT concentration, and which is expressed in units per milligram of protein, was determined using the SOD standard curve.

Apoptosis Rate Detection
The apoptosis assay was carried out using a fluorescent cyanine Oxazole Yellow (YP1). YP1 does not penetrate the plasma membrane of viable cells. However, during apoptosis, apoptotic processes cause the cell membrane to become slightly permeable. This allows Oxazole Yellow (YP1) to enter these cells and bind to nucleic acids so as to detect apoptotic cells. Notably, IEC-6 cells were collected after washing with PBS and digesting with trypsin. Then, they were centrifuged to keep the cells in suspension. Next, 50 µL of Oxazole Yellow were added and mixed. The cells were then cultured at 20-25 • C for 5-10 min and protected from light. The apoptosis cells observed under fluorescence microscopy represent small, bright green dots.

Conclusions
To sum up, a sulfated polysaccharide was successfully extracted from the red seaweed Gelidium spinosum. This study evaluated the protective effect of PSGS on H 2 O 2induced injury cells. The result indicates that the PSGS pre-incubation not only hinders oxidative stress in intestinal epithelial cells, but also inhibits H 2 O 2 -induced apoptosis by scavenging ROS.
Similar to other active polysaccharides, this study prompts that the PSGS may have an important value to prevent and cure oxidation-related diseases, and it may be well applied in medical health care in the future.
Finally, it is worth noting that more diligent efforts should be performed in this area to further explore the functions and mechanisms of PSGS in relation to intestine function through additional in vitro and in vivo studies.