Application of caulerpa lentillifera polysaccharide

Abstract

The application belongs to the technical field of polysaccharide extraction and application, and specifically discloses a use of a caulerpa lentillifera polysaccharide.The caulerpa lentillifera polysaccharide is sulfated galactan, and the relative molecular mass is 100-1000 kDa; the caulerpa lentillifera polysaccharide has a triple helix structure, can prevent and improve vascular endothelial glycocalyx damage by inhibiting heparanase, and is expected to become a drug for treating vascular lesions caused by vascular endothelial glycocalyx damage.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction and application technology, specifically relating to a method for extracting Pteris polysaccharide from green algae of the genus Pteris, its preparation method, and the use of Pteris polysaccharide extracted by this method in the preparation of a protective agent for vascular endothelial glycocalyx damage. Specifically, it relates to the application of Pteris polysaccharide in pharmaceuticals and biomaterials for the prevention or treatment of symptoms / diseases related to vascular endothelial glycocalyx damage. Background Technology

[0002] The vascular endothelium is the first line of defense for blood vessels. It comes into direct contact with the blood, sensing changes in hemodynamics and various substances. Through its physiological barrier function and the secretion of a series of vasoactive substances such as nitric oxide (NO), prostacyclin (PGI2), and endothelin (ET-1), it protects vascular physiological function and the stability of the internal environment. Endothelial damage and dysfunction are major causes of vascular diseases.

[0003] The luminal surface of vascular endothelial cells is covered by a glycocalyx composed of polysaccharides and transmembrane proteins, forming the vascular endothelial barrier. The glycocalyx is a porous, hair-like, regularly organized layer. It carries a negative charge, and its growth and shedding reach equilibrium under physiological conditions. The glycocalyx is composed of proteoglycans (PGs), glycoproteins (endothelial cell adhesion molecules, vascular cell adhesion molecules, and platelets), and glycolipids. Proteoglycans are composed of glycosaminoglycans (GAGs) and protein ligands (syndecans). The main component of glycosaminoglycans in the glycocalyx is heparan sulfate (HS), while chondroitin sulfate and hyaluronic acid account for a smaller proportion, with a 4:1 ratio of heparan sulfate to chondroitin sulfate in the vascular endothelium. The non-sulfated glycosaminoglycan hyaluronic acid (HA) is non-covalently linked to glycoprotein (CD44) and can be exchanged with blood flow, while heparan sulfate and chondroitin sulfate are covalently linked to the proteoglycan core protein. The fundamental functions of endothelial glycocalyx are to create a space between the blood and endothelial cells, controlling vascular permeability by inhibiting or slowing the entry and exit of proteins and macromolecules; inhibiting platelet and leukocyte adhesion to endothelial cells, reducing inflammation and thrombosis; and responding to changes in blood flow through mechanosensing. As a skeletal structure on the surface of endothelial cells, the glycocalyx is a key platform for integrating signal transduction of shear stress, cytokines, and chemical factors, a crucial regulator of vascular endothelial function, and a key link in the regulation of intravascular and extravascular fluid balance. As a "sensor" of blood flow shear stress, the glycocalyx can reduce or counteract the shear stress on the endothelial surface to a theoretically negligible level.

[0004] Glycocalyx shedding is regulated by glycocalyx-degrading / synthetic enzymes. Heparanase, hyaluronidase, matrix metalloproteinases, neuraminidase, and hyaluronic acid synthase are directly involved in glycocalyx degradation. These enzymes can trigger glycocalyx shedding. Glycocalyx shedding leads to vascular endothelial dysfunction, which can further cause vascular complications. Therefore, reducing glycocalyx shedding can prevent vascular complications. Currently, there are no effective drugs for treating vascular glycocalyx damage and its complications. Therefore, inhibitors of glycocalyx-degrading enzymes are a promising research direction for treating vascular glycocalyx damage and its complications.

[0005] *Caulerpa*, belonging to the phylum Chlorophyta and family Cauleraria, is a typical marine multinucleate tubular green algae, mainly distributed in tropical seas, with approximately 100 species worldwide, including 12 species found in the South my country Sea. However, its potential has not been fully realized. The sulfated polysaccharides in *Caulerpa* are mainly sulfated arabinogalactan and sulfated galactan. Current research on the structure and function of sulfated polysaccharides in *Caulerpa* is very limited. This invention discloses for the first time that *Caulerpa* polysaccharides have an inhibitory effect on heparanase, inhibiting the shedding of vascular endothelial glycocalyx and thus suppressing vascular lesions. Summary of the Invention

[0006] One objective of this invention is to provide a novel sulfated polysaccharide extracted from green algae of the genus *Pteris*. It is primarily composed of galactose, with a weight-average molecular weight of 100-1000 kDa and a sulfate content of 20-30%. Its main chain structure is β-D-galactose linked by a 1→3 linkage, with branches mainly consisting of →6)-β-D-Galp-(1→), and also containing →3,6-, →4,6-, and a small amount of →4- linkage units. Sulfation is concentrated at the C-2 / C-4 positions of the galactose.

[0007] The polysaccharides mentioned are derived from green algae of the genus *Fragaria*, including *Fragaria septemlobata*, *Fragaria racemosa*, *Fragaria longstemosa*, or *Fragaria fischeri*.

[0008] The aforementioned *Fragariae* polysaccharide is prepared through the following steps: (1) Degreasing: Dry and crush the algae, soak and stir it in an organic solvent to obtain degreased algae powder; (2) Water extraction: The defatted algae powder is extracted with hot water and stirred to obtain an aqueous extract; the obtained extract is filtered, desalted, concentrated, and centrifuged to remove the precipitate; the supernatant is precipitated with alcohol and centrifuged; the precipitate is washed and dried to obtain crude polysaccharide of *Fragaria spp.* (3) Grading: The crude polysaccharide of *Fragaria spp.* was dissolved in distilled water. Using distilled water and sodium chloride aqueous solution as the mobile phase, it was separated and purified by anion exchange resin and detected by the sulfuric acid phenol method. It was eluted sequentially with water and 0.05–2.0 M NaCl solution. The eluted fraction of 0.9 M NaCl solution was collected, desalted, concentrated, precipitated with ethanol, and the precipitate was washed and dried to obtain *Fragaria spp.* polysaccharide.

[0009] In a further improvement to the above technical solution, the volume ratio of the organic solvent added in step (1) to the mass ratio of the algae powder is 10 to 20:1, and the degreasing is carried out by soaking or intermittent stirring for 2 to 8 hours. The organic solvent used is one or more of ethanol, acetone or methanol solution.

[0010] This invention also provides the application of the aforementioned *Fragaria spp.* polysaccharide in pharmaceuticals, health products, foods, and biomaterials for the prevention or treatment of symptoms / diseases related to vascular endothelial glycocalyx damage.

[0011] The polysaccharide from *Fragaria spp.* is a heparinase inhibitor. By inhibiting heparinase, *Fragaria spp.* reduces or improves vascular endothelial glycocalyx damage.

[0012] The present invention also includes compositions in which the aforementioned ferns polysaccharide is used as an active ingredient, the compositions optionally further comprising food, health product or pharmaceutically acceptable excipients.

[0013] The present invention has the following beneficial effects: This invention discloses a sulfated polysaccharide derived from *Fragaria spp.* and its preparation method. The polysaccharide is obtained through a simple and efficient water extraction, alcohol precipitation, and anion exchange chromatography purification process, resulting in a novel sulfated galactan. Experimental results show that *Fragaria spp.* polysaccharide can significantly inhibit heparinase and vascular endothelial calyx damage in vitro, thus preventing and treating vascular lesions. As a natural polysaccharide derived from green algae, *Fragaria spp.* polysaccharide is safe, non-toxic, and has low immunogenicity, demonstrating good potential application value. Attached Figure Description

[0014] Figure 1 FT-IR spectra of CSGS polysaccharide and its desulfurization product DSCS.

[0015] Figure 2 Total ion chromatogram (TIC) of GC-MS analysis of DSCS, a desulfurization product of ferruginous polysaccharide CSGS.

[0016] Figure 3 DSCS, a desulfurization product of ferns polysaccharide CSGS 13 C10 NMR spectrum.

[0017] Figure 4 DSCS, a desulfurization product of ferns polysaccharide CSGS 1 H NMR spectrum.

[0018] Figure 5. NMR spectrum of DSCS, the desulfurization product of *Fragaria spp.* polysaccharide CSGS. Note: a AF: T-β-D-Gal p →3)-β-D-Gal p -(1→; →6)-β-D-Gal p -(1→; →4)-β-D-Gal p -(1→; →3,6)-β-D-Gal p -(1→; →4,6)-β-D-Gal p -(1→ Figure 6 Fern polysaccharide CSGS 1 H NMR spectrum.

[0019] Figure 7 Fern polysaccharide CSGS 13 C10 NMR spectrum.

[0020] Figure 8. Two-dimensional NMR spectrum of CSGS (a polysaccharide from *Fragaria spp.*) Note: a LS: T-β-D-Gal p →3)-β-D-Gal p -(1→; →6)-β-D-Gal p -(1→; →4)-β-D-Gal p -(1→; →3,6)-β-D-Gal p -(1→; →3)-β-D-Gal p (2S)-(1→; →3)-β-D-Gal p (4S)-(1→; →6)-β-D-Gal p (4S)-(1→).

[0021] Figure 9 Fern polysaccharide can inhibit heparinase-induced glycocalyx shedding in HUVEC cells.

[0022] Figure 10 SPR experiments showed that the polysaccharide CSGS from *Fragaria spp.* interacts with heparanase.

[0023] Figure 11 CSGS can protect the integrity of the glycocalyx structure of the aorta and kidneys in mice fed a high-sugar, high-fat diet and reduce glycocalyx shedding.

[0024] Figure 12 Western blot results showed that CSGS could downregulate the expression level of heparanase in the kidneys of mice fed a high-sugar, high-fat diet. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1: Needle-leaved fern ( Caulerpa sertularioides Preparation of polysaccharide CSGS a. Polysaccharide extraction: After naturally drying the raw material of *Nephrolepis cordifolia*, it was chopped, ground using a high-speed mixer, and passed through a 60-mesh sieve. 1 kg of algal powder was accurately weighed, and 20 L of 95% ethanol was added. The mixture was stirred and defatted at room temperature for 3 hours. The ethanol was discarded after centrifugation, and the extraction was repeated once using ethanol. The resulting defatted algal powder was dried to obtain 850 g.

[0027] Take 800g of defatted algae powder, add purified water at a material-to-liquid ratio of 1:30, extract at 100℃ for 2 h, take the supernatant and concentrate by rotary evaporation, dialyze, and use a dialysis bag to retain polysaccharides with a molecular weight of more than 14000 Da. After concentrating to a small volume, add ethanol to a final concentration of 75%, stir to precipitate alcohol, centrifuge at 4000 rpm for 10 min to collect the precipitate, wash twice with 95% ethanol, and vacuum dry to obtain 95g of crude polysaccharide from *Pteris vittata*.

[0028] b. Polysaccharide purification: The fern polysaccharide prepared in step a was separated and purified by DEAE-Sepharose Fast Flow anion exchange chromatography.

[0029] 10 g of crude polysaccharide from *Fragaria spp.* prepared in step a was dissolved in distilled water and separated using a DEAE Sepharose FastFlow anion exchange column. The solution was eluted sequentially with deionized water, 0.5 M, 0.7 M, 0.9 M, 1.2 M, and 2.0 M NaCl solutions. The sulfuric acid-phenol method was used for detection, and elution curves were plotted. The 0.9 M NaCl eluent was collected and combined according to the elution curves, dialyzed, concentrated, centrifuged, and the supernatant was freeze-dried to obtain 2.6 g of purified *Fragaria spp.* polysaccharide CSGS.

[0030] Example 2: *Vitis thunbergii* (long-stemmed *Vitis thunbergii*) Caulerpa lentillifera Preparation of polysaccharide CLGS a. Polysaccharide extraction: The naturally air-dried *Vitis vinifera* was dried in the sun, ground using a high-speed mixer, and passed through a 60-mesh sieve. 400 g of algal powder was accurately weighed, and 8 L of 95% ethanol was added. The mixture was stirred and defatted at room temperature for 4 hours. The ethanol was discarded by centrifugation, and the extraction was repeated once using ethanol. The resulting defatted algal powder was dried to obtain 325 g.

[0031] Take 300g of defatted algae powder, add purified water at a material-to-liquid ratio of 1:20, extract at 100℃ for 2 h, take the supernatant and concentrate by rotary evaporation, dialyze, and retain the polysaccharide with a molecular weight of more than 14000 Da using a dialysis bag. After concentrating to a small volume, add ethanol to a final concentration of 75%, stir to precipitate alcohol, centrifuge at 4000 rpm for 10 min to collect the precipitate, wash twice with 95% ethanol, and vacuum dry to obtain 43 g of crude polysaccharide from *Vitis thunbergii*.

[0032] b. Polysaccharide purification: 10 g of crude polysaccharide from *Vitis thunbergii* prepared in step a was dissolved in deionized water and separated using a DEAE Sepharose Fast Flow anion exchange column. The solution was eluted sequentially with deionized water, 0.5 M, 0.7 M, 0.9 M, 1.2 M, and 2.0 M NaCl solutions. The elution was detected using the sulfuric acid-phenol method, and elution curves were plotted. The 0.9 M NaCl eluent was collected and combined according to the elution curves, dialyzed, concentrated, centrifuged, and the supernatant was freeze-dried to obtain 2.4 g of purified *Vitis thunbergii* polysaccharide CLGS.

[0033] Example 3: Structural analysis of *Cypripedium spp.* polysaccharide CSGS (1) The relative molecular mass of the CSGS polysaccharide prepared in Example 1 was determined to be 851.61 kDa by high performance gel permeation chromatography (HPGPC), and its purity was 94%.

[0034] (2) The monosaccharide composition of the completely hydrolyzed sample of *Fragaria spp.* polysaccharide CSGS was determined by PMP derivatization. It was found that *Fragaria spp.* polysaccharide CSGS mainly contained galactose and trace amounts of glucose, xylose, arabinose, etc., with a molar ratio of 100: 2: 3: 2. Using galactose as the standard, the total sugar content of CSGS was determined to be 72.3%, and the sulfate content was 26.3%.

[0035] (3) Infrared spectral analysis of the polysaccharide CSGS from *Fragaria spp.* is shown in the following figure. Figure 1 CSGS at 3380–3390 cm -1 Broad and strong absorption peaks appear throughout the range, corresponding to the O–H stretching vibration of the hydroxyl group, 2924–2956 cm⁻¹. -1 The C–H stretching vibration appears at 1618–1635 cm⁻¹, which is a characteristic peak for methyl / methylene groups. -1 The peak at 1214–1220 cm⁻¹ represents the stretching vibration of the C–O bond. -1 S=O stretching vibration and 817–821 cm -1 The C–O–S stretching vibration indicates the presence of a sulfate group. Simultaneously, at 820 cm⁻¹... -1The nearby signal is a stretching vibration caused by the substitution of a sulfate group at position 2.

[0036] (4) Glycosidic residue linkage analysis: To further analyze the glycosidic bond linkage type and branching characteristics of CSGS, CSGS was desulfurized and then methylated. After multiple methylation processes, the desulfurized polysaccharide DSCS was examined by infrared spectroscopy at 3200 cm⁻¹. -1 The disappearance of the nearby -OH signal and 2920 cm -1 A triplet signal was generated near the -CH3 group. Partially methylated alcohol-sugar acetate was obtained through acid hydrolysis, reduction, and acetylation. The product was analyzed by GC-MS. The total ion chromatogram and corresponding mass spectra after DSCS methylation are shown below. Figure 2 As shown in Table 1, a total of seven major residue types were detected, including terminal Galp (T-Galp), →6)-Galp-(1→, →3)-Galp-(1→, →4)-Galp-(1→, →3,6)-Galp-(1→, →4,6)-Galp-(1→, and →3,4)-Galp-(1→). The mass spectra of each fragment are shown in Figure 4 (4G-H). The relative content results after optimization by relative molar response factor (Rf) are shown in Table 4-4. Among them, β-D-galactose with a main chain structure of 1→3 linkage and →6)-β-D-Galp-(1→ as the main chain linkage are predominant, and a high proportion of 3,6-substituted residues were also detected, indicating that it has a highly branched structure. In addition, the detection of 4,6- and 3,4-linked galactose further indicates that there is a multi-branched structure in DSCS.

[0037] Table 1. GC-MS analysis of DSCS, a desulfurization product of *Fragaria spp.* polysaccharide CSGS.

[0038] (5) Nuclear magnetic resonance spectroscopy was used to analyze the DSCS desulfurization product of CSGS of ferns polysaccharide.

[0039] In DSCS 13 In the 10⁻⁶ C NMR spectrum, six distinct signals were detected in the anomeric carbon region, located at 101.15, 102.07, 103.38, 103.74, 104.16, and 104.55 ppm, corresponding to β-galactopyranose (β-Gal). p )unit( Figure 3 The H-1 signals include 4.35, 4.37, 4.45, and 4.55 ppm, while the proton signals for H2–H6 are concentrated between 3.35 and 4.21 ppm. Figure 4 ).

[0040] 1 H– 1H COSY and TOCSY spectra provide short-range and long-range HH signals within sugar residues, while HSQC spectra reveal information about C–H direct linkages. Figure 5 The H1 signal of 4.35 ppm and its corresponding C1 signal of 101.15 ppm were identified as residue A, belonging to the terminal β-Gal. p The assignment of this unit is based not only on its consistent chemical shift position in both DSCS and CSGS, but also on the high-field characteristics of its carbon signal. Further analysis of the anodic region signal and the correlation of cross-peaks in the two-dimensional spectrum, combined with the demasking effect of the glycosidic bond linkage site, confirmed the structural characteristics of each residue. Residue B exhibits →3)-β-D-Gal p -(1→ unit, with a deshielding effect at C3 (75.47 ppm), indicates substitution in the O-glycosidic bond. Similarly, residue C is attributed to →6)-β-D-Gal p -(1→ unit, with a low-field shift at C6 site at 68.99 ppm. Residue D is →4)-β-D-Gal p -(1→ unit, whose C4 signal shifts to 4.20 ppm. The H1 signal (4.45 ppm) of residue E corresponds to the C1 signal (104.16 ppm), and combined with the low-field shifts of its C3 and C6 signals (74.08 and 68.99 ppm), it is determined to be →3,6)-β-D-Gal p -(1→ unit. The H1 signal (4.55 ppm) of residue F corresponds to the C1 signal (104.55 ppm), and its C4 and C6 signals are located at 74.08 and 67.20 ppm, respectively, and therefore it is classified as →4,6)-β-D-Gal p -(1→ unit. The complete spin system of H2–H6 was resolved by using the coupling information of TOCSY and COSY.

[0041] Regarding the determination of repeating sequences, 1 H– 1 H ROESY and 1 H– 13 The C HMBC spectrum provides crucial evidence. In ROESY, the correlation signals between H1(B)–H3(E) and H1(C)–H6(E) confirm →3)-β-D-Gal p →6)-β-D-Gal p With →3,6)-β-D-Gal p The connections between units reveal the presence of →3)-β-D-Gal on the main chain. p -(1→3,6)-β-D-Gal p -(1→6)-β-D-Gal p-(1→ branching motif [2] Similarly, the correlation signals of H1(D)–H4(F) and H1(C)–H6(F) further support →4)-β-D-Gal p -(1→4,6)-β-D-Gal p -(1→6)-β-D-Gal p The sequence -(1→. Long-range correlations in the HMBC spectrum also corroborate the above connections, with H3(B)–C1(B) and H6(C)–C1(C) signals clearly supporting β-D-Gal connections at the 3- and 6-sites. p The chain segment is present. The cross peaks of H1(B)–C3(E) and H1(E)–C3(B) further confirm the presence of →3)-β-D-Gal. p -(1→3,6)-β-D-Gal p -(1→3)- β-D-Gal p -(1→ sequence.→6)-β-D-Gal p With →3,6)-β-D-Gal p The connection between them is confirmed by the H1(C)–C6(E) signal, showing its branched structure characteristics. Furthermore, the H1(F)–C6(C) and H1(C)–C6(F) signals reveal →6)-β-D-Gal p -(1→4,6)-β-D-Gal p -(1→6)-β-D-Gal p -(1→ sequence, while the long-range correlation of H1(D)–C4(F) indicates →4)-β-D-Gal p Primarily replaces →6)-β-D-Gal p The C4 position of the residue forms a secondary branch.

[0042] In summary, through comprehensive analysis of multidimensional NMR spectra, the types of sugar residues, linkage modes, and branching characteristics of DSCS were clarified, revealing the complex main chain and branched structure of this type of sulfated polysaccharide. Specific signal attribution information is shown in Table 2. This structural information not only validates the GC-MS analysis results but also provides a solid basis for further investigation into the impact of desulfurization on the structure and function of polysaccharides.

[0043] Table 2. NMR assignment data of DSCS, a desulfurization product of *Fragaria spp.* polysaccharide CSGS.

[0044] Note: a AF: T-β-D-Gal p →3)-β-D-Gal p -(1→; →6)-β-D-Galp -(1→; →4)-β-D-Gal p -(1→; →3,6)-β-D-Gal p -(1→; →4,6)-β-D-Gal p -(1→).

[0045] (6) Nuclear magnetic resonance spectroscopy was used to analyze the CSGS of *Fragaria spp.* polysaccharide. A systematic nuclear magnetic resonance spectroscopy method was used for the structural analysis of CSGS. 1 In the H NMR spectrum, anomeric proton signals were clearly detected, located at 4.35, 4.36, 4.41, 4.55 and 4.57 ppm, respectively. Figure 6 Correspondingly. 13 The anomeric carbon signals in the C10 NMR spectrum were concentrated at 101.10, 101.65, 102.75, 103.57, and 104.42 ppm. Figure 7 These chemical shifts all fall within the typical β-anomeric configuration range, indicating that the galactose residues in CSGS are mainly in the β-D-Gal configuration. p It exists in form.

[0046] Compared to CSGS, DSCS exhibits an overall high-field shift in the C2–C5 region, and the anomaly proton signal in the HSQC spectrum also shows a significant high-field shift, reflecting the increased electron cloud density and enhanced shielding effect caused by desulfurization. The signal attenuation at 4.26 / 78.38 ppm further indicates the removal of sulfate groups at the C-2 and C-4 positions. It is noteworthy that in DSCS… 13 The signal at C significantly weakens at 101.65 ppm, suggesting that the corresponding galactose unit is a sulfated residue. Simultaneously, the anoproton at 4.57 ppm shows a marked high-field shift after desulfurization, which is consistent with the β-residue reduction after sulfate removal. C / H The site electron shielding effect is enhanced consistently. In summary, the sulfate substitution effect exhibits a dual characteristic at different C-H sites: α C / H Site substitution typically leads to low-field shift ( 13 C +6–10 ppm, 1 H +0.4–0.8 ppm), while β C / H Site substitution manifests as a high-field shift of the carbon signal and a low-field shift of the proton signal.

[0047] Through the system's two-dimensional spectra (COSY, TOCSY, HSQC, and HSQC-TOCSY), Figure 8Based on literature data, eight structural units (residues L–S, Table 3) were successfully resolved and identified. HSQC spectra provided direct correlations between anomeric protons and carbon: L (4.35 / 101.10 ppm), M (4.35 / 103.57 ppm), N (4.55 / 103.57 ppm), O (4.36 / 104.42 ppm), P (4.41 / 102.75 ppm), Q (4.55 / 101.65 ppm), R (4.57 / 101.65 ppm), and S (4.57 / 101.65 ppm). The remaining protons on the ring (H2–H6) were located between 3.32–4.34 ppm, with corresponding C2–C6 position signals distributed between 61.42–82.36 ppm. It is noteworthy that the strong correlation between anoprotons and H2 (4.24–4.28 ppm) in the COSY spectrum disappeared after desulfurization, further confirming that these signals belong to C-2 sulfation residues.

[0048] TOCSY and HSQC-TOCSY further revealed the spin system and scalar correlation of each residue. Residue Q was assigned to →3)-β-D-Gal p (2S)-(1→, its H2 / C2 signal shows a significant low-field shift (4.31 / 76.21 ppm); residue R is identified as →3)-β-D-Gal p (4S)-(1→, its characteristic H4 / C4 signal appears at 4.34 / 78.37 ppm; residue S shows a characteristic signal at δ 4.31 / 76.21 ppm and is identified as →6)-β-D-Gal p (2S)-(1→). In the HSQC spectrum, the inverted signal at 61.42 ppm corresponds to the unmodified C6 position, which is attributed to the terminal or 3-, 4-connected β-D-Gal. p Residues. Among them, the 3.59 / 61.42 ppm signal corresponds to residue M(→3)-β-D-Gal. p -(1→) C6 remains abundant even after desulfurization; while the 4.09 / 69.50 ppm signal corresponds to C6 of residue R, showing a sulfation effect. In addition, H3 / C3 (4.27 / 75.49 ppm) of residue M, H3 / C3 (4.26 / 76.21 ppm) and H6 / C6 (4.09 / 69.50 ppm) of residue P all show low-field shifts, suggesting substitution at C3 or C6 sites, reflecting branching or sulfation modification characteristics.

[0049] HMBC spectra revealed key glycosidic bond linkage information. Long-range correlations between H1(L)-C3(M) and H1(M)-C3(M) indicate the presence of a typical 3-linked structure in the main chain; correlations between H1(N)-C6(N) and H4(O)-C1(O) confirm the presence of 6- and 4-linked structures. Cross-peaks between H1(N)-C6(P), H1(M)-C3(P), and H1(P)-C3(M) suggest branching of the C6 side chain. A correlation between H1(M)-C3(Q) indicates that the C2-sulfated galactose residue is involved in the main chain structure. Conversely, the absence of H1(M)-C4(O) and H1(O)-C3(M) signals suggests that 4-linked units are more likely present in the 6-linked side chain than in the main chain.

[0050] In summary, based on the NMR spectral analysis of *Fragaria spp.* CSGS and its desulfurization product DSCS, it is indicated that CSGS is converted into →3)-β-D-Gal p -(1→ is the main backbone, with sulfation modifications at C-2 and C-4 sites. Its branches are mainly composed of 6-linked β-D-Gal p It forms a complex secondary side chain, and further forms a 4-connection branch at the C-4 position.

[0051] Table 3. NMR assignment data of CSGS for *Fragaria spp.* polysaccharide

[0052] Note: a LS: T-β-D-Gal p →3)-β-D-Gal p -(1→; →6)-β-D-Gal p -(1→; →4)-β-D-Gal p -(1→; →3,6)-β-D-Gal p -(1→; →3)-β-D-Gal p (2S)-(1→; →3)-β-D-Gal p (4S)-(1→; →6)-β-D-Gal p (4S)-(1→).

[0053] (7) Spatial structure analysis of CSGS polysaccharide from *Fragaria spp.* Prepare a 2 mg / mL polysaccharide solution and mix it with an equal volume of 80 μM Congo red solution. Perform a full-wavelength scan (300-600 nm) using a microplate reader to obtain the absorption curve of the mixture. Gradually add 1 M NaOH to the system concentrations of 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5 M. Perform full-wavelength scans at each concentration, record and compare the redshift of the maximum absorption wavelength. For the negative control, mix an equal volume of dd water with Congo red. Prepare a 1% agarose gel (containing 25 μg / mL purified β-D-galactosyl Yariv reagent and 1% NaCl). After cooling to form a gel, punch holes. Add an equal volume of polysaccharide solution to the gel wells to achieve final concentrations of 50, 100, and 200 μg. Use an equal volume of gum arabic as a positive control and 1% NaCl as a negative control. Incubate at 4℃ for 24 h and observe the formation of red blots.

[0054] Congo red binding assays showed that after CSGS formed a complex with Congo red, the λmax shifted from 488 nm to 497 nm and remained stable under alkaline conditions, proving that its sugar chain has a stable triple helix structure. Yariv reagent blot results showed that CSGS exhibited a positive red blot ring, indicating the presence of a β-D-1,3-galactan structure, further demonstrating that CSGS possesses a triple helix spatial conformation formed by β-1,3-galactose, exhibiting unique structural characteristics.

[0055] Example 4: Fructus lycosanol has a protective effect against heparinase-induced glycocalyx shedding in HUVEC endothelial cells. HUVEC cells were treated with 0.5 U / mL recombinant heparinase to establish an in vitro model of glycocalyx damage. The blank control group was treated with serum-free culture medium plus an equal volume of enzyme dilution buffer. HUVEC glycocalyx sections were prepared using lanthanum nitrate staining / chemical fixation / low-temperature dehydration. Results showed that after heparinase treatment, the glycocalyx on the cell surface of HUVEC cells was significantly detached. The *Fragaria spp.* polysaccharide CSGS prepared in Example 1 and the *Fragaria spp.* polysaccharide CLGS prepared in Example 2, added at 100 μg / mL to heparinase-treated HUVEC cells, inhibited the glycocalyx detachment caused by heparinase and restored the normal morphology of the glycocalyx. (See attached figures). Figure 9 .

[0056] The interaction between the polysaccharide CSGS from *Fragaria spp.* and heparanase was studied using the SPR experiment. The results showed that there is an interaction between CSGS and heparanase. Figure 10 The equilibrium dissociation constant (KD) is 3.075 μM, and the binding rate constant (Ka) is 2018 M. - 1 S -1The dissociation rate constant (Kd) is 0.006204 s. -1 .

[0057] In vitro enzyme activity inhibition experiments also showed that *Cypripedium spp.* polysaccharide CSGS could inhibit heparinase activity in vitro. CSGS exhibited strong heparinase inhibitory activity in vitro, with an inhibition rate of 51.2% at a concentration of 50 μg / mL and 83.1% at a concentration of 100 μg / mL. Figure 10 ).

[0058] Example 5: *Cypripedium spp.* polysaccharide CSGS can reduce the shedding of glycocalyx in the aortic / renal vascular endothelium of mice with disordered glucose and lipid metabolism and inhibit vascular lesions. Four-week-old male C57BL / 6J mice were divided into four groups of eight each and fed a high-sugar, high-fat diet. Blood glucose and lipid levels were monitored regularly. After four weeks, blood glucose and lipid levels significantly increased. From this point onward, the treatment groups were administered *Cyclocarya paliurus* polysaccharide CSGS prepared in Example 1 via gavage daily. The dosages for the two treatment groups were 50 mg / kg and 150 mg / kg body weight, respectively, while the model group was administered the same volume of physiological saline via gavage. The high-sugar, high-fat diet was continued during gavage, and blood glucose levels were monitored. After four weeks of treatment, blood samples were collected for blood biochemistry and inflammatory factor analysis. Mouse kidney / aortic glycocalyx sections were prepared using lanthanum nitrate staining / chemical pre-fixation / low-temperature dehydration and analyzed by electron microscopy. The results showed that high-sugar, high-fat diets significantly increased blood glucose and lipid levels in mice, while administration of *Cyclocarya paliurus* polysaccharide CSGS for four weeks significantly reduced blood glucose and lipid levels (Table 4).

[0059] Table 4 Blood Biochemical Indicators

[0060] abc Groups with non-identical letters in a row are considered significantly different, as determined by a one-way ANOVA test. p The level is <0.05. Feeding mice with a high-sugar, high-fat diet causes glycocalyx loss in the aorta and glomeruli of mice, while the CSGS prepared in Example 1 can reduce glycocalyx loss in the aorta and kidneys of mice. Figure 11The results showed that the glycocalyx villi structure of normal mice was intact and clear, with obvious needle-like structures. WGA-labeled extracellular glycocalyx layers of the mouse aorta / kidney showed strong and continuous green fluorescence expression. In contrast, the glycocalyx of the aorta and kidney of the high-sugar, high-fat model mice was completely detached, and WGA-labeled extracellular glycocalyx of the kidney / aorta showed almost no green fluorescence expression. Glycocalyx-degrading enzymes play an important role in glycocalyx formation and can protect the integrity of the glycocalyx by affecting glycocalyx synthesis and degradation enzymes. Immunoblot analysis of the protein expression of glycocalyx-degrading enzymes showed a significant upregulation of heparanase expression levels in the aorta and kidney of model mice. CSGS had a protective effect on the vascular endothelial glycocalyx of mice fed a high-sugar, high-fat diet. The glycocalyx structure of the aorta / kidney of CSGS-treated high-sugar, high-fat mice was restored to a clear and intact state, with visible needle-like structures, and WGA restored strong green fluorescence expression. Figure 11 Western blot analysis showed that CSGS administration significantly downregulated the expression levels of heparanase in the aorta and kidneys. Figure 12 ).

Claims

1. The application of a fern polysaccharide in pharmaceuticals and biomaterials for the prevention or treatment of symptoms / diseases related to vascular endothelial glycocalyx damage, characterized in that: The *Fragariae* polysaccharide was prepared through the following steps: (1) Degreasing: Dry and crush the algae, soak and stir it in an organic solvent to obtain degreased algae powder; (2) Water extraction: The defatted algae powder is extracted with hot water and stirred to obtain an aqueous extract; the obtained extract is filtered, desalted, concentrated, and centrifuged to remove the precipitate; the supernatant is precipitated with alcohol and centrifuged; the precipitate is washed and dried to obtain crude polysaccharide of *Fragaria spp.* (3) Grading: The crude polysaccharide of *Fragaria spp.* was dissolved in distilled water. Using distilled water and sodium chloride aqueous solution as the mobile phase, it was separated and purified by anion exchange resin and detected by the sulfuric acid phenol method. It was eluted sequentially with water and 0.05-2.0M NaCl solution. The eluted fraction of 0.9M NaCl solution was collected, desalted, concentrated, precipitated with ethanol, and the precipitate was washed and dried to obtain *Fragaria spp.* polysaccharide.

2. The application according to claim 1, characterized in that, The polysaccharide described is mainly composed of galactose, with a weight-average molecular weight of 100 kDa to 1000 kDa and a sulfate content of 20-30%. Its main chain structure is β-D-galactose with 1→3 linkages, and the side chains are mainly →6)-β-D-Galp-(1→, with →3,6-, →4,6- and a small amount of →4- linkage units. Sulfation is concentrated at the C-2 / C-4 position of galactose.

3. The application according to claim 1, characterized in that, The green algae of the genus *Fragaria* are *Fragaria stenoptera*, *Fragaria racemosa*, *Fragaria longstemosa*, or *Fragaria fischeri*.

4. The application according to claim 1, characterized in that, The volume ratio of the organic solvent added in step (1) to the mass ratio of the algae powder is 10 to 20:

1. Degreasing is carried out by soaking or intermittent stirring for 2 to 8 hours. The organic solvent used is one or more of ethanol, acetone or methanol solution.

5. The application according to claim 1, characterized in that, The polysaccharide from *Fragaria spp.* is used as an active ingredient to prepare heparinase inhibitors. By inhibiting heparinase, *Fragaria spp.* reduces or improves vascular endothelial glycocalyx damage.

6. A composition comprising the polysaccharide of *Fragaria spp.* according to claims 1-5 as an active ingredient, the composition optionally further comprising food, health product or pharmaceutically acceptable excipients.

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