Polysaccharides from Porphyra haitanensis: A Review of Their Extraction, Modification, Structures, and Bioactivities

Porphyra haitanensis (P. haitanensis), an important food source for coastal residents in China, has a long history of medicinal and edible value. P. haitanensis polysaccharides are some of the main active ingredients in P. haitanensis. It is worth noting that P. haitanensis polysaccharides have a surprising and satisfactory biological activity, which explains the various benefits of P. haitanensis to human health, such as anti-oxidation, immune regulation, anti-allergy, and anticancer properties. Hence, a systematic review aimed at comprehensively summarizing the recent research advances in P. haitanensis polysaccharides is necessary for promoting their better understanding. In this review, we systematically and comprehensively summarize the research progress on the extraction, purification, structural characterization, modification, and biological activity of P. haitanensis polysaccharides and address the shortcomings of the published research and suggest area of focus for future research, providing a new reference for the exploitation of polysaccharides from P. haitanensis in the fields of medicine and functional foods.


Introduction
Porphyra haitanensis (P.haitanensis), a temperate plant belonging to the genus Porphyra of the family Bangiaceae, is mainly distributed along the coasts of the Zhejiang, Fujian, and Guangdong provinces in China [1,2].P. haitanensis, commonly known as Tan-Zi-Cai, is an artificially cultivated seaweed unique to China [3]; it is not only an important food source for coastal residents but also has good medicinal value.According to classic ancient historical books of Chinese medicine, such as "Compendium of Materia Medica" (Ben-Cao-Gang-Ben, the Ming Dynasty) and "Thoroughly Revised Materia Medica" (Ben-Cao-Cong-Xin, the Qing Dynasty), P. haitanensis can treat beriberi, edema and cough.In addition, P. haitanensis is sweet and salty, cold, and naturally nontoxic and has the ability to resolve phlegm, soften firmness, and clear heat to eliminate toxicity and promote diuresis.Modern research has shown that P. haitanensis is rich in proteins, polysaccharides, vitamins, and essential amino acids [4][5][6].Therefore, P. haitanensis has a reputation as a "treasure house of nutrition" and is a high-grade nutritional and health food [7].
Polysaccharides, which are large molecules found in animal cell membranes, as well as in plant and microbial cell walls, are prevalent in biological systems [8,9].Previous studies have shown that polysaccharides are significant bioactive compounds in P. haitanensis, making up approximately 20% to 40% of its composition [10].P. haitanensis polysaccharides are composed primarily of galactose, glucose, mannose, arabinose, fucose, and other monosaccharides.The typical structure of P. haitanensis polysaccharides mainly includes repeating units of galactose and 3,6-anhydro galactose, as well as sulfate groups [11][12][13].In the past two decades, various in vivo and in vitro experimental studies have shown that P. haitanensis polysaccharides display various biological activities, including antioxidant [14,15],

Extraction of Polysaccharides from P. haitanensis
Extraction is the first indispensable step in the study and utilization of natural polysaccharides [25].Due to their polar macromolecular nature and intricate structures [26], polysaccharides can undergo glycosidic bond cleavage and conformational changes when subjected to various extraction methods and conditions, ultimately impacting their structure and biological functions [27].Therefore, scholars have developed and optimized polysaccharide extraction methods to obtain more bioactive polysaccharides from P. haitanensis.The known extraction methods for P. haitanensis, including hot water extraction (HWE), microwave-assisted extraction (MAE), and ultrasonic-microwave-assisted extraction (UMAE), are shown in Table 1.
Generally, to begin the extraction of P. haitanensis polysaccharides, a sample of P. haitanensis after crushing and drying needs to be soaked in ethanol [13] or treated with methanol/dichloromethane/water for 24 h in a shaking incubator at ambient temperature [11,28] to remove pigments and low-molecular-weight impurities.The detailed extraction and purification steps, structural characteristics, and biological activity of polysaccharides from P. haitanensis are shown in Figure 1.According to the literature, the polysaccharide yield of P. haitanensis obtained by conventional HWE is generally between 3.3% and 20.33% [11,29,30].Dong et al. obtained the best extraction conditions for P. haitanensis through a Box-Behnken response surface design, in which the ratio of material to water was 0.04, the extraction time was 3 h, and the temperature was 80 • C.This condition had little effect on the structure and function of the polysaccharides, and the extraction rate of PHP-XJ was optimized to 20.33% (±0.15) [31].Presently, the primary method of extracting P. haitanensis polysaccharides is HWE, which is favored for its affordability, ease of use, and low requirement for specialized equipment.The HWE technique can obtain a high yield of polysaccharides via an optimized process, but as a traditional extraction method, HWE still has the disadvantages of a long extraction time, difficult temperature adjustment, and high energy consumption [32].
polysaccharides was 20.98% when the temperature was controlled at 80 °C and the ratio of material to liquid was 1:42 g/mL for 30 min [37].UMAE, as a complementary technology with the combined advantages of MAE and UAE, reduces the extraction temperature, heats the material more evenly, reduces the extraction time, and greatly improves the extraction efficiency.In recent years, to simplify the extraction conditions of P. haitanensis polysaccharides, new extraction methods, such as MAE, have been adopted.By fully consulting the literature, it was found that when the liquid to solid ratio is 30:1-50:1 mL/g, the extraction time is 8-14 min, and the power is 78-300 W, the rate of PHP extraction by the MAE method is 3.6-5.1% [20,32].The MAE method uses high-frequency electromagnetic waves to penetrate the cell wall, resulting in cell rupture, so that the effective components can be extracted from the medium under uniform heating, eliminating the thermal gradient in the material, thus improving the extraction quality and effectively protecting the functional components in the sample [33,34].Compared with the conventional HWE method, the MAE method offers benefits such as an adjustable temperature, quick reaction time, and reduced solvent usage [35,36].Furthermore, Xu et al. used the UMAE method to extract P. haitanensis polysaccharides and optimized the extraction method by a response surface methodology.The results showed that the maximum yield of polysaccharides was 20.98% when the temperature was controlled at 80 • C and the ratio of material to liquid was 1:42 g/mL for 30 min [37].UMAE, as a complementary technology with the combined advantages of MAE and UAE, reduces the extraction temperature, heats the material more evenly, reduces the extraction time, and greatly improves the extraction efficiency.
In summary, the current methods for extracting polysaccharides from P. haitanensis are HWE, MAE, and UMAE, but there are few studies on the extraction optimization of the MAE method and UMAE method.In future research, the extraction optimization process of these two methods needs to be further studied.Moreover, although there have been recent developments in polysaccharide extraction technology, we found that there are few methods for polysaccharide extraction from P. haitanensis, which undoubtedly limits its research scope.Therefore, new extraction techniques should be investigated in subsequent studies, such as dilute lye and acid extraction [38], enzyme extraction [39], supercritical fluid extraction [40] and ultrahigh-pressure extraction [41].Regardless of the extraction method, the solvent ratio, temperature, and time affect the relative molecular weight and sulfuric acid content of P. haitanensis polysaccharides and subsequently affect their biological activity [8].Therefore, to increase polysaccharide extraction efficiency, new extraction methods and processes that are highly efficient and obtain high yields with high biological activity need to be explored through scientific research.20.53% [37] N/A means not mentioned.

Purification of P. haitanensis Polysaccharides
The crude polysaccharides obtained by preliminary extraction often contain pigments, inorganic small molecules, proteins, and other impurities, which may affect the analysis and identification of polysaccharides and even cause polysaccharide pollution [43].Purification is an indispensable part of the study of natural polysaccharides, and its purpose is to purify isolated mixed polysaccharides to obtain various uniform polysaccharides [44].In particular, highly biologically active nontoxic or low-toxicity components purified from food-borne natural polysaccharides are widely used in food, biological materials, and cosmetics [45].Therefore, after extracting crude polysaccharides from P. haitanensis using the above methods, it is essential to further separate and purify the polysaccharides to obtain high-quality refined polysaccharides to study their structural characteristics and biological activity more accurately.We summarize the steps for separating and purifying P. haitanensis polysaccharides in Figure 1, referencing the existing research.First, as mentioned above, P. haitanensis is pretreated before extraction to remove pigments and small molecular impurities.Second, the Sevag method [29,32] or Sevag enzymatic (papain) hydrolysis method [13,17] is used to remove macromolecular impure proteins with complex spatial structures, to prevent the combination of proteins and polysaccharides and reduce the difficulty of polysaccharide separation.Then, small molecular impurities (3-10 kDa molecular weight cut-off membrane) are further removed by distilled water dialysis [11,12,15].
In summary, high-purity and different kinds of P. haitanensis polysaccharides can be obtained by the above methods.However, different column chromatography methods are used in the purification process, and the biological activities of the purified components are also different.In addition, the loss of polysaccharides will inevitably occur during the purification process, and the cost of column chromatography is high, which is challenging for the subsequent development of P. haitanensis polysaccharides in functional foods and pharmaceutical products.Therefore, in future research, on the basis of ensuring the rich activity of P. haitanensis polysaccharides, green, efficient and economical purification methods for P. haitanensis polysaccharides that can be used in industrial production should be sought and optimized to further accelerate the application of P. haitanensis polysaccharides in practical markets and increase their use in functional foods and medicine.

Physicochemical and Structural Characteristics of Polysaccharides from P. haitanensis
In recent decades, studies on the physicochemical and structural characteristics of P. haitanensis polysaccharides have focused mainly on their monosaccharide composition, relative molecular weight, monosaccharide sequence, chemical structure, type of glycosidic bond, and configuration of glycosidic bonds.The relative molecular mass of P. haitanensis polysaccharides is determined by high-performance size exclusion chromatography (HPSEC) and high-performance gel permeation chromatography (HPGPC), while the monosaccharide composition is generally analyzed by gas chromatography-mass spectrometry (GC-MS) or high-performance liquid chromatography (HPLC).The basic chemical structure characteristics of the purified polysaccharide products are further identified by a series of analytical methods, such as gas chromatography (GC), Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR).The primary structural characteristics of P. haitanensis polysaccharides, including their sulfate content, monosaccharide composition, molecular weight, and chemical structure, are listed in Table 2.

Sulfate Content
Due to the special environment of the ocean, polysaccharides extracted from marine organisms often contain more sulfate groups than those extracted from terrestrial animals, plants, and microbial polysaccharides [49].Among seaweed plants, red algae are the main source of sulfate polysaccharides.Thus, as a red algae plant, P. haitanensis generally contains sulfate galactose polysaccharides [50].The sulfate content of polysaccharides is generally determined by the BaCl 2 -gelatine turbidimetric technique [11,19].In the polysaccharides of P. haitanensis, sulfate replaces the hydroxyl group on galactose on the polysaccharide chain, mainly in the form of →4)-α-L-galactose-6-sulfate linkages [12].Research has shown that sulfate accounts for a large proportion of polysaccharide components in P. haitanensis and that the sulfate content of various P. haitanensis polysaccharides generally ranges from 3.7-14.7%,as shown in Table 2.In addition, Ji et al. investigated the effects of different production areas and harvesting periods on the sulfate content in polysaccharides from P. haitanensis.The results showed that the polysaccharide PHPR03 extracted from Porphyra haitanensis picked in the third harvest period (late December) in Raoping had the highest sulfate content (14.70%), indicating that the sulfate content of P. haitanensis polysaccharides was also affected by the production area and harvesting period [51].Recent research indicates that the sulfate content is closely linked to the biological activity of polysaccharides derived from P. haitanensis.For example, Wang et al. reported that as the harvest period increased, the sulfate content in P. haitanensis polysaccharides decreased gradually, while the anti-allergic properties increased gradually [13].Moreover, Wu et al. reported that among the three polysaccharides isolated from P. haitanensis, the purified component PHP3 eluted with 0.7 M NaCl on a Sephadex G-100 column had the highest level of sulfate and the most potent antioxidant properties, suggesting that as the sulfate content increases, so does the antioxidant activity of polysaccharides [14].In summary, the sulfate group, as an important component of P. haitanensis polysaccharides, affects the structure and activity of polysaccharides.In the future, it will be highly important to isolate new active sulfated P. haitanensis polysaccharides for development and utilization.

Monosaccharide Composition
Generally, the monosaccharide composition of P. haitanensis polysaccharides is analyzed by acid hydrolysis and derivatization, followed by detection via GC-MS and HPLC.According to the published studies, a significant portion of the monosaccharide composition of polysaccharides from P. haitanensis is galactose.The galactose content of P. haitanensis polysaccharide obtained via different methods generally accounts for 24.35% to 95.60% of its monosaccharide composition.Chen et al. separated and purified waterextracted PHPs using a DEAE-Sepharose FF column and Sephacryl S-400HR column and obtained the homogeneous polysaccharide, PHP1, with only galactose in its monosaccharide composition [52].Furthermore, the extraction, separation, and purification techniques for P. haitanensis vary depending on the source, resulting in differing types and ratios of monosaccharides in the polysaccharides, leading to varying biological activities.For example, through water extraction, a probiotic active polysaccharide from P. haitanensis, known as PHP, was acquired.The composition of the substance was primarily galactose, glucose, and mannose, determined through high-performance liquid chromatography, with a molar ratio of 94.85:3.18:1.97[30].Moreover, Wang et al. further purified P. haitanensis polysaccharides by DEAEseult52 column chromatography and Sephadex G-100 gel column chromatography to isolate a polysaccharide, PH, with anti-colon cancer activity.GC-MS analysis revealed that PH was composed of Gal, Glu, Man, Ara, Fuc, Xyl, and Rha in a molar ratio of 98.66:0.23:0.49:0.07:0.05:0.07:0.44 [22].Three antioxidant polysaccharides, PHP1, PHP2, and PHP3, were obtained from PHPs by cellulose DEAE-52 and Sephadex G-100 column chromatography.GC-MS analysis revealed that these polysaccharides contained Rha, Xyl, Gal, Glc, and Man.Among them, the proportions of Rha and Xyl in PHP3 were greater than those in the other two polysaccharides, and the antioxidant activity of PHP3 was also the strongest, suggesting that the presence of Rha and Xyl in polysaccharides from P. haitanensis may affect their antioxidant properties [14].In addition to the above monosaccharides, P. haitanensis polysaccharides also contain 3,6-anhydrogalactose (3,6-AG), a crucial bioactive compound found in red algae polysaccharides [30].In the study of P. haitanensis polysaccharides, some researchers have regarded 3,6-AG as a separate component different from the composition of monosaccharides [19], but others classify 3,6-AG as a monosaccharide [29].However, regardless of the type of division, 3,6-AG is an important component of P. haitanensis polysaccharides.Furthermore, the variation in 3,6-AG levels and monosaccharide makeup in P. haitanensis polysaccharides is likely due to the source and timing of harvest of the raw materials, as well as the methods used for extraction and purification [32].Table 2 contains a comprehensive breakdown of the monosaccharide compositions found in the polysaccharides of P. haitanensis.
In summary, the different types and proportions of monosaccharides in the monosaccharide composition of P. haitanensis polysaccharides potentially affect their bioactivities.Hence, in future research, a more efficient and accurate ion chromatography method is needed to accurately determine the monosaccharide composition of P. haitanensis polysaccharides, laying a reliable foundation for subsequent research.In addition, for the diversification of the monosaccharide composition of P. haitanensis polysaccharides from different sources and different harvests, the fingerprint of their monosaccharide composition should be established in subsequent studies, and a reasonable quality evaluation, as well as biological activity assessment, of P. haitanensis polysaccharides should be carried out.

Molecular Weight
The average molecular weight (Mw), as a vital parameter for chemical compounds, is closely related to the structure and functional biological activity of polysaccharides.The molecular weight of P. haitanensis polysaccharides is analyzed mainly by HPGPC [12][13][14]53] and HPSEC [11].However, even for the same polysaccharide, the Mw detection results are different due to different extraction and purification processes and analysis methods.For example, the Mw of PHPs extracted from hot water was measured to be 201 kDa by HPSEC [11]; interestingly, for PHPs extracted from P. haitanensis using the same extraction method and analyzed by HPSEC-RID, their molecular weight was 250 kDa [54].In addition, Wu et al. used HPGPC to determine molecular weights, obtaining three polysaccharides, namely, PHP1, PHP2, and PHP3, with relative molecular weights of 499, 523, and 797 kDa, respectively, by DEAE-cellulose column separation and the purification of PHPs [14].Additionally, Wang et al. extracted and purified P. haitanensis from different harvest periods in November, December, and January and obtained three polysaccharides, namely, PHP1 (November), PHP2 (December), and PHP3 (January), with each exhibiting anti-allergic activity; the molecular weights were 567.05, 414.09, and 323.80 kDa, respectively.Notably, with the extension of the harvest period, the relative molecular mass of PHPS gradually decreased, but the anti-allergic activity gradually increased [13].The process, separation method, and detection conditions affect the molecular weight of polysaccharides, and the P. haitanensis itself (such as its source or harvest period) has a significant effect on the molecular weight, which in turn affects the biological activity of the polysaccharides.

Morphological Traits
The surface microstructure is also an important parameter for analyzing polysaccharides and is helpful for observing the diversity of polysaccharide structures and verifying the relationship between chemical groups and the conformation of polysaccharides.In recent years, the molecular morphology of polysaccharides from P. haitanensis has been analyzed by atomic force microscopy (AFM) and scanning electron microscopy (SEM).For example, the microstructure of PHSP (hp) was found to have an irregular geometric shape, a gap on the surface, and a loose overlap under SEM [58].In addition, Khan used SEM technology to determine that PHP had hexagons and a small number of rectangular pores, in addition to an irregular network structure and a loose microstructure.This loose and highly porous structure had good water absorption and water retention.Additionally, AFM once again confirmed the hollow structure of PHP-KC-AC.Under an atomic force microscope, some irregular fiber networks with hexagonal network structures can be observed, and the fiber height ranges from 0.23 to 0.92 nm, with an average of 0.45 ± 0.17 nm [11].Moreover, by using SEM and AFM, Ji et al. reported that PHPR03 exhibited a high level of antioxidant activity due to its loose microstructure, strong aggregation, and branching structure [51].The loose and dense porous structure of PHP03 suggested that it possesses excellent hydration properties, allowing complete absorption by the human body to enhance its antioxidant effects.Chen et al. analyzed two purified sulfated polysaccharides, PHP1 and PHP2, by AFM.The 3D AFM image of PHP1 showed a relief map of the aggregated polysaccharide chains, indicating that there was a strong interaction between the polysaccharide chains [52].In addition, as viewed with nano-analysis software, PHP2 exhibited a nonuniform spherical chain structure, indicating that the aggregates were significantly wider and longer than the individual polysaccharide chains [57].From the above microstructure, it can be inferred that the presence of sulfate groups between PHP1 and PHP2 can cause distortion and mutual transformation between sugar ring conformations and that hydroxyl groups can also interact with the branched chain structure.In short, the study and analysis of the surface ultrastructure characteristics of P. haitanensis polysaccharides can aid in understanding their chemical structure and spatial conformation, and the microstructural characteristics of polysaccharides can also reflect their physical and chemical properties and be used to identify potential biological activities.Therefore, in future development, the surface characteristics of the polysaccharides of P. haitanensis should be modified so that they can be used as bioactive materials, thus expanding the research and development scope of P. haitanensis.

Molecular Modifications of Polysaccharides from P. haitanensis
Polysaccharides, diverse large biological molecules, exhibit biological functions that are intricately linked to their structural composition [59].The methods used to extract and modify polysaccharides directly affect their composition and biological properties.The chemical modification of polysaccharides can significantly increase structural diversity, promote biological activity, and even increase new biological activity [60].P. haitanensis polysaccharides have rich functional groups and a variety of bioactivities.Molecular modification may improve the biological activity of P. haitanensis polysaccharides by changing their chemical structure.Common methods include polysaccharide degradation, phosphorylation, sulfation, acetylation, and the creation of drug-loaded nanoparticles.Previous studies have confirmed that chemical modification can effectively improve the structural and functional properties of polysaccharides from Porphyra haitanensis.Table 3 lists the modifications of P. haitanensis polysaccharides.

Degradation Modification
Degradation modification can reduce the molecular weight of polysaccharides and enhance their water solubility, so as to solve the problem that polysaccharides with too large a molecular weight have difficulty entering cells and cannot exert their activity [61].Currently, the methods used to degrade polysaccharides in P. haitanensis include the ascorbate (vitamin C) and hydrogen peroxide methods [7, 29,62] and the pectinase degradation method [17], among which the ascorbate and hydrogen peroxide methods are the most common.Based on the published studies, we found that degraded polysaccharides of P. haitanensis display rich biological activities, such as antioxidation [29,62], immune regulation [7, 17,53], anti-allergy [18] and antidepressant [63] properties, neurotoxin inhibition, and memory improvement [64].Furthermore, studies have shown that the activity of the degraded polysaccharide is different from that of the original polysaccharide.For example, after treatment with ascorbate and hydrogen peroxide, degraded polysaccharides of P. haitanensis have a greater ability to reduce iron ions and eliminate free radicals than do the original polysaccharide, showing stronger antioxidant activity [53,62].Similarly, Gong et al. reacted the purified polysaccharide PP3 with different proportions of H 2 O 2 -VC for different durations and obtained polysaccharides with different Mw values.The ability of the degraded polysaccharides to scavenge DPPH radicals, superoxide anion radicals, and hydroxyl radicals was greater than that of PP3.In particular, the polysaccharide PP3-4 (20 kDa), which has the smallest molecular weight, showed the highest antioxidant activity [29].In addition, using RSM, Li et al. determined the ideal parameters for the degradation of Porphyra haitanensis polysaccharide with pectinase: 50 U/mL pectinase, a temperature of 47.5 • C, and a pH of 5.0.In this scenario, CPH (Mw = 524 kDa) was degraded into DCPH (Mw = 217 kDa).DCPH demonstrated greater efficacy than CPH in decreasing malondialdehyde and reactive oxygen species production in RAW264.7 cells exposed to hydrogen peroxide, as indicated by in vitro studies.Moreover, DCPH showed the potential to boost the growth, phagocytosis, and nitric oxide secretion of RAW264.7 cells [17].In summary, numerous studies have shown that the antioxidant activity [29,53], anti-allergic activity [18] and immune regulation ability [7,17] of P. haitanensis polysaccharides improve after degradation, with the activity increasing as the molecular weight of the degraded polysaccharide decreases.Degradation can improve some of the biological activities of polysaccharides from P. haitanensis, which is undoubtedly an effective means to realize the full utilization and high-value development of polysaccharides from P. haitanensis and to develop new ideas and increase the potential application value of polysaccharides from P. haitanensis in food and medicine.

Sulfation Modification
Sulfation is a commonly used polysaccharide modification method.Polysaccharides have been shown to have excellent biological activity after sulfation, and studies have shown that sulfate derivatives exhibit good antioxidant, anticoagulant, anti-tumor, antibac-terial, and immunological activities [65][66][67].The sulfation of P. haitanensis polysaccharides was studied as early as 2009.For example, Zhang et al. used the sulfated reagent SO 3 -DMF produced from chlorosulfonic acid (HClSO 3 ) and N,N-dimethylformamide (DMF) to sulfate polysaccharides from P. haitanensis [68].FT-IR analysis revealed that the sulfated polysaccharides had two characteristic absorption peaks, which were located at 817 cm −1 and 1225 cm −1 , indicating that the sulfate group was connected to the primary hydroxyl group.In addition, compared with that of the original polysaccharide, the S=O vibration of the modified polysaccharide at 1249 cm −1 was increasingly wider, and the sulfate content of the modified polysaccharide increased from 15.6% to 27.6%.The above data indicated that the sulfation of polysaccharides was successful.In addition, the antioxidant activity test results indicated that the ability of sulfated P. haitanensis polysaccharides to scavenge superoxide anion radicals and hydroxyl radicals was greater than that of the original polysaccharides, which showed stronger antioxidant activity [68].Furthermore, the biological activity of sulfated polysaccharides is influenced by factors such as the level of substitution, position of substitution, molecular weight, and spatial conformation [69].Zhang et al. used 4,4 ′ -dimethoxytrityl chloride (DMT-Cl) to replace the hydroxyl groups at different positions on the disaccharide unit in P. haitanensis polysaccharides, introduced a DMT protective group, sulfated and modified it with SO 3 -DMF reagent, and deprotected it to obtain fully sulfated porphyrans, i.e., 2,2 ′ ,4-O-sulfated derivatives and 6-O-sulfated derivatives [70].The specific disaccharide unit structure is shown in Figure 3.The results showed that the sulfate contents of the total sulfated derivatives, 6-O-sulfated derivatives, and 2,2 ′ ,4-O-sulfated derivatives were 39.20%, 31.43%, and 22.72%, respectively.Among them, the completely sulfated and 6-O-sulfated derivatives exhibited a superior ability to scavenge superoxide anion radicals compared to the original polysaccharides and 2,2 ′ ,4-O-sulfated derivatives.Interestingly, the reducing power of the fully sulfated product with a higher degree of substitution was much weaker than that of the 6-O-sulfation product.Obviously, the sulfation of P. haitanensis polysaccharides at the O-6 position seems to increase their reducing power.The anticoagulant activity test results confirmed that the anticoagulant activity of completely sulfated derivatives significantly increased, while the anticoagulant activity of 6-O-sulfate derivatives was lower than that of 2,2 ′ ,4-O--sulfate derivatives, indicating that the sulfate group on C-6 was not necessary for anticoagulant activity [70].The above conclusions indicate that the biological activity of sulfated P. haitanensis polysaccharide derivatives depends not only on the degree of sulfation but also on the substitution position of the sulfate group.In summary, sulfation improves the anticoagulant and antioxidant characteristics of P. haitanensis polysaccharides, broadening the scope of their development and utilization.However, in general, there are few studies on the sulfation of P. haitanensis polysaccharides, the modification methods are relatively simple, and the structure-activity relationships of polysaccharides are not clear.Future research on the sulfation of P. haitanensis polysaccharides is expected to improve our understanding of the correlation between the chemical structure and biological effects of sulfated P. haitanensis polysaccharides, to develop new P. haitanensis polysaccharide products and expand the scope of their market application.

5-Fluorouracil Polysaccharide
5-Fluorouracil (5-FU) is a first-line chemotherapeutic drug with a wide range of activities against various types of cancers, and it can be used to treat solid tumors such as breast cancer, colorectal cancer, and head and neck cancer [71].Nevertheless, its anticancer effects are often accompanied by adverse side effects such as reproductive toxicity [72] and cardiac toxicity [73].Currently, studies have shown that the combination of 5-fluorouracil and natural plant polysaccharides can effectively alleviate and treat the serious side effects caused by the anticancer drug 5-fluorouracil [74][75][76].In 2009, Zhang et al. conducted a chloroacetylation reaction between P. haitanensis polysaccharide P and chloroacetic anhydride under the catalysis of DMAP, and then, the product after chloroacetylation was substituted with 5-fluorouracil to obtain the conjugate P-5FU (the reaction equation is shown in Figure 4).A release mechanism study revealed that this 5fluorouracil conjugate drug exhibited typical Fickian diffusion [77].Moreover, Zhang et al. fixed the 6th position of the unit structure of low-molecular-weight polysaccharide (LP) with 5-FU to obtain LP-5FU and then treated different groups of S180 tumor mice with 5-FU and LP-5FU in in vivo experiments.LP-5FU significantly reversed the decrease in the lymphocyte proliferation rate and TNF-α and NO levels induced by 5-FU, indicating that the conjugate can increase the anti-tumor activity of 5-FU and improve the immune function disrupted by 5-FU [78].Therefore, we speculate that P. haitanensis polysaccharides are good adjuvants for the treatment of cancer and that 5-FU can also be used as a new potential anticancer drug with low toxicity and side effects.

5-Fluorouracil Polysaccharide
5-Fluorouracil (5-FU) is a first-line chemotherapeutic drug with a wide range of activities against various types of cancers, and it can be used to treat solid tumors such as breast cancer, colorectal cancer, and head and neck cancer [71].Nevertheless, its anticancer effects are often accompanied by adverse side effects such as reproductive toxicity [72] and cardiac toxicity [73].Currently, studies have shown that the combination of 5-fluorouracil and natural plant polysaccharides can effectively alleviate and treat the serious side effects caused by the anticancer drug 5-fluorouracil [74][75][76].In 2009, Zhang et al. conducted a chloroacetylation reaction between P. haitanensis polysaccharide P and chloroacetic anhydride under the catalysis of DMAP, and then, the product after chloroacetylation was substituted with 5-fluorouracil to obtain the conjugate P-5FU (the reaction equation is shown in Figure 4).A release mechanism study revealed that this 5-fluorouracil conjugate drug exhibited typical Fickian diffusion [77].Moreover, Zhang et al. fixed the 6th position of the unit structure of low-molecular-weight polysaccharide (LP) with 5-FU to obtain LP-5FU and then treated different groups of S180 tumor mice with 5-FU and LP-5FU in in vivo experiments.LP-5FU significantly reversed the decrease in the lymphocyte proliferation rate and TNF-α and NO levels induced by 5-FU, indicating that the conjugate can increase the anti-tumor activity of 5-FU and improve the immune function disrupted by 5-FU [78].Therefore, we speculate that P. haitanensis polysaccharides are good adjuvants for the treatment of cancer and that 5-FU can also be used as a new potential anticancer drug with low toxicity and side effects.

Other Modification Methods
Phosphorylation and acetylation are also common modification methods of polysaccharides from P. haitanensis.Phosphorylation is a frequently utilized chemical modification technique that can improve the physical and chemical properties of natural polysaccharides, thus enhancing their anti-tumor, antioxidant, and other biological activities [79].P. haitanensis polysaccharides have been dissolved in formamide and treated with tributylamine and polyphosphoric acid to obtain phosphorylated P. haitanensis polysaccharides [68,80].FT-IR analysis revealed that phosphorylated P. haitanensis polysaccharides have two special absorption peaks: one at 1268 cm −1 , which represents the stretching vibration related to P=O, and the other at 988 cm −1 , which represents the vibration related to P-O [80].The introduction of phosphate groups can activate the hydrogen atoms on the isomer carbon, thereby enhancing the scavenging effect of DPPH free radicals and resulting in good antioxidant capacity [68].Moreover, acetylation can also effectively improve the biological activity of polysaccharides [81].For example, P. haitanensis polysaccharides are acetylated using acetic anhydride as an acylating agent, with a distinct peak appearing at 1726 cm −1 or 1730 cm −1 in the infrared spectrum as a result of C=O stretching vibrations [68,82].Compared with natural P. haitanensis polysaccharides, acetylated P. haitanensis polysaccharides have a stronger free scavenging ability and reducing energy for superoxide anions [44,58,59].The reason may be that acetylated derivatives have a greater hydrogen supply capacity, which can prevent the formation of hydrogen peroxide, thus affecting their antioxidant capacity and

Other Modification Methods
Phosphorylation and acetylation are also common modification methods of polysaccharides from P. haitanensis.Phosphorylation is a frequently utilized chemical modification technique that can improve the physical and chemical properties of natural polysaccharides, thus enhancing their anti-tumor, antioxidant, and other biological activities [79].P. haitanensis polysaccharides have been dissolved in formamide and treated with tributylamine and polyphosphoric acid to obtain phosphorylated P. haitanensis polysaccharides [68,80].FT-IR analysis revealed that phosphorylated P. haitanensis polysaccharides have two special absorption peaks: one at 1268 cm −1 , which represents the stretching vibration related to P=O, and the other at 988 cm −1 , which represents the vibration related to P-O [80].The introduction of phosphate groups can activate the hydrogen atoms on the isomer carbon, thereby enhancing the scavenging effect of DPPH free radicals and resulting in good antioxidant capacity [68].Moreover, acetylation can also effectively improve the biological activity of polysaccharides [81].For example, P. haitanensis polysaccharides are acetylated using acetic anhydride as an acylating agent, with a distinct peak appearing at 1726 cm −1 or 1730 cm −1 in the infrared spectrum as a result of C=O stretching vibrations [68,82].Compared with natural P. haitanensis polysaccharides, acetylated P. haitanensis polysaccharides have a stronger free scavenging ability and reducing energy for superoxide anions [44,58,59].The reason may be that acetylated derivatives have a greater hydrogen supply capacity, which can prevent the formation of hydrogen peroxide, thus affecting their antioxidant capacity and resulting in stronger antioxidant activity.In addition, drug-loaded polysaccharide metal nanoparticles have also been used as a promising drug delivery system.The principle involves using polysaccharides with an enhanced biological tolerance to encapsulate drug-loaded molecules and load them onto the surface of nanoparticles, so that the conjugates have both a physiological affinity and anti-efflux properties, thereby addressing treatment failure due to the limited delivery of drugs to the target site [83].Hence, to overcome the poor bioavailability and photostability of resveratrol (RES), Xu et al. prepared OPRs with ovalbumin (OVA) and a P. haitanensis polysaccharide (PHP) as encapsulation agents.In vitro simulated digestion experiments showed that compared with OVA-RES nanoparticles (ORs), OPR was released more slowly in the stomach and faster in the intestine.In addition, OPR nanoparticles were shown to inhibit the growth of HeLa and HepG2 tumor cells and effectively deliver Res to biological targets, indicating that the OVA-PHP system may enhance the bioavailability and stability of resveratrol [84].N/A means not mentioned.

Biological Activities of P. haitanensis Polysaccharides
P. haitanensis is a kind of medicinal and edible plant from China, and polysaccharide is one of the important bioactive components in P. haitanensis.In recent years, numerous studies have validated the diverse biological and pharmacological effects of P. haitanensis polysaccharides, such as antioxidative, immunomodulatory, anti-allergic, anticancer, antiaging, hypoglycemic, anticoagulant, anticomplement, anti-diarrheal, and other effects.The biological activities and health benefits of P. haitanensis polysaccharides are discussed in the following paragraphs and summarized in Table 4.Typical and representative molecular mechanisms of pharmacological activity-for example, their immunomodulatory, antiallergic, anti-tumor, and anti-aging activities-are also summarized and presented in Figures 5-8.[15]

P1 P2 (S) P3 (A)
In vitro DPPH superoxide radicals and ferric ion reducing 0 to 5 mg/mL The sulfated derivative with certain DS showed stronger antioxidant activity.The acetylated derivative showed the most excellent antioxidant activity. [82]

AP (A) PP (P) BP (B)
In vitro Hydroxyl, superoxide anion radicals, and reducing powers N/A The acetylation, phosphorylation, and benzoylation derivatives of P. haitanensis polysaccharides showed stronger antioxidant activity than the original polysaccharides. [80]

DCPH-TZ-AO (D)
In vitro Ferric reducing power 1 to 5 mg/mL The total antioxidant capacity is significant.

Antioxidant Activity
Oxidative stress refers to an imbalance between oxidation and antioxidant systems in the body [87].The results of recent studies suggest that oxidative stress may lead to numerous severe illnesses, including chronic obstructive pulmonary disease, atherosclerosis, cancer, and Alzheimer's disease [88].As biomacromolecules with good biocompatibility [89], natural polysaccharides, which are widely used in the biomedical and pharmaceutical fields, have significant antioxidant effects and can reduce oxidative stress damage [90].In biological systems, the inappropriate production of endogenous and exogenous reactive oxygen species (ROS) leads to conformational and oxidative changes in key biomolecules, resulting in oxidative damage to cells [91].Khan et al. reported that a P. haitanensis polysaccharide (PHP-KC-AC) was most effective at scavenging ABTS free radicals, followed by DPPH radicals and hydroxyl radicals, and that the antioxidant ability of PHP-KC-AC increased in a dose-dependent manner.As the concentration of PHP-KC-AC increased from 0.0625 mg/mL to 2.0 mg/mL, the scavenging rates of ABTS, DPPH, and hydroxyl radicals increased gradually.Moreover, this dose dependence was more obvious at high concentrations, and the highest activity clearance rates at 2.0 mg/m L were 34.63%, 23.80%, and 53.16% for ABTS, DPPH, and hydroxyl radicals, respectively [11].Subsequently, Wu et al. isolated PHP using cellulose DEAE-52 ion exchange and Sephadex G-100 column chromatography, yielding three sulfated polysaccharide fractions (PHP, PHP2, and PHP3).Through an assessment of antioxidant activity in vitro, it was found that the three components showed strong superoxide anion free radical, hydroxyl radical, and DPPH free radical scavenging ability and reducing ability; in general, the order of activity was PHP3 > PHP2 > PHP1, and this order was also the order of sulfate content, indicating that the antioxidant activity of the three components was positively correlated with the sulfate content.In addition, in RAW264.7 cells treated with H 2 O 2 , the ability of PHP3 to reduce intracellular ROS levels was the strongest among the three components, and at a dose of 400 µg/mL, the ROS level decreased by 182.37% compared with the level observed in normal cells.Similarly, PHP3 can also reduce the ROS level and MDA content in H 2 O 2 -treated RAW264.7 cells and enhance the activities of catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD), thus resulting in better antioxidant activity [14].Furthermore, a degraded P. haitanensis polysaccharide (DCPH, Mw = 217 kDa) showed stronger free radical scavenging activity and iron ion reduction ability than did another P. haitanensis polysaccharide (CPH, Mw = 524 kDa).CPH and DCPH decreased the levels of propylene glycol and ROS inside RAW264.7 cells treated with H 2 O 2 , and this effect was dependent on the dose.DCPH exhibited a more potent effect than CPH.The reason may be that the degradation process exposes more hydroxyl groups on DCPH, and that low-molecularweight DCPH is more conducive to penetrating the cell membrane, thereby facilitating interactions with intracellular substances [17].
In addition, through animal experiments, Zhang et al. found that P. haitanensis polysaccharides also showed significant antioxidant activity in vivo.After the intragastric administration of 50, 100, and 200 mg/kg P. haitanensis (F1) polysaccharides for 28 days, the activities of SOD in the serum, liver, heart, and brain of aged Kunming mice increased in a dose-dependent manner.Compared with that in the aging model group, the level of GSH-Px in the administration group was also obviously increased.The dose that resulted in the greatest increase in GSH-Px activity in the liver and brain was 5.0 mg/kg, and the greatest increase in GSH-Px activity in the heart was observed at a dose of 100 mg/kg.At doses of 100 and 200 mg/kg, F1 evidently inhibited the production of MDA in the liver and heart of aging mice, and the inhibitory effect of a 200 mg/kg dose of F1 on MDA was comparable to or stronger than that of the same dose of vitamin C (a positive control drug) [56].In conclusion, P. haitanensis polysaccharides have good antioxidant effects, are a good natural antioxidant, and can be used for nutritional and health food development and utilization.However, there are few studies on the in vivo antioxidant activity of polysaccharides from P. haitanensis, and the mechanism of in vivo activity is not clear.Therefore, it is necessary to further carry out scientific research on antioxidation in vivo, and fully analyze and summarize the antioxidation effects of polysaccharides from P. haitanensis, in vitro and in vivo, so as to provide a more valuable theoretical basis for the clinical application of P. haitanensis polysaccharides.

Immunomodulatory Activity
The body's first line of defense against threats is the immune system; it can not only identify hosts and pathogens, to eliminate infection and maintain the body's dynamic balance, but it can also protect the body from bacteria, fungi, viruses, and other infections and eliminate abnormal cells such as tumor cells through cellular and humoral immunity [92,93].Research has shown that natural plant polysaccharides have good immunomodulatory effects and are good immune enhancers.Polysaccharides can attach to receptors on the surface of cells, which can increase engulfment by macrophages and stimulate the release of cytokines and chemokines by macrophages [94,95].P. haitanensis polysaccharides can also facilitate the proliferation of immune cells and the production of cytokines by immune cells, thereby improving the immune response and enhancing the body's immune system.In recent years, in vivo and in vitro studies have shown that P. haitanensis polysaccharides have good immunomodulatory effects; their possible effects and mechanisms of action are shown in Figure 5.
Macrophages, a crucial component of the immune system, serve as the initial barrier against both internal and external pathogens [96], regulating a variety of dynamic balances and evolutionary host defense immune responses [97].In vitro experiments have shown that P. haitanensis polysaccharides can greatly improve the survival rate of RAW 264.7 cells, increase phagocytic activity in a dose-dependent manner, and stimulate the release of TNF-α, IL-6, and IL-10.Moreover, P. haitanensis polysaccharides activate the JNK and JAK2 signaling pathways in RAW 264.7 cells, induce increased iNOS mRNA expression, and promote NO production [16].Similarly, Gong et al. treated RAW 264.7 cells with different concentrations of purified polysaccharide (PHPD-IV-4) from 12.5 µg/mL to 200 µg/mL.Their findings indicated notable increases in the phagocytic capacity, intracellular acid phosphatase activity, and NO generation of RAW 264.7 cells, and the phagocytic activity (phagocytosis index = 1.57), acid phosphatase activity (vitality index = 1.37), and NO production ability (28.67 µg/mL) were greatest when the concentration of PHPD-IV-4 was 200 µg/mL.Furthermore, Western blot results demonstrated that PHPD-IV-4 notably increased the phosphorylation levels of three components of the MAPK signaling pathway, ERK1/2, P38, and JNK, in a dose-dependent fashion, suggesting that PHPD-IV-4 modulates macrophage immune activity by enhancing the phosphorylation of MAPK signaling molecules [7].
Dendritic cells (DCs) are antigen-presenting cells that are crucial for initiating and controlling both innate and adaptive immune reactions [98].CD4+ regulatory T cells are important for maintaining immune self-tolerance and a dynamic balance [99].Liu et al. studied the immunomodulatory activity of P. haitanensis sulfated polysaccharides (PHSPs) in BALB/c mouse models.According to the flow cytometry results, PHSPs enhanced the generation of TNF-α and IL-10 in mice, with CD4+ spleen T lymphocytes, DCs, and Tregs showing notable increases in mice treated with PHSPs [16].Combined with the experimental results of Liu et al., these findings confirmed that PHSPs play a key role in immune regulation.Subsequently, Fu et al. reported that a P. haitanensis polysaccharide (PH) can activate the NF-κB signaling pathway in mouse spleen cells, thereby inducing Treg proliferation.In addition, PH may promote the production and release of Th1 cytokines (IFN-γ, TNF-α and IL-2) and promote and inhibit the secretion and expression of the Th2 cytokines IL-4 and IL-5, respectively.This demonstrates that PH has the ability to control the equilibrium between Th1 and Th2 cells by regulating the secretion and gene expression of Th1 and Th2 cytokines, thus maintaining a dynamic immune balance [10].In summary, the immunomodulatory effect of polysaccharides from P. haitanensis is mainly achieved by regulating the activity of immune cells such as lymphocytes, macrophages, and DCs and promoting or inhibiting the expression of immune factors to maintain the Th1/Th2 balance.This indicates that P. haitanensis polysaccharides can be used as immunomodulators or immune supplements in food and medicine to treat immune diseases.However, the available studies on the immunomodulatory effects of P. haitanensis are insufficient, and there is a lack of research and discussion on the mechanisms of action involved.Therefore, it is necessary to further study the immunomodulatory effects and mechanisms of action of P. haitanensis polysaccharides, especially in vivo, to establish a strong theoretical foundation for their development and application.

Anti-Allergy Activity
Food allergy is an immune system response that occurs shortly after eating certain foods, affecting more than 10% of the global population and causing severe harm to individuals with allergies [100].Studies have shown that natural polysaccharides have a good ability to mitigate food allergies and can be used as potential products for preventing and treating food allergies [101].Shi et al. studied the anti-allergic ability of P. haitanensis sulfated polysaccharide (PHSPs) in a tropomyosin (TM)-induced model of allergy in mice.Mice were sensitized through the intraperitoneal administration of TM (100 µg/mouse) at 0 and 14 days.The sensitized mice received intraperitoneal injections of PHSPs three times weekly for 15 days, starting either 7 days prior to sensitization (PHSP prevention group) or 1 day after the initial immunization (PHSP treatment group).These findings indicated that PHSPs significantly decreased the expression of Th2 cytokines (including IL-4, IL-5, and IL-13), while enhancing the release of Th1 cytokines (such as IFN-γ and IL-10).This regulatory change affected the levels of various specific immunoglobulins in serum.PHSP intervention before TM sensitization significantly decreased the level of TM-specific immunoglobulin IgE in serum and increased the level of TM-specific IgG2a.After TM sensitization, there was no significant change in TM-specific IgE or IgG2a in the serum after PHSP treatment, but the TM-specific IgG1 level was significantly decreased.In addition, further studies have shown that PHSPs can activate the JNK and JAK2 pathways to induce IFN-γ secretion [19].These results strongly suggest that PHSPs prevent or ameliorate TM-induced allergic reactions by regulating the Th1/Th2 immune imbalance.Moreover, Wei et al. studied the antigen-specific immunity of a P. haitanensis polysaccharide (PP) in ovalbumin (OVA)-sensitized mice.Mice received injections of 40 and 80 µg/mouse OVA on days 4 and 8, respectively, and were treated with 25, 150, or 250 mg/kg PP daily for 30 days.PP treatment decreased the levels of the two immunoglobulin subclasses, IgG1 and IgG2a, which assist Th2 and Th1 immune responses, and PP significantly decreased the concentration of the OVA-specific immune protein IgE at doses of 150 and 250 mg/kg.Additionally, PP inhibited the production of Th17 cytokines (IL-17) and Th2 cytokines (IL-4 and IL-2) and inhibited and promoted the expression of the Th1 cytokines IFN-γ and IL-10b, respectively, leading to a notable increase in the IL-10/IL-4 ratio [85].In summary, PP can shift the Th1/Th2 balance to Th1 polarization, thereby regulating antigen-specific immune responses and reducing allergic reactions.In addition, research was conducted on the anti-allergic properties of degraded polysaccharides from P. haitanensis and polysaccharides from P. haitanensis harvested at various times.The lower the molecular weight and sulfate content were, the better the anti-allergic activity was [13,18].
In conclusion, P. haitanensis polysaccharides can inhibit allergic reactions by regulating Th1/Th2 immune imbalances.We summarize various possible relationships involved in this regulatory process in Figure 6.However, the regulatory effect of P. haitanensis polysaccharides on allergic reactions is complex and changeable.For example, the effects of P. haitanensis polysaccharides on Th1 cytokines (IFN-γ) and the immunoglobulin subclass IgG2a are different in different environments.For example, mice were treated with an intraperitoneal injection of P. haitanensis polysaccharides, which promoted the secretion of IFN-γ and IgG2a, while Wei et al. found that the intragastric administration of P. haitanensis polysaccharides reduced the levels of IFN-γ and IgG2.The reason for the differences may be that the different routes of administration affect the absorption, distribution, and metabolism of polysaccharides in the body, which in turn alters their effects.In addition, the application range of P. haitanensis polysaccharides is different, and their mechanisms of action are also different.As shown above, the effects of P. haitanensis polysaccharides on immunoglobulin in allergic reactions are also different.Therefore, future studies should further clarify the anti-allergic mechanism of P. haitanensis polysaccharides and explore the effects of different administration routes or drug dosage forms on their activity, to provide a theoretical basis for the development of anti-allergic health foods and drugs containing P. haitanensis polysaccharides and to broaden the research scope of P. haitanensis polysaccharides.

Anti-Tumor Activity
Cancer is a highly dangerous disease that has become the most serious threat to human life and health, solidifying it as a major public health problem worldwide [102].Polysaccharides are bioactive macromolecules that are considered potential anticancer candidates because of their anti-tumor activity and nontoxic properties [103].Seaweed polysaccharides have been shown to be important active substances in seaweed and to display anticancer activity [104,105].Polysaccharides of P. haitanensis, a food homologous to traditional Chinese medicines derived from algae, show potential as candidate drugs for cancer treatment due to their anticancer activity.Chen et al. studied a polysaccharide (PHP) extracted from P. haitanensis by MAE and found that it had obvious anti-tumor effects in vitro and in vivo.The suppressive impact of PHP on the proliferation of human gastric carcinoma SGC-7901 cells was confirmed through in vitro experimentation.PHP was found to suppress the growth of SGC-7901 cells in a dose-dependent manner.With an increasing PHP concentration (10-500 µg/mL), the inhibition rate also gradually increased, and the inhibition rate of cell proliferation increased from 8.25% to 70.40%.In addition, PHP induced apoptosis in SGC-7901 cells.At 500 µg/mL, the percentage of apoptotic cells was 19.65%, and the percentage of apoptotic cells was positively correlated with the PHP concentration.In vivo experiments showed that PHP had a strong anticancer impact on mice with SGC-7901 tumors.At doses of 80 and 160 mg/kg/d, the tumor inhibition rates were 67.55% and 77.04%, respectively, which were better than that of the positive control drug 5-Fu (58.3157%) [20].Furthermore, Yao et al. isolated and purified three components (PHP-F1, PHP-F2, and PHP-F3) from the polysaccharides of P. haitanensis.In vitro studies revealed that the three components inhibited the proliferation of human colon cancer cells (HT-29, LoVo, and SW-480), with PHP-F2 and PHP-F3 demonstrating particularly potent inhibitory effects on HT-29 cells.Further studies revealed that PHP-F2 and PHP-F3 can effectively trigger oxidative stress in HT-29 cells and promote apoptosis.Moreover, PHP-F2 and PHP-F3 can arrest cells in the G0-G1 phase and prolong cell cycle progression, thereby inhibiting cell division and ultimately leading to apoptosis [21].Moreover, a P. haitanensis polysaccharide (PH) exhibited a notable suppressive effect on CMT93 colon cancer cells.PH inhibited the expression of the core transcription coactivator and regulator YAP1 in a dose-dependent manner, activated the Hippo pathway, caused crosstalk between the Hippo and mTOR pathways, and activated the mTOR pathway, thereby inhibiting the expression of the CTGF and Ankrd1 genes and thus inhibiting the proliferation of intestinal epithelial cells [22].core transcription coactivator and regulator YAP1 in a dose-dependent manner, activated the Hippo pathway, caused crosstalk between the Hippo and mTOR pathways, and activated the mTOR pathway, thereby inhibiting the expression of the CTGF and Ankrd1 genes and thus inhibiting the proliferation of intestinal epithelial cells [22].Based on the above findings, we summarized and analyzed the possible anti-tumor mechanism of P. haitanensis polysaccharides, as shown in Figure 7. P. haitanensis polysaccharides can achieve anti-tumor effects by interfering with the cell cycle and the Hippo/mTOR pathway.However, the existing anti-tumor research on P. haitanensis polysaccharides is still relatively basic, and there is a lack of in vivo experimental studies.The immune system is crucial for monitoring cancer cells [93], and P. haitanensis polysaccharides are good immunomodulatory substances.Whether the immunomodulatory effect of P. haitanensis in vivo affects its anti-tumor activity remains to be determined.Therefore, the relationship between the immune activity and anti-tumor activity of polysaccharides from P. haitanensis should be further explored in future studies.We hope to develop a product that can inhibit tumor growth without side effects, while enhancing the body s immune system.Based on the above findings, we summarized and analyzed the possible anti-tumor mechanism of P. haitanensis polysaccharides, as shown in Figure 7. P. haitanensis polysaccharides can achieve anti-tumor effects by interfering with the cell cycle and the Hippo/mTOR pathway.However, the existing anti-tumor research on P. haitanensis polysaccharides is still relatively basic, and there is a lack of in vivo experimental studies.The immune system is crucial for monitoring cancer cells [93], and P. haitanensis polysaccharides are good immunomodulatory substances.Whether the immunomodulatory effect of P. haitanensis in vivo affects its anti-tumor activity remains to be determined.Therefore, the relationship between the immune activity and anti-tumor activity of polysaccharides from P. haitanensis should be further explored in future studies.We hope to develop a product that can inhibit tumor growth without side effects, while enhancing the body's immune system.

Anti-Aging Activity
Aging is considered one of the greatest known risk factors for morbidity and mortality.As early as 2008, some scholars found that polysaccharides from P. haitanensis could effectively enhance the vitality of middle-aged Drosophila melanogaster and prolong its life span.In particular, polysaccharides with lower molecular weights may be more conducive to increasing the growth of Drosophila melanogaster [23].The oxidative stress theory is considered a famous aging mechanism theory.Mitochondrial ROS are thought to significantly contribute to the aging process, as oxidative stress-induced mitochondrial dysfunction is recognized as a key factor in aging [106].In addition, the aging of the brain is linked to oxidative damage to nuclear and mitochondrial DNA, which involves the regulation of various systems by reactive oxygen species (ROS) through different mechanisms [107].The effect of P. haitanensis polysaccharides on premature senescence induced by H2O2 in a WI-38 model was studied in vitro.P. haitanensis polysaccharides had obvious growth-promoting effects on cells.At a polysaccharide concentration of 10 μg/mL, the viability of prematurely senescent WI-38 cells increased from 70.3% to 88.5%; additionally, P. haitanensis polysaccharides significantly restored the destruction of cell morphology caused by H2O2.Moreover, polysaccharides from P. haitanensis suppressed the activation of the p53 and p21 proteins and decreased ROS production in cells, thus preventing oxidative stress and minimizing DNA damage in aging cells [24].In recent years, it has been recognized that aging is a sign of the aging of the nervous system, manifesting as memory loss and a deterioration in mental state [108].Zhang et al. reported that the degradation of a P. haitanensis polysaccharide (DPPH) improved the

Anti-Aging Activity
Aging is considered one of the greatest known risk factors for morbidity and mortality.As early as 2008, some scholars found that polysaccharides from P. haitanensis could effectively enhance the vitality of middle-aged Drosophila melanogaster and prolong its life span.In particular, polysaccharides with lower molecular weights may be more conducive to increasing the growth of Drosophila melanogaster [23].The oxidative stress theory is considered a famous aging mechanism theory.Mitochondrial ROS are thought to significantly contribute to the aging process, as oxidative stress-induced mitochondrial dysfunction is recognized as a key factor in aging [106].In addition, the aging of the brain is linked to oxidative damage to nuclear and mitochondrial DNA, which involves the regulation of various systems by reactive oxygen species (ROS) through different mechanisms [107].The effect of P. haitanensis polysaccharides on premature senescence induced by H 2 O 2 in a WI-38 model was studied in vitro.P. haitanensis polysaccharides had obvious growth-promoting effects on cells.At a polysaccharide concentration of 10 µg/mL, the viability of prematurely senescent WI-38 cells increased from 70.3% to 88.5%; additionally, P. haitanensis polysaccharides significantly restored the destruction of cell morphology caused by H 2 O 2 .Moreover, polysaccharides from P. haitanensis suppressed the activation of the p53 and p21 proteins and decreased ROS production in cells, thus preventing oxidative stress and minimizing DNA damage in aging cells [24].In recent years, it has been recognized that aging is a sign of the aging of the nervous system, manifesting as memory loss and a deterioration in mental state [108].Zhang et al. reported that the degradation of a P. haitanensis polysaccharide (DPPH) improved the cognitive function and memory deficits caused by neurotoxic amyloid β peptide (Aβ) in mice with Alzheimer's disease (AD).AD mice were continuously treated with DPPH (75, 150, 300 mg/kg) for 16 days.The results from the water maze test indicated that the number of times the mice entered the maze and the time it took for them to find the exit on the third day were notably shorter in the 300 mg/kg group than in the control group (p < 0.05).Furthermore, the number of errors and time to exit decreased significantly in all the dose groups after the fifth day (p < 0.01).In addition, further studies have shown that DPPH can increase acetyltransferase (CHAT) activity in the cerebral cortex and hippocampus, and reduce acetylcholinesterase (AChE) activity and increase acetylcholine content in brain tissue, thereby improving Aβ1-40-induced learning and memory impairment in mice [64] (the possible detailed mechanism of action is shown in Figure 8).The above results indicate that P. haitanensis polysaccharides can delay aging and improve memory, suggesting that they can be developed as a new type of anti-aging health food to prevent neurodegenerative diseases.

Prebiotic Activity
In recent years, there has been increased focus on the impact of the gastrointestinal microflora on human health and diseases.The human gastrointestinal microbiota is a highly diverse microbial population, whose composition and diversity are associated with a variety of pathologies [109].Moreover, immune cells and symbiotic microorganisms in the human intestinal tract constantly communicate and react with each other in a stable environment to maintain healthy immune activities [110].Recent studies have indicated that natural active polysaccharides can regulate gastrointestinal function, contribute to the growth of probiotics in the intestine, and inhibit the formation of harmful bacteria in the intestine.Through in vitro fermentation experiments with intestinal microorganisms, Ying et al. fermented a polysaccharide from P. haitanensis (PHP) in the feces of healthy adults and found that PHP significantly promoted the growth of beneficial bacteria such as Bacteroides thetaiotaomicron, Bacteroides ovatus, Defluviitalea saccharophila, and Faecalibacterium prausnitzii; additionally, this selective influence on the intestinal flora led to an increase in the diversity of the human gut microbiota [30].Chen et al. isolated and purified two polysaccharide components (PHP1 and PHP2) from PHP and then examined the fermentation of PHP1 and PHP2 in rat intestinal microbiota in vitro.PHP1 enhanced the development of bacteria that produce propionic acid and the synthesis of propionic acid; promoted the growth of Ruminococcaceae_NK4A214, Christensenellaceae_R-7, Fusicatenibacter, Ruminiclostridium_5, Blautia, E. coprostanoligenes, Desulfovibrio, Lactobacillus, and Parasutterella; regulated microbial diversity; and increased the abundance of beneficial microflora.In addition, the molecular weight of PHP1 decreased from 6.68 × 10 6 g/mol to 5.46 × 10 5 g/mol during fermentation.The reason was that the increase in Bacteroides, Ruminococcaceae, Bifidobacterium, and other genera caused the cleavage of glycosidic bonds in PHP1.For example, Bifidobacteria glycosyl hydrolases can hydrolyze the α-or β-galactose glycosidic bond in PHP1 [52].Additionally, through metabolic studies, it was found that the microbial flora consumed galactose and mannose in PHP2 during in vitro fermentation, resulting in less metabolism of microbial fructose, mannose, and galactose.PHP2 also reduced amino acid metabolism and carbohydrate metabolism in the gut microbiota.In addition, PHP2 inhibited the biosynthesis of secondary bile acids and primary bile acids in the microflora and reduced the level of intestinal bile acids.Moreover, PHP2 regulated the composition of the fecal microbiota in rats and greatly increased the abundance of the dominant genera Escherichia-Shigella and Lactobacillus.The analysis showed that a decrease in bile acid levels could promote the conversion of cholesterol to bile acid in the liver, suggesting that PHP2 regulates cholesterol metabolism [57].The above results show that P. haitanensis polysaccharides have great potential for development into new probiotic products for use in functional foods or by the pharmaceutical industry.However, research on the effect of P. haitanensis polysaccharides on the intestinal flora is limited to in vitro experiments.More studies on the effect of P. haitanensis polysaccharides on intestinal flora in in vivo treatments are needed to increase the efficacy of P. haitanensis polysaccharides in improving or regulating microbial flora.

Other Activities
In addition to the above biological activities, P. haitanensis polysaccharides also have other pharmacological effects, including hypoglycemic, hypolipidemic, anti-complement, anti-diarrheal, and anticoagulation effects.For example, Mesocricetus auratus fed a high-fat diet was also fed PHP at doses of 100 mg and 200 mg/kg/d for 4 weeks.PHP in both dose groups significantly suppressed an increase in serum total cholesterol (TC), total triglyceride (TG), and low-density lipoprotein cholesterol (LDL) levels.PHP (100 mg/kg) downregulated the expression of a fat chain extension gene (Elovl6), a fatty acid synthesis gene (FASN and Pnpla3), a fat degradation gene (Abhd5), and an adipogenesis gene (ME1), thereby reducing triglyceride and blood lipid levels.PHP (200 mg/kg) also inhibited the expression of a fatty acid oxidation gene (Acacb) and fatty acid transporter gene (CD36) by activating the liver PPARg signaling pathway, thereby reducing liver triglyceride and blood lipid levels [86].Additionally, Cao et al. reported that a polysaccharide (APHP) extracted from waste P. haitanensis stimulated the proliferation of β-cells in a mouse model of diabetes induced by alloxan, promoted the regeneration of damaged islet cells, and significantly reduced blood glucose levels.Additionally, APHP improved antioxidant activity, reduced ROS levels, reduced the degree of damage to β-cells, reduced LDL, TG, and TC levels, increased high-density lipoprotein (HDL) cholesterol levels, and restored blood lipid levels [42].These results indicate that P. haitanensis polysaccharides have good hypoglycemic and hypolipidemic activities and can potentially be used as functional foods for the treatment of diabetes and its complications.
Moreover, in vitro anticoagulation experiments showed that sulfated P. haitanensis polysaccharides could inhibit the endogenous coagulation pathway, thrombin-mediated fibrin formation pathway, and exogenous coagulation pathway to achieve anticoagulant effects [70].Zhang et al. reported that degraded sulfated heterogalactan (LP-G2) can inhibit the hemolysis of sheep red blood cells (CH50 = 3.08 ± 0.25 mg/mL) through the classical pathway and can also inhibit the hemolysis of rabbit red blood cells (AP50 = 2.23 ± 0.20 mg/mL) through the alternative pathway.Further studies have shown that the anti-complement bioactivity of LP-G2 is reflected in its ability to exert anti-complement effects through the above two pathways and that LP-G2 selectively interacts with C1q, C2, C4, and C9 [12].
Furthermore, in BALB/c mice infected with the enterotoxin Escherichia coli (ETEC-K88), the rate of infectious diarrhea decreased within the first four days following the oral intake of P. haitanensis polysaccharide (PHSP (hp) ) at a concentration of 10 mg/kg, with all affected mice recovering to normal by the fourth day.Compared with the weight loss of the mice in the diarrhea group after 5 consecutive days, the weight loss of the mice in the PHSP (hp) treatment group rebounded on the second day, suggesting that PHSP (hp) could reduce the diarrhea rate and increase body weight, thus relieving diarrhea symptoms.Additionally, PHSP (hp) has been shown to decrease nitroblue tetrazolium levels in mouse serum and decrease the number of B cells, thereby suppressing the production of proinflammatory cytokines and immunoglobulin A secretion and reducing the inflammatory response to diarrhea induced by ETEC-K88 through specific and nonspecific immunity [58].
In summary, P. haitanensis polysaccharides show rich biological activity and have an important practical significance, warranting further exploration and application.However, the relationship between the unique structure and structure-activity relationship of the polysaccharides of P. haitanensis and their biological activity needs to be further elucidated, which would provide a favorable basis for promoting the creation of health products and clinical therapeutic drugs containing P. haitanensis polysaccharides.

Conclusions and Future Prospects
As early as in ancient China, P. haitanensis was shown to have good medicinal value and has been an important food source for coastal residents.Polysaccharides, as important active components of P. haitanensis, have important research value.Studies have confirmed that the polysaccharides of P. haitanensis display rich biological activity, revealing that the polysaccharides of P. haitanensis have considerable development and utilization value.
In recent years, research on P. haitanensis has focused mainly on extraction, purification, modification, structural identification, and biological activity.The above aspects have been reviewed and discussed in detail, but some conclusions and viewpoints need to be further elaborated.
First, the methods for extracting polysaccharides from P. haitanensis mainly include the HWE method, MAE method, and UMAE method, but at present, the extraction methods are relatively basic, with low extraction rates.In the future, in the development of products from P. haitanensis polysaccharides and in putting P. haitanensis polysaccharides into industrial production, the extraction and purification yield of P. haitanensis polysaccharides will be problematic, because their yield will directly affect the economic benefits of the product.Therefore, it is necessary to explore and innovate high-yield, environmentally friendly, economical, and practical extraction and purification process routes, while standardizing the extraction and purification of P. haitanensis polysaccharides.
Second, the monosaccharide composition, monosaccharide sequence, molecular weight and type, and location of glycosidic bonds are the main focus of structural analyses of P. haitanensis polysaccharides.P. haitanensis polysaccharides generally contain more sulfate (3.7-14.7%),and the main monosaccharide is Gal.The main linkage mode of P. haitanensis polysaccharides is β-or α-glycosidic bonds, and the basic structural skeleton is 3) β-D-galactose (1→4), 3,6-anhydro-α-L-galactose (1→, and→3), β-D-galactose (1→4), α-L-galactose-6-S (1→).However, their detailed structural composition and conformational characteristics, especially the relationship between the structure and biological activity of P. haitanensis polysaccharides and their derivatives, have not been fully studied and need to be further explored.In the future, it will be necessary to use more advanced analytical techniques, such as X-ray diffraction, SEM, AFM, capillary electrophoresis and Raman spectroscopy, to analyze the advanced structure of P. haitanensis polysaccharides.In addition, the molecular modification of polysaccharides from P. haitanensis can change the structural characteristics and biological functions of polysaccharides.For example, the introduction of sulfate groups could enhance the anticoagulant and antioxidant activities of P. haitanensis polysaccharides, and different substitution degrees and substitution sites alter the biological activity and activity intensity of P. haitanensis polysaccharides.P. haitanensis polysaccharides can also be used as drug carriers.The combination of P. haitanensis polysaccharides and 5-FU can alleviate the toxicity and side effects of 5-FU, and the introduction of P. haitanensis polysaccharides into the OVA-RES nanoparticle system can improve the stability and bioavailability of resveratrol.Moreover, the basic structure of P. haitanensis polysaccharides does not change after degradation and modification, but their antioxidant activity, immune activity regulation, and anti-allergic activity improve.The reason may be that degraded small molecules are more conducive to absorption by the body.Therefore, we speculate that the basic skeleton of P. haitanensis polysaccharides is likely one of the key factors determining their activity; therefore, further studies on the composition of the basic skeleton of P. haitanensis or its modification are likely to reveal additional biological activities.However, the substitution site and mechanism of action involved in the modification process of P. haitanensis polysaccharides are still unclear.In particular, the impacts of the addition of new functional groups on chemical structure and conformational characteristics, as well as the degree of derivatization and safety, need to be further clarified.
Third, as a biomolecule, the molecular structure of P. haitanensis polysaccharides is highly diverse and complex; therefore, it is very important to study their advanced structural characteristics and the relationships of those characteristics with biological activity.Furthermore, P. haitanensis polysaccharides, as a traditional Chinese food and medicine, could be studied and identified by querying ancient records and using intestinal flora analysis, metabolomics, proteomics, genomics, and other technologies to investigate and discover additional pharmacological activities of P. haitanensis polysaccharides.Overall, there will be many opportunities and challenges in future research on polysaccharides from P. haitanensis.Researchers can reveal the diverse biological activities of P. haitanensis

Figure 1 .
Figure 1.Schematic diagram of extraction, purification, structural features, and biological activities of polysaccharides from P. haitanensis.

Figure 3 .
Figure 3. Structure and synthesis of disaccharide units and their derivatives of P. haitanensis polysaccharides [70].

Figure 3 .
Figure 3. Structure and synthesis of disaccharide units and their derivatives of P. haitanensis polysaccharides [70].

Figure 5 .
Figure 5. Potential immunomodulatory mechanism of P. haitanensis polysaccharides and their derivatives.Figure 5. Potential immunomodulatory mechanism of P. haitanensis polysaccharides and their derivatives.

Figure 5 .
Figure 5. Potential immunomodulatory mechanism of P. haitanensis polysaccharides and their derivatives.Figure 5. Potential immunomodulatory mechanism of P. haitanensis polysaccharides and their derivatives.

Figure 6 .
Figure 6.Potential anti-allergic mechanism of P. haitanensis polysaccharides and their derivatives.

Figure 6 .
Figure 6.Potential anti-allergic mechanism of P. haitanensis polysaccharides and their derivatives.

Figure 7 .
Figure 7. Potential anti-tumor mechanism of P. haitanensis polysaccharides and their derivatives.

Figure 7 .
Figure 7. Potential anti-tumor mechanism of P. haitanensis polysaccharides and their derivatives.

Figure 8 .
Figure 8. Potential anti-aging mechanism of P. haitanensis polysaccharides and their derivatives.Figure 8. Potential anti-aging mechanism of P. haitanensis polysaccharides and their derivatives.

Figure 8 .
Figure 8. Potential anti-aging mechanism of P. haitanensis polysaccharides and their derivatives.Figure 8. Potential anti-aging mechanism of P. haitanensis polysaccharides and their derivatives.

Table 1 .
A summary of all the extraction methods of Porphyra haitanensis polysaccharides is given in the table below.

Table 2 .
Monosaccharide composition and chemical structure of polysaccharides from Porphyra haitanensis.

Table 3 .
Summary of chemical modification methods of Porphyra haitanensis polysaccharide.