This method used hot water to break the cell wall component of the algae to release the intracellular molecules into the solvent. According to Cho et al. (31), the collected algae were washed with distilled water and dried at 60°C. The dried samples were then minced to get homogenate powder and filtered using a sieve (<0.5 mm). Next, the powder was soaked in hot water (100°C, alga density 33.3 g/L) for 3 h (20) or (1:20 algae to water ratio, at 65°C for 2 h) with constant mechanical stirring (31). According to Chi et al. (25), the extraction conditions were: ratio of algae to water, 1:30; extraction temperature, 100°C; and extraction time, 2 h. The solution was cooled, centrifuged at 18,500 × g for 10 min, and the supernatant was collected and precipitated with 95% ethanol (1:4, v/v) for 24 h at 4°C. The precipitate was dried at room temperature (24 h) overnight to obtain crude polysaccharides. The crude polysaccharide was deproteinized by sevage method [chloroform: butyl alcohol, 4:1 (v/v)] (35). Depending on the purpose, different fractions of polysaccharides could be obtained by various treatments of crude polysaccharides. For example, Cho et al. (31) dissolved 100 mg crude polysaccharide with 10 mL distilled water and fractionated using ion-exchange chromatography on a DEAE Sepharose fast flow column. Then, they washed the column and eluted the polysaccharide with distilled water (F1), 0.5 (F2), and 1.0 M (F3) NaCl. This method has been reported to yield 10% (20), 21.3% (25), and 25.1% (31) crude polysaccharides. The inconsistency in yield may be attributed to the difference in extraction conditions. However, fractionating and elution solvent concentrations can also affect the extraction yield of polysaccharides. For example, the yields of polysaccharides eluted with distilled water, 0.5 and 1.0 M NaCl were 7.3, 32, and 46.7%, respectively (31). The average molecular weight of crude polysaccharides obtained in this method was 966 kDa (25).

Alkali extraction techniques used NaOH to isolate polysaccharides from E. prolifera (36). The principle of this method is that the hydroxyl ions (OH⁻) of the base interfere with hydrogen linkages in the polysaccharides to release it into the solvent. Briefly, the algae were dried and subjected to extraction with 95% ethanol at 60°C for 2 h and distilled water at 90°C for 2 h. According to Zhao et al. (37), the algae were further washed, dried (overnight at 60°C), and treated with NaOH solution (0.30 mol/L) at room temperature for 3 h with 1:10 (w/v) algae to solvent ratio. However, the extraction conditions reported by Chi et al. (25) were: 0.5 M NaOH solution; 1:40 algae to solvent ratio; temperature, 60°C and time, 3 h. The residue was then filtered, and the alkali was neutralized with 0.1 mol HCl. The crude extract was then centrifuged (4,800 rpm, 10 min), the supernatant was collected and precipitated with 95% ethanol (1:4, v/v) for 24 h at 4°C. Following centrifugation, the precipitated solution was further washed with 95% ethanol and freeze-dried (−50°C for 24h). The extracted crude polysaccharide was deproteinized by chloroform: butyl alcohol (4:1, v/v) (38). Next, the polysaccharide was fractioned in DEAE-52 cellulose and eluted with distilled water (500 mL) and 0.3, 0.5, 0.7 M NaCl at a flow rate of 1.0 mL/min (37). Finally, the fractionated polysaccharides were purified through the Sephadex G-100 column (2.5 × 60 cm) and then concentrated, dialyzed, and lyophilized. According to Zhao et al. (37), average molecular weights of polysaccharides extracted with distilled water (500 mL), 0.3, 0.5, 0.7 M NaCl were 34.4, 64.2, 120, and 48.2 kDa, respectively. In addition, E. prolifera extracted with this method was found to yield 10.7% crude polysaccharide with an average molecular weight of 47.7 kDa (25). These results demonstrated alkali method is less efficient and produced a low yield (10.7%) compared with acid (24.7%) and enzyme-assisted extraction methods (36%) (25).

The principle of this method is that the acid (HCl) penetrates the algae cell component and then the H⁺ of the acid interferes with hydrogen linkages in polysaccharide to release it into the solvent. Different researchers isolated polysaccharides using this method in different time-temperature combinations. Briefly, Liu et al. (33) reported that polysaccharides could be extracted from E. prolifera using 0.1 N HCl at room temperature for 4 h in 1:30 (w/v) algae: water ratio. Recently, Chi and his colleagues isolated polysaccharides from E. prolifera with 0.1 M HCl solution at 80°C for 1h and sample to solvent ratio of 1:20 (w/v) (25). Following filtration, the collected crude polysaccharide was treated with 6 N NaOH to neutralize the acid. After centrifugation (4,800 rpm, 10 min), the supernatant was recovered and precipitated with absolute ethanol. The crude polysaccharide was then deproteinized by treating with chloroform: butyl alcohol (4:1, v/v) (35). Next, polysaccharide was purified using anion exchange chromatography on a DEA Bio-Gel Agarose FF gel and eluted with 1 M NaCl. Then, the polysaccharide was precipitated by 95% ethanol (1:4, v/v) for 24 h at 4°C, dialyzed with distilled water, and freeze-dried (−50°C for 24 h) (25). The acid extraction method was found to produce of 24.7% yield with an average molecular weight of 41.1 kDa (25). Liu et al. (33) reported that the extraction yield and molecular weight of the polysaccharide obtained by this method were 36.0% and 17.3 kDa, respectively. As compared with hot water and alkali extraction methods, the acid extraction method was found to produce better yield, high molecular weight E. prolifera polysaccharides with iron chelating capacity (25).

The principle of this method is that the enzymes degrade the algae's cell wall structure, thereby releasing the intracellular molecules to the solvent (39, 40). Currently, various enzymes have been used for the extraction of polysaccharides from Enteromorpha prolifera, including xylanase, viscozyme, kojizyme, cellucast, ultraflo, flavourzyme termamyl, protamex, cellulase, and neutrase (41). However, the selection of appropriate hydrolytic enzymes significantly affects the extraction efficiency. Furthermore, enzymes work at a specific pH, and the time-temperature combination also influences the rate of enzyme reaction. The optimum pH and time-temperature requirements of different enzymes were summarized previously (40).

In an enzyme-assisted extraction method, the E. prolifera samples were washed, 2 g sample was dispersed in 50 mL distilled water and incubated in an agitated water bath for 10 min, dried in an oven at 60°C, and milled to <1 mm particle size. Appropriate enzymes were then selected, and the time-temperature combination was adjusted. According to Michalak et al. (34), for enzyme-assisted extraction using cellulase (Trichoderma reesei ATCC 26921; Sigma-Aldrich Chemie GmBh, Schnelldorf, Germany), the recommended optimal extraction conditions were; the algae: enzyme: water ratio was 1 g:25 μL:50 mL, pH 4.6 and 8 h extraction time. The enzyme was prepared by dissolving in sterile deionized (DI) water in the presence of 0.15% polyhexamethylene biguanide at 5 mg/mL concentration. In another study by Chi et al. (25) for E. prolifera extraction using cellulase (Novozymes Co., Ltd., Copenhagen, Denmark), the enzyme amount was 2% (w/v), the ratio of algae to water was 1:30 (w/v), and the extraction was performed at 50°C for 1.5 h at pH 5.0. The enzyme reaction was then inactivated by heating the reaction at 90–100°C for 10 min, and then immediately cooled on an ice bath. Finally, the solutions were centrifuged (4,800 rpm, 10 min) and filtered to collect the crude polysaccharides (24). The crude polysaccharides could be further degraded with hydrogen peroxide and ascorbic acid to produce polysaccharides of different molecular weights (24, 36). The yield obtained using enzyme assisted method was found to be 36% (34). Chi et al. (25) reported the yield and average molecular weight of E. prolifera polysaccharide extracted by this method to be 21.45% and 1,327.5 kDa, respectively. Compared with the above-mentioned methods, this method is efficient in terms of extracting high molecular weight polysaccharides because of less off-target degradation. However, consideration should be given to select appropriate enzymes and optimize influencing factors to improve the extraction efficiency and the quality of extracted molecules.

This method used microwave energy to heat solvents in contact with a sample to partition analytes from the sample matrix into the solvent (30). Briefly, the algae sample was prepared by washing, air drying, and trituration/grinding. Then, the algae powder was suspended in 0.01 HCl solution (1:20, algae: solvent ratio), thoroughly mixed, and placed into a 200 mL microwave tube. The solution was then exposed to irradiation (2.45 GHz) using a UWave-2000 reactor at 120°C for 15 min. The solution was agitated at 300 rpm using a magnetic stirring bar during irradiation. Following irradiation, the residue was separated by centrifugation (6,000 × g for 20 min) and dried at 80°C. The supernatant was collected and neutralized by 1 M NaOH. The solution was then precipitated with 95% ethanol (1:2 v/v), washed, and lyophilized to obtain crude polysaccharides. The yield and average molecular weight of polysaccharides obtained in this method were 36.38% and 156 kDa, respectively (30). However, extraction temperature and acid concentration significantly influenced the yield and molecular weight of extracted material. For example, at 150°C and 0.1 M HCl extraction conditions, the yield and average molecular weight of the polysaccharide were 6.09% and 10.5 kDa, respectively (30). Therefore, the extraction condition should be optimized to get maximum yield. Generally, if the extraction conditions are optimized, this method is more efficient than the other methods in terms of yield.

The role of sulfated E. prolifera polysaccharide as an immune stimulator has been recognized previously by different studies. Sulfated polysaccharides were extracted from E. prolifera using hot water and their immunomodulatory activity was explored on RAW 264.7 macrophages cells and in mice. It has been demonstrated that RAW 264.7 macrophages cell that received sulfated polysaccharides exhibited upregulated macrophage proliferation, as well as increasing nitric oxide secretion (21). The in vivo results further confirmed that the administration of sulfated E. prolifera polysaccharide to mice could enhance cell proliferation besides increasing interferon-alpha (IFN-α) and interleukin-2 (IL-2) secretions (21). Similarly, Liu et. (33) demonstrated the immunomodulatory effect of E. prolifera polysaccharide on RAW 264.7 macrophages and cyclophosphamide (CYP)-induced immunosuppression mouse models, found that E. prolifera polysaccharide could promote the secretion of interleukin-1beta (IL-1β), interleukin (IL-6), and tumor necrosis factor-alpha (TNF-α) via activation of TLR4/MAPK/NF-κB signaling pathway in RAW 264.7 macrophages cells and mice.

In another study to prove the immunomodulatory effects of E. prolifera polysaccharide, the sulfated polysaccharide was extracted by a hot water method and administered to mice. The result showed that E. prolifera polysaccharide treatment could stimulate splenocyte proliferation (13, 21). Moreover, administration of mice with low molecular weight sulfated E. prolifera polysaccharide was found to increase splenocytes interferon-γ (INF-γ) and interleukin-2 (IL-2) production (56).

Further immune-related enzyme analysis showed that the treatment of mice with E. prolifera polysaccharide could enhance Nuclear factor-kappa B (NF-κB) expression and alkaline phosphatase (AKP), superoxide dismutase (SOD), and lactate dehydrogenase (LDH) production in a dose-independent manner, suggesting that the immunomodulatory effect of E. prolifera polysaccharide might be closely related to the upregulation of NF-κB transcription factor (13). Overall, this study confirmed the effect of E. prolifera polysaccharides on the humoral and cell-mediated immune response in mice.

In addition to studies in vitro and mice, it has also been reported that the administration of E. prolifera polysaccharides could enhance the non-specific immunity of sea cucumbers, thereby protecting sea cucumbers from splenic vibrio infection (57). Taken together, studies have confirmed that polysaccharide isolated from E. prolifera is a potential immunomodulator that can induce both humoral and cellular immunity.

On the other hand, a study showed that E. prolifera polysaccharide treatment protects human cardiac microvascular endothelial cells from oxygen-glucose deprivation-induced viability loss, proliferation inhibition, apoptosis, inflammatory cytokine expression via up-regulation of HIF-1α, and inactivation of the NF-κB pathway (58). Overall, these studies suggest that E. prolifera polysaccharides might play both immunomodulatory and anti-inflammatory roles depending on the physiological condition of experimental animals or cells.

Abnormally high levels of lipids or fats can cause hyperlipidemia, a chronic disease that elevates the risk of heart disease and stroke (59). The anti-hyperlipidemic activity of E. prolifera polysaccharide was evaluated in high-fat diet-induced hyperlipidemic mice. Oral administration of hot water extract of E. prolifera polysaccharide (300 mg kg⁻¹ body weight) to high fat-fed mice successfully reduced serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and inhibited lipid metabolism in a dose-dependent manner (46). Similarly, Teng et al. (60) reported that dietary supplementation of E. prolifera polysaccharide to rats markedly decreased plasma and liver triacylglycerol, total cholesterol (TC), and low-density lipoprotein content.

In another study, a polysaccharide isolated from E. prolifera was administered to high-fat diet-induced hyperlipidemic rats. The hypolipidemic activity was assessed in terms of serum and liver TG levels and mRNA expression of hepatic acetyl-CoA carboxylase (ACC). It was observed that the administration of E. prolifera polysaccharide to hyperlipidemic rats improved glucose tolerance and insulin resistance, reduced plasma and liver TG and TC levels, and showed the abundance of ACC mRNA expression (61, 62). Moreover, studies have shown that the administration of 200 mg kg⁻¹ sulfated E. prolifera polysaccharides could attenuate high-fat diet-induced non-alcoholic fatty liver disease in rats (63). It was proposed that the anti-hyperlipidemic effect of E. prolifera polysaccharides might be associated with an inhibition of sterol regulatory element-binding protein-1c (SREBP-1c) that resulted in the suppression of biosynthesis of cholesterol (61).

Cells undergo an oxidation reaction during the process of energy production. However, this oxidation process may generate excessive reactive oxygen species (ROS, oxygen-derived free radicals), which may adversely affect cells (64, 65). The antioxidant glutathione peroxidase (GSH), superoxide dismutase (SOD), catalase (CAT), and total anti-oxidant (T-AOC) play a decisive role in maintaining body health by removing overproduced ROS. Therefore, it is vital to increase the production of antioxidants in the body to get rid of these free radicals.

Recently, marine algae, including Enteromorpha prolifera, have attracted great interest owing to their free radical scavenging activities (66–68). Xu and his colleagues isolated polysaccharides from E. prolifera and evaluated their antioxidant activities in terms of their ability to scavenge free radicals. The results showed that E. prolifera polysaccharides have antioxidant activity (32). In another study, a water-soluble polysaccharide was extracted from E. prolifera using hot water methods and administered to mice fed a high-fat diet to evaluate the antioxidant activities. The results obtained from this study demonstrated that supplementation of E. prolifera polysaccharide increased the serum SOD, CAT, GSH-Px activities, and decreased serum malondialdehyde (MDA) content (46). Similarly in chicken, dietary supplementation of E. prolifera polysaccharides increased antioxidant levels of T-SOD, GSH-Px, CAT, and, GST and reduced MDA contents in the bursa of Fabricius (69). This may be partially attributed to the activation of the nuclear-related factor 2 (Nrf2) signaling pathway in response to E. prolifera polysaccharide supplementation (70).

Research evidence has shown that the antioxidant activities of E. prolifera can be influenced by the molecular weight and a sulfate group. In this regard, Li et al. extracted polysaccharides from E. prolifera by hot-water method and degraded them into low molecular weight by P. pabuli, and then sulfonated with chlorosulfonic acid/pyridine method. The antioxidant activities results showed that the lower molecular weight and sulfated polysaccharides were found to have higher free radical scavenging activities than the higher molecular weight un-sulfated polysaccharides (20).

Furthermore, E. prolifera polysaccharide could ameliorate reactive oxygen species accumulation and DNA damage via up-regulation of genes, such as protein skinhead-1(SKN-1) and DAF-16, and down-regulation of miR-48, miR-51, and miR-186, suggesting that it has a vigorous antioxidant activity (47). Altogether the above studies suggest that E. prolifera polysaccharide has an intense antioxidant activity, and its activity can be enhanced by modifying its chemical composition and molecular weight.

The antidiabetic effect of E. prolifera has been explored recently in diabetic-induced rats and mice. Intragastric administration of E. prolifera polysaccharides to diabetic rats ameliorated glucose metabolism by decreasing the fasting serum blood glucose and insulin levels. This effect might be attributed to promoting antioxidant levels and upregulating the mRNA abundance of glucose and insulin metabolism-related genes, such as glucokinase, insulin receptor, glucose transporter type 4 (GLUT-4), and adiponectin (71). In a recent study, polysaccharide was extracted from E. prolifera and enzymatically degraded to produce low molecular weight oligomers. The administration of these oligomers to diabetic mice has shown to relieve diabetic symptoms and reduce pancreatic inflammation and apoptosis, thereby ameliorating streptozotocin-induced diabetes mellitus (72). In addition, a recent study has demonstrated that E. prolifera polysaccharide prevents high-fat diet-induced obesity and ameliorated HFD-induced metabolic dysfunction in hamsters (73). Furthermore, administration of E. prolifera-chromium (III) complex to mice fed a high-fat and high-sucrose diet could improve glucose tolerance and reduce serum insulin levels via activation of the IR/IRS-2/PI3K/PKB/GSK-3β signaling pathway, suggesting that it could be a potential therapeutic agent against type 2 diabetes (74).

It is well established that diet and nutritional factors have a direct effect on the microbial colonization of the gut (75–77). A growing body of evidence suggests that gut microbiota is involved in the digestion and utilization of fibers such as polysaccharides, which otherwise cannot be utilized by the host. These microbes produce short-chain fatty acids as an end product from the diet, which has an important role in energy metabolism (78). Apart from this, gut microbiota play a decisive role in maintaining the homeostasis and health of the host via direct involvement in gut structure and morphology, regulation of immune responses, and protection from luminal pathogens. Therefore, any factor that leads to microbiota dysbiosis may affect the health status and immune response of the host. Recently, Kong et al. (79) extracted sulfated polysaccharides from E. prolifera with hot water method and fermented them in vitro for 48 h by human fecal cultures. Those authors observed that human fecal cultured with E. prolifera polysaccharides produced more short-chain fatty acids (SCFAs), including butyrate, acetate, and lactic acid, and increased the production of beneficial intestinal Lactobacillus bacteria (79). Thus, it was concluded that E. prolifera polysaccharides could exert a prebiotic effect in humans by promoting SCFAs production and regulating the intestinal microbiota.

Similarly, the modulatory effects of E. prolifera polysaccharide on gut microbiota were also investigated in mice. For this purpose, water extracted E. prolifera polysaccharides were administered to mice for 2 weeks, and then the fecal pellets were analyzed using the 16S-rRNA sequencing approach. The results showed that E. prolifera polysaccharide could modulate intestinal microbiota (80). Furthermore, a metagenomic sequencing analysis study in the intestinal microbiota of rabbit fish S. oramin showed that E. prolifera diet could increase the abundance of Bacteroidetes bacteria, suggesting that this bacterium is responsible for the digestion of E. prolifera (81). All results indicated that E. prolifera polysaccharides could modulate gut microbiota and enhance SCFAs production, thereby regulating the health and homeostasis of the host. Further research is required to elucidate the influence of these polysaccharides on the immune-microbiome interactions at a cellular and molecular level.

Apart from the above-mentioned health benefits, E. prolifera polysaccharides were found to have other biological properties, such as removal of environmental pollutants, anticoagulant, antibacterial, anticancer, anti-tumor drug delivery, iron-chelating, moisture retention, gelling property, and growth-promoting effect.