Salicornia polysaccharide, and preparation method and application thereof

Abstract

The application discloses a Salicornia polysaccharide as well as a preparation method and application thereof. The Salicornia polysaccharide is composed of 11 kinds of monosaccharides, wherein arabinose (24.96%), galactose (30.39%) and galacturonic acid (23.20%) are main monosaccharides of the polysaccharide, and the relative molecular weight is 3.24*10 4 Da. The Salicornia polysaccharide has a very special surface morphological structure, and presents a regular sawtooth distribution. The anti-tumor activity of the Salicornia polysaccharide is researched, and it is found that the polysaccharide can inhibit the growth of HepG2 cells, and the molecular mechanism of the anti-tumor activity is that the polysaccharide can induce the apoptosis of HepG2 cells. The Salicornia polysaccharide has significant anti-tumor activity, can be used as a candidate drug for developing an anti-tumor drug, and has a wide development prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polysaccharide from sea spirulina, its preparation method, and its application. Background Technology

[0002] Sea sedge (S. bigelovii) is a halophyte belonging to the genus S. bigelovii in the family Chenopodiaceae. It is leafless with fleshy stems and primarily grows on salt marshes. Sea sedge exhibits strong salt tolerance, able to withstand concentrations exceeding 1000 mM sodium chloride, and can be directly irrigated with seawater. As a dual-purpose food and medicinal resource, sea sedge has wide applications. It is highly nutritious, containing various substances such as fatty acids, vitamins, proteins, and minerals, and is therefore consumed as an organic vegetable. Furthermore, sea sedge contains various bioactive components such as flavonoids, alkaloids, polysaccharides, and saponins, possessing anti-inflammatory, antioxidant, anti-tumor, hypoglycemic, and hypolipidemic effects. In Xinjiang, my country, it is mainly used to treat scurvy. In countries like South Korea and India, sea sedge is frequently used to treat obesity, diabetes, scabies, and gastrointestinal imbalances.

[0003] Polysaccharides are polymeric carbohydrates widely distributed in plants, animals, and microorganisms, and are important biomolecules. Polysaccharides possess various effects, including anticancer, immunomodulatory, and hypoglycemic activity. With their advantages of safety, high efficiency, low toxicity, wide availability, and low cost, they have gradually become a hot topic in the development and research of antitumor drugs. Polysaccharides inhibit tumor cell proliferation and are closely related to tumor cell apoptosis. Studies have shown that polysaccharides can significantly increase the expression of Caspase-3 / 9 proteases by upregulating the Bax / Bcl-2 ratio, enhancing the accumulation of reactive oxygen species in cells, inducing a decrease in mitochondrial membrane potential and mitochondrial degradation, thereby inducing tumor cell apoptosis. Numerous studies have shown that polysaccharides with different structures, molecular weights, and degrees of polymerization have different biological activities. The anticancer activity of polysaccharides is directly related to the monosaccharide residue sites, monosaccharide sequences, and glycosidic bond types. High molecular weight polysaccharides have better anticancer effects than low molecular weight polysaccharides. Therefore, elucidating the structure and activity of natural plant polysaccharides is of great significance. Compared to common soil plants, halophytes grow in areas with high salinity. High salinity causes oxidative stress, leading to an excessive production of free radicals in plant cells, which damage cell membranes, DNA, proteins, and other biomolecules. The unique chemical structure of polysaccharides enables them to resist this oxidative stress. Therefore, polysaccharides from halophytes possess strong antioxidant properties, and their structures may exhibit higher specificity. This characteristic makes halophyte polysaccharides a highly promising alternative to antioxidants.

[0004] Current research on *Sargassum fusiforme* polysaccharides is limited. Existing reports mainly focus on optimizing the extraction process of crude polysaccharides and their biological activities, such as immunomodulatory, antioxidant, and lipid-lowering effects. However, there are no reports on the structure and antitumor activity of *Sargassum fusiforme* polysaccharides. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a polysaccharide from *Salvia splendens*, its preparation method, and its applications. The composition and chemical structure of the monosaccharides in the polysaccharide were characterized using optical, chromatographic, and spectroscopic methods. The inhibitory mechanism of the polysaccharide on HepG2 cells was also investigated. The research results will provide a useful reference for the development and utilization of polysaccharides from halophytes.

[0006] This invention is achieved through the following technical solution:

[0007] A polysaccharide from *Salvia splendens* is composed of 11 monosaccharides: fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, guluronic acid, glucuronic acid, and mannuronic acid. The monosaccharide ratio is 1.12:24.96:6.21:30.39:1.54:4.99:1.69:23.20:1.03:3.12:1.75; the relative molecular weight is 3.24 × 10⁻⁶. 4 The structure of the polysaccharide from *Salvia splendens* is shown below:

[0008]

[0009] Where n is a natural number ≥ 1.

[0010] A method for preparing polysaccharides from sea spirulina includes the following steps:

[0011] Step 1) Raw material pretreatment: Collect fresh sea sedges, wash them, air dry them, bake them, and crush them to obtain dried sea sedge powder;

[0012] Step 2) Treat the dried sea sedge powder with ethanol solution overnight. Extract the residue after drying with hot water at a material-to-water ratio of 1:10 and extract the precipitate. Repeat the extraction operation twice and combine the supernatants. Concentrate the extracted solution and precipitate it with 4 times the volume of anhydrous ethanol to obtain the crude polysaccharide sample.

[0013] Step 3) The crude polysaccharide sample was redissolved in water, deproteinized using the Sevag method, dialyzed with water, concentrated and freeze-dried to obtain crude polysaccharide;

[0014] Step 4) After redissolving the crude polysaccharide, separate the crude polysaccharide using a 26×500mm DEAE seplife FF cellulose column, distilled water, and NaCl solution at a rate of 1.5 mL / min.

[0015] Step 5) The crude polysaccharide was eluted with a 26×1000mm Sephacryl S-400HR dextran gel column and distilled water at a flow rate of 1.0 mL / min to obtain the sea sedge polysaccharide.

[0016] Preferably, the drying temperature in step 1) is 55°C.

[0017] Preferably, the extraction temperature in step 2) is 60°C and the time is 4 hours; the precipitation temperature is 4°C.

[0018] Preferably, the molecular weight of the dialysis sample in step 3) is 3000 Da.

[0019] Preferably, the concentration gradient of the NaCl solution in step 4) is 0, 0.1, 0.2, and 0.3 mol / L.

[0020] The application of the above-mentioned *Sargassum fusiforme* polysaccharide, or the *Sargassum fusiforme* polysaccharide prepared by the above method, in the preparation of antitumor drugs.

[0021] Preferably, when the concentration of sea sedge polysaccharide is ≥100μg / mL, the inhibitory effect on tumor cells is significantly enhanced, and the inhibitory effect is dose-dependent.

[0022] Preferably, the tumor is liver cancer.

[0023] An antitumor pharmaceutical composition comprising the above-mentioned *Sargassum fusiforme* polysaccharide, or *Sargassum fusiforme* polysaccharide prepared by the above-mentioned method, and pharmaceutically acceptable excipients.

[0024] The beneficial effects of this invention are as follows:

[0025] The *Sargassum fusiforme* polysaccharide of this invention possesses a very unique surface morphology, exhibiting a regular serrated distribution. This invention studies the antitumor activity of the *Sargassum fusiforme* polysaccharide and finds that it can inhibit the growth of HepG2 cells. The molecular mechanism of its antitumor effect is that the polysaccharide can induce apoptosis in HepG2 cells. The *Sargassum fusiforme* polysaccharide of this invention exhibits significant antitumor activity and has broad development prospects as a candidate drug for antitumor therapy. Attached Figure Description

[0026] Figure 1 For the extraction, purification, molecular weight composition, and monosaccharide composition analysis of the polysaccharides from *Sargassum fusiforme* in Example 1: A is the elution curve of DEAE cellulose column purification; B is the elution curve of dextran gel purification; C is the absolute molecular weight analysis diagram of the polysaccharides; D is the molecular configuration analysis diagram of the polysaccharides; E is the ion chromatogram of the monosaccharide standard; F is the ion chromatogram of the monosaccharide composition of the sample.

[0027] Figure 2Infrared (A) and electron (B) images of the polysaccharide from *Sargassum fusiforme* in Example 1;

[0028] Figure 3 The polysaccharide from sea peltaceus in Example 1 1 H-NMR(A), 13 C-NMR (B), HSQC (C), HMBC (D), COSY (E) and NOESY (F) spectral analysis diagrams;

[0029] Figure 4 The main chain repeating unit and branch chain structure of the sea pelt polysaccharide in Example 1;

[0030] Figure 5 The effect of sea sedge polysaccharide on HepG2 cell viability in Example 2;

[0031] Figure 6 Microscopic images of HepG2 cells treated with different concentrations of SabPS-1 in Example 2: A is the Control group cells; B is the cells treated with 50 μg / mL 5-Fu; C is the cells treated with 50 μg / mL SabPS-1 polysaccharide; D is the cells treated with 100 μg / mL SabPS-1 polysaccharide; E is the cells treated with 200 μg / mL SabPS-1 polysaccharide; F is the cells treated with 400 μg / mL SabPS-1 polysaccharide.

[0032] Figure 7 Fluorescence micrographs of HepG2 cells treated with different concentrations of *Sargassum fusiforme* polysaccharide in Example 2: A is the Control group cells; B is the cells treated with 50 μg / mL 5-Fu; C is the cells treated with 50 μg / mL SabPS-1 polysaccharide; D is the cells treated with 100 μg / mL 1SabPS-1 polysaccharide; E is the cells treated with 200 μg / mL SabPS-1 polysaccharide; F is the cells treated with 400 μg / mL SabPS-1 polysaccharide.

[0033] Figure 8 The effect of *Sargassum fusiforme* polysaccharide on HepG2 cell apoptosis in Example 2: A shows the flow cytometry analysis spectra of V-FITC and PI-labeled HepG2 cells: a is the Control group cells, b is the cells treated with 50 μg / mL 5-Fu, c is the cells treated with 50 μg / mL SabPS-1 polysaccharide, d is the cells treated with 100 μg / mL SabPS-1 polysaccharide, e is the cells treated with 200 μg / mL SabPS-1 polysaccharide, and f is the cells treated with 400 μg / mL SabPS-1 polysaccharide; B shows the cell apoptosis rate.

[0034] Figure 9 For the protein expression analysis in Example 2: A shows the results of different protein expression analysis; B shows the grayscale value of protein expression. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0036] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] The HepG2 liver cancer cell line used in the following examples was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0039] Example 1: Preparation and structural analysis of SabPS-1, a polysaccharide from the sea sedge.

[0040] A method for preparing polysaccharides from sea spirulina, the specific steps of which are as follows:

[0041] 1. Raw material pretreatment: Collect fresh sea pelts, wash away the mud and sand, and dry them. Dry them at 55℃, and then crush them with a pulverizer to obtain dried sea pelt powder for later use.

[0042] 2. The dried *Spatholobus suberectus* powder was treated with ethanol solution overnight for defatting and decolorization. The dried residue was extracted with hot water at a material-to-water ratio of 1:10 at 60°C for 4 hours. The precipitate was extracted again (twice) as described above, and the supernatants were combined. The extracted solution was concentrated and precipitated with 4 times its volume of anhydrous ethanol at 4°C to obtain the water extract (crude polysaccharide sample).

[0043] 3. The crude polysaccharide sample was redissolved in water, deproteinized using the Sevag method, dialyzed with water (3000 Da) sequentially, concentrated and freeze-dried to obtain crude (aqueous) polysaccharide.

[0044] 4. After redissolving the crude polysaccharide, separate it using a DEAE Seplife FF cellulose column (26×500 mm) with distilled water and NaCl (0.00, 0.10, 0.20, 0.30 mol / L) solutions at a flow rate of 1.5 mL / min. Determine the polysaccharide content in each fraction (5 mL / tube) using the phenol-sulfuric acid method. Transfer the major polysaccharide to a Sephacryl S-400HR dextran gel column (26×1000 mm) on an AKTA exlorer system (GE Healthcare), elute with distilled water at a flow rate of 1.0 mL / min, and determine the content using the phenol-sulfuric acid method to obtain the *Sargassum fusiforme* extract sample.

[0045] 5. Dissolve the above sample in a 0.1M NaNO3 aqueous solution containing 0.02% NaN3 at a concentration of 1 mg / mL, and filter through a filter with a pore size of 0.45 μm. Dissolve the sample in a DMSO solution containing 1 mg / mL lithium bromide (0.5% w / w) (DMSO / LiBr), and filter through a filter with a pore size of 0.45 μm. Measure the homogeneity and molecular weight of each component using SEC-MALLS-RI. Measure the weight and number-average molecular weight (Mw and Mn) and polydispersity index (Mw / Mn) of each component using a DAWN HELEOS-II laser spectrophotometer equipped with three tandem columns (300×8 mm, Shodex OH pak SB-805, 804, and 803), maintaining a column temperature of 45°C and a flow rate of 0.4 mL / min.

[0046] 6. Determination of monosaccharide composition of pure *Sargassum fusiforme* polysaccharides: In a sealed tube, approximately 5 mg of sample was hydrolyzed with trifluoroacetic acid (2M) at 121 °C for 2 h. The sample was dried under nitrogen, washed with methanol, and then dried. This methanol washing was repeated 2-3 times. The residue was redissolved in deionized water and filtered through a 0.22 μm microporous membrane for measurement. The sample extract was analyzed by high performance anion exchange chromatography (HPAEC) using a pulsed current detector (PAD; Dionex-ICS 5000+ system) on a CarboPac PA-20 anion exchange column (3 × 150 mm). The flow rate was 0.5 mL / min, the injection volume was 5 μL, and the solvent system A consisted of ddH₂O, solvent system B consisted of 0.1 M NaOH, and solvent system C consisted of 0.1 M sodium hydroxide and 0.2 M NaAc. A gradient program was used, with the volume ratios of solutions A, B, and C being 95:5:0 at 0 min, 85:5:10 at 26 min, 85:5:10 at 42 min, 60:0:40 at 42.1 min, 60:40:0 at 52 min, 95:5:0 at 52.1 min, and 95:5:0 at 60 min. Data were acquired using ICS5000+ (Thermo Scientific) and processed using Chromeleon 7.2 CDS (Thermo Scientific).

[0047] 7. Methylation was used to reduce and analyze the uronic acid content of polysaccharides. The sample was reduced with NaBH4 and NaBD4, dialyzed, and freeze-dried to obtain the reduced product. The reduced product was then methylated with CH3I in NaOH / DMSO. After complete methylation, the product was hydrolyzed with 2 mol / L TFA at 121 °C for 1.5 h, reduced with NaBD4, and acetylated with acetic anhydride for 2.5 h (100 °C). The acetate was dissolved in chloroform and analyzed by GC-MS on an Agilent 6890A-5975C column equipped with an Agilent BPX70 column (30 m × 0.25 mm × 0.25 μm, SGE, Australia). High-purity helium (split ratio 10:1) was used as the carrier gas, with an injection volume of 1 μL. Mass spectrometry was performed at an initial temperature of 140 °C for 2.0 min, and the temperature was increased to 230 °C at a rate of 3 °C / min for 3 min. The scanning mode is scan, and the scanning range (m / z) is 30 to 600.

[0048] 8. The Fourier transform infrared (FT-IR) spectrum of *Sargassum fusiforme* polysaccharide was determined using a spectrometer (Nicolet iZ-10, Thermo Nicolet, USA). The sample was mixed with KBr powder and then pressed into 1 mm particles. The particles were then analyzed at 4000-400 cm⁻¹. -1 FT-IR measurements were performed within the specified range. The molecular morphology of the polysaccharide was observed using a scanning electron microscope (Zeiss-Merlin Compact, Germany). Samples coated with a thin gold layer were placed on a substrate, and images were observed at 100x and 4000x magnification under high vacuum at a voltage of 1.0 kV.

[0049] 9. NMR Analysis of Polysaccharides: Seaweed polysaccharides were dissolved in 0.5 mL of D₂O, resulting in a final concentration of 40 mg / mL. 1D-NMR and 2D-NMR were recorded and analyzed at 500 MHz using a Bruker AVANCE NEO 500M spectrometer system at 25 °C. 1 H-NMR, 13 C-NMR, COSY, NOESY, HMBC, and HSQC.

[0050] 10. Experimental Results

[0051] (1) In this embodiment, 27.35g of crude polysaccharide containing impurities was extracted from 500g of *Sargassum fusiforme* powder, with a yield of 5.46%. Impurities were removed from 20g of the crude polysaccharide, yielding 5.64g of crude polysaccharide. The crude polysaccharide was separated by elution with DEAE FF anion exchange resin and sodium chloride at concentrations of 0, 0.1, 0.2, and 0.3 mol / L, and divided into four fractions ( Figure 1(A) : SabPS-0, SabPS-0.1, SabPS-0.2, and SabPS-0.3, with yields of 15.96%, 10.64%, 2.66%, and 0.89%, respectively. Figure 1 As shown in Figure B, SabPS-0 was further purified using a Sephacryl S-400HR gel column to obtain homogeneous polysaccharide SabPS-1 (tubes 35-41). The yield of SabPS-1 obtained by the sulfuric acid method was 66.67%. The molecular weight of the purified polysaccharide was determined using GPC-RI-MALS technology. Figure 1 As shown in Figure C, the absolute molecular weight analysis of the polysaccharide is performed using multi-angle laser scattering signals, differential signals, and molecular weights fitted from these two signals. The solvent peak of the mobile phase is approximately 65 min for the inorganic salt phase and approximately 102 min for the DMSO phase. Figure 1 Figure D shows the molecular configuration diagram, with the slope representing the molecular configuration of different polysaccharides. Analysis of the chromatographic data revealed that the molecular weight of the homogeneous polysaccharide from *Salvia splendens* (SabPS-1) is 3.24 × 10⁻⁶. 4 The slope of the α value is 0.08 ± 0.03, indicating that SabPS-1 is composed of densely cross-linked macromolecules with a highly branched structure. The composition of SabPS-1 was analyzed using ion chromatography with an electrochemical detector. The ion chromatograms of the monosaccharide standard and the SabPS-1 sample are shown below. Figure 1 Figures E and F show the results. Based on the retention time and concentration standard curves of the standards on the analytical column, the monosaccharide composition and content of SabPS-1 were analyzed and calculated. The results showed that the monosaccharide composition included Fuc (1.12%), Ara (24.96%), Rha (6.21%), Gal (30.39%), Glc (1.54%), Xyl (4.99%), Man (1.69%), Gal-UA (23.20%), Gul-UA (1.03%), Glc-UA (3.12%), and Man-UA (1.75%), indicating that SabPS-1 is a multiphase polysaccharide mainly composed of Gal, Gal-UA, and Ara.

[0052] (2) Gas chromatography-mass spectrometry (GC-MS) was used to analyze characteristic fragments of methylated SabPS-1 to confirm the polysaccharide binding modes. The names, molecular weights, and relative molar ratios of the derivatives with different binding structures obtained are shown in Table 1. The binding modes of arabinose in SabPS-1 were: t-Ara(f), 3-Ara(f), 5-Ara(f), 2,5-Ara; the binding modes of galactose were: t-Gal(p), 3-Gal(p), 4-Gal(p-), 6-Gal(p-), 3,4-Gal(p-), 2,3,4-Gal(p-), 4,6-Gal. The binding mode of mannose was t-Man(p). The binding modes of glucose were 2-Glc(p), 2,4-Glc(p), and 4-Glc(p)-UA. The methylation results indicated that SabPS-1 belongs to the arabinogalactan family.

[0053] Table 1. Linkage patterns and GC-MS data of SabPS-1 methylation

[0054]

[0055]

[0056] (3) The FT-IR spectrum of SabPS-1 is as follows: Figure 2 As shown in Figure A, the results indicate that the absorption band is in the range of 3600-3200 cm⁻¹. -1 The absorption peak at this point is due to the stretching vibration of the -OH group; this region's absorption peak is a characteristic peak of sugars. 3435.59 cm⁻¹ -1 This is the absorption peak of the stretching vibration of OH, which is a characteristic peak of sugars. 2929.55 cm⁻¹ -1 The absorption peak at 1625.99 cm⁻¹ is attributed to the CH stretching vibration of the CH₂ group in the carbohydrate. -1 The absorption peak at 1418.91 cm⁻¹ is attributed to the C=O stretching vibration. -1 The vibrations at this location are CH2 bending and COO tensile, 1384.32 cm. -1 (Bending vibration of CH), 1249.84cm -1 (COC stretching) and 1074.38cm -1 The position is attributed to the stretching vibration of CO, suggesting the presence of a β-configuration of the dextran due to O-substituted glucose residues. 765.96 cm -1 The absorption peak at that point indicates the presence of β-pyranoside in the molecular structure of the polysaccharide. Figure 2Scanning electron microscopy (SEM) results from the sample revealed that SabPS-1 possesses a unique and complex planar structure with regularly spaced serrated protrusions. This structure is significantly different from other cylindrical and network polysaccharides, which may be closely related to the fact that *Sargassum fusiforme* lives in saline-alkali regions. This unique structure may enable *Sargassum fusiforme* to adapt to extreme soil environments.

[0057] (4) In this embodiment, nuclear magnetic resonance (NMR) spectroscopy was used to analyze the bonding structure of SabPS-1, and combined with one-dimensional... 1 H spectrum, 13 C-spectroscopy, two-dimensional COSY spectroscopy, HSQC spectroscopy, HMBC spectroscopy, and NOESY spectroscopy were used to analyze the configurations and related data of various bonding structures. For example... Figure 3 As shown in Figure A, the hydrogen spectral signal of SabPS-1 is mainly concentrated between δ 3.0-5.4 ppm. Multiple coupling signal peaks were found in the heteropolymer signal region of δ 4.3-5.4 ppm, indicating the presence of multiple sugar residues in SabPS-1, corresponding to the chemical shifts of heteropolymer hydrogen at δ 5.18, 5.02, and 4.41 ppm. The non-exotic hydrogen signal is mainly concentrated in the δ 3.2-4.2 ppm region. Due to the severe overlap of individual signals, it is necessary to combine HSQC and COSY spectra to separately assign the H2-H6 chemical shifts of each sugar residue. Figure 3 (C and E in the middle). The strong signal peak near δ4.71 ppm is a solvent peak. The signal peak for O-CH3 hydrogen is near δ3.66 ppm. (Compared to...) 1 Compared to H NMR, polysaccharides are 13 The chemical shift signal distribution is broader in C10 NMR. 13 Cross peaks in the anodic region of the C NMR and HSQC spectra were used to determine the anodic signals in the sample as: δ4.41 / 103.16, δ5.18 / 109.37, δ5.02 / 107.48, δ5.03 / 99.59 and δ4.61 / 103.84 ppm, which were recorded as sugar residues A, B, C, D and E, as shown in Table 2.

[0058] Table 2 Sugar residues of sea pelt polysaccharides 1 H and 13 Chemical shift of C

[0059]

[0060]

[0061] In Table 2: nd indicates not detected.

[0062] Based on the information in Table 2, the sample binding structure (methylation) information, and the anodic signal, it is inferred that sugar residue A is →4)-β-D-GalpA-(1→), residue B is α-L-Araf-(1→), residue C is →5)-α-L-Araf-(1→, residue D is →3,6)-β-D-Galp-(1→, and residue E is →4)-β-D-Galp-(1→). The signal peak near δ59.38ppm is the carbon signal of O-CH3. Combined with HSQC, a total of three methoxy signals were identified in the spectrum. Figure 3 The concentrations (C) in the samples, specifically δ3.66 / 59.38 ppm, δ3.41 / 59.96 ppm, and δ3.73 / 52.86 ppm, indicate a higher concentration of methoxy groups. This is based on the sugar residues in SabPS-1. 13 C and 1 The chemical shift of H, combined with HMBC spectral analysis of the polysaccharide structure and linkage pattern, revealed a cross-peak at δ 4.41 / 69.22 ppm between sugar residues A-H1 and D-C6. Due to the relatively weak cross-peak signal in the HMBC spectrum... Figure 3 Therefore, it was further combined with NOESY spectroscopy to determine the linkage sequence of residues in the polysaccharide. Figure 3 (F). A cross peak exists between sugar residues A-H1 and D-H6 at δ 4.41 / 3.82 ppm. A cross peak exists between sugar residues B-H1 and D-H3 at δ 5.18 / 3.68 ppm. A cross peak exists between sugar residues C-H1 and E-H4 at δ 5.02 / 3.65 ppm. A cross peak exists between sugar residues D-H1 and C-H5 at δ 5.03 / 3.73 ppm. A cross peak exists between sugar residues E-H1 and A-H4 at δ 4.61 / 3.82 ppm.

[0063] In summary, based on the analysis of one-dimensional and two-dimensional NMR information, it is inferred that the polysaccharide backbone is mainly composed of →4)-β-D-GalpA-(1→, →5)-α-L-Araf-(1→ and →4)-β-D-Galp-(1→), and the side chains are mainly composed of α-L-Araf-(1→ linked to the O-3 positions of the sugar residue →3,6)-β-D-Galp-(1→). Therefore, the structure of the SabPS-1 chain is as follows: Figure 4 As shown, where n is a natural number ≥ 1.

[0064] Example 2: Analysis of the antitumor activity of SabPS-1 polysaccharide from the sea sedge.

[0065] 1. Effects of sea pelt polysaccharides on tumor cell activity and morphology

[0066] HepG2 cells were cultured in DMEM medium supplemented with 10% (v / v) heat-inactivated FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured at 37°C, 5% CO2, and 95% air. The medium was changed 2-3 times weekly. Cell viability was assessed using the MTT assay. After complete digestion of cells in the logarithmic growth phase, 10 μL was transferred to a hemocytometer and cultured at 6 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates. After 24 h of culture, cells were treated with different concentrations (25, 50, 100, 200, 400 μg / mL) of SabPS-1 for 48 h. 5-Fluorouracil (5-FU, 50 μg / mL) was used as a positive control, while untreated cells served as a negative control (Control). Five wells were set up for each group. After 48 h, the original culture medium was removed, and 100 μL of MTT solution (1 mg / mL) was added to each well. Cells were then incubated for another 4 h in a cell culture incubator. The MTT solution was then removed, and the cells were incubated with 100 μL of LDMSO per well for 1 h. The absorbance at 570 nm was measured using a microplate reader. Cell morphology was observed and photographed using an inverted microscope at randomly selected fields of view.

[0067] 2. DAPI (4',6-diamidinyl-2-phenylindole) staining is used to detect cell nuclear morphology.

[0068] HepG2 cells in the logarithmic growth phase were digested and then subjected to a process involving 4 × 10⁻⁶ cells. 4 HepG2 cells were seeded at a density of 500 μL / mL into 24-well plates. After 24 h of culture, the cells were treated with different concentrations (50, 100, 200, 400 μg / mL) of SabPS-1 polysaccharide for specific durations. The culture medium was removed, the cells were washed twice with PBS, and fixed with 4% PFA for 15 min at room temperature. DAPI staining solution was added and the cells were incubated in the dark at room temperature for 10 min. The staining solution was removed, the cells were washed twice with PBS, and nuclear changes were observed under fluorescence.

[0069] 3. Flow cytometry detection of apoptosis

[0070] Apoptosis rate was detected using Annexin V-FITC and PI staining. HepG2 cells were treated with different concentrations (50, 100, 200, 400 μg / mL) of SabPS-1 for specific time periods, and 1×10⁻⁶ cells were collected. 6 Cells were washed twice with cooled PBS, and 400 μL of buffer solution was added and gently mixed. 5 μL of Annexin V-FITC and 5 μL of PI solution were added, and the cells were incubated in the dark for 15 min. The apoptosis rate was then detected by flow cytometry.

[0071] 4. Protein expression analysis

[0072] After treatment with different concentrations (50, 100, 200, 400 μg / mL) of SabPS-1 for 24 h, HepG2 cells were removed, washed twice with ice-cold PBS, and centrifuged at 12000×g for 10 min at 4 °C. The cells were then incubated on ice for 20 min using lysis buffer. The lysis solution was transferred to a new EP tube and centrifuged at 12000×g for 5 min. The protein concentration in the supernatant was determined using a BCA protein assay kit. Proteins were separated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a PVDF membrane (0.45 μm). Subsequently, the PVDF membrane was placed in blocking buffer (0.3 g BSA, 20 mL PBST, and 5% skim milk) and sealed at room temperature for 2 h. Then, the protein bands were incubated overnight at 4°C with caspase 3 (1:1000), caspase 8 (1:1000), Bax (1:1000), Bcl-2 (1:1000), and internal control GAPDH (1:1000). The mixture was then eluted with PBST solution to remove non-specific bindings, three times for 15 min each time, followed by incubation with secondary antibody dilution buffer (1:2000) for 2 h. Protein bands were detected using enhanced chemiluminescence reagents. Chemiluminescence signals were detected and analyzed using a Chemi-Doc XRS imaging system (Bio-Rad, CA, USA).

[0073] 5. Experimental Results

[0074] (1) The effect of SabPS-1 polysaccharide on the proliferation of HepG2 cells was analyzed using the MTT assay. Figure 5 As shown, after treating HepG2 cells with different concentrations (25, 50, 100, 200, and 400 μg / mL) of SabPS-1 for 24 h, the inhibitory effect on cell proliferation increased with increasing SabPS-1 concentration. The viability of HepG2 cells was 97.24±1.16 (25 μg / mL), 94.32±1.73 (50 μg / mL), 90.05±4.84 (100 μg / mL), 83.36±2.78 (200 μg / mL), and 72.05±2.14 (400 μg / mL). Compared with the positive control group (5-Fu, 50 μg / mL) of 61.03±2.24, when the concentration of SabPS-1 was 100 μg / mL, it had a significant inhibitory effect on the proliferation of HepG2 cells (P<0.05), and showed a dose-dependent effect.

[0075] (2) To study the effect of SabPS-1 on HepG2 cell apoptosis, morphological changes in HepG2 cells treated with different concentrations (50, 100, 200, and 400 μg / mL) of SabPS-1 were observed using an inverted microscope. Figure 6 As shown, HepG2 cells treated with polysaccharides underwent a series of changes compared to untreated cell tissue, and these changes increased with increasing SabPS-1 concentration. Figure 6 As shown in the CF, the cell tissue exhibited severe shrinkage, loose arrangement, and a significant reduction in cell number. The positive control group (5-Fu, 50 μg / mL) showed... Figure 6 The control group (B) showed more significant changes, while the untreated group (Control) showed more significant changes. Figure 6 The cells in group A) showed no change.

[0076] (3) The changes in cell nuclear morphology after treatment with SabPS-1 (50, 100, 200, and 400 μg / mL) were observed using DAPI staining. For example... Figure 7 As shown in Figure A, the untreated (Control) cell nuclei exhibit a regular oval shape. Figure 7 As shown in the CF, characteristic signs of apoptosis were observed, including nuclear pyknosis and karyorrhagia, accompanied by bright blue fluorescence. The positive control group (5-Fu, 50 μg / mL) showed similar characteristics. Figure 7 (B) showed a stronger effect. The results indicated that SabPS-1 could induce apoptosis in HepG2 cells and inhibit cell growth.

[0077] (4) To verify the effect of SabPS-1 on HepG2 cell apoptosis, Annexin V-FITC / PI staining combined with flow cytometry was used for analysis. After HepG2 cells were treated with different concentrations (50, 100, 200, and 400 μg / mL) of polysaccharide for 24 h, the early and late apoptosis rates compared to normal cells were significantly lower. Figure 8 As shown, the apoptosis rate of cells was 12.38±2.03% and 15.18±3.21% (…). * P < 0.05), 21.72 ± 5.31% ** P < 0.01), 30.35 ± 4.91% *** (P < 0.001) The apoptosis rate of the positive control group (5-Fu, 50 μg / mL) was 34.51 ± 3.46%, and the apoptosis rate of each treatment group was significantly higher than that of the untreated group (Control). These results further confirm that SabPS-1 can induce apoptosis in HepG2 cells.

[0078] (5) Caspase family proteins are the main protease components in the proteolytic system involved in apoptosis. These enzymes trigger a cascade of enzymatic degradation reactions under the promotion of pro-apoptotic signals. Based on structural and functional differences, caspase family proteins can be divided into two categories: initiating caspases (caspase 2 / 8 / 9 / 10) and executing caspases (caspase 3 / 6 / 7). In the extrinsic apoptosis pathway, the precursor of caspase 8 recognizes the FAS ligand (FASL) through the intracellular adaptor protein FAS-associated death domain (FADD), forming the death-inducing signal complex (DISC). DISC catalyzes the activation of caspase 8. Caspase 8 induces apoptosis indirectly by directly activating the effector caspase 3. To further understand the mechanism of SabPS-1-induced apoptosis, we examined the expression and activation of caspase 3 and caspase 8. Figure 9 As shown in Figures A and B, compared with the untreated group (Control), the expression levels of caspase 3 and caspase 8 increased significantly with the increase of polysaccharide SabPS-1 concentration.

[0079] Studies have shown that the Bcl-2 protein family is closely related to apoptosis. Bcl-2 family proteins can control the release of mitochondrial apoptosis-inducing factors and are mainly divided into pro-apoptotic proteins (Bax, Bak, Bid, etc.) and anti-apoptotic proteins (Bcl-2, Bcl-xL, Bcl-w, etc.). Therefore, this example also analyzes the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2, such as... Figure 9 As shown in Figures A and B, the results indicate that with the increase of SabPS-1 concentration, the expression level of Bxa significantly increases, while the expression level of Bcl-2 significantly decreases, and the Bax / Bcl-2 ratio increases. This is an important factor in apoptosis and promotes the release of cytochrome C into the cytoplasm in HepG2 cells.

[0080] The experimental results of this embodiment demonstrate that the polysaccharide SabPS-1 can significantly inhibit the proliferation of HepG2 cells and promote apoptosis. The mechanism by which SabPS-1 induces apoptosis in HepG2 cells is related to the increased expression of caspase 3 and caspase 8, as well as the increased expression of Bax and the decreased expression of Bcl-2, indicating that SabPS-1 has anti-tumor activity.

[0081] The above embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the protection scope of the present invention.

Claims

1. The application of a polysaccharide from *Sargassum fusiforme* in the preparation of an anti-liver cancer drug, characterized in that... The polysaccharide from *Salvia splendens* is composed of 11 monosaccharides: fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, guluronic acid, glucuronic acid, and mannuronic acid. The monosaccharide ratio is 1.12:24.96:6.21:30.39:1.54:4.99:1.69:23.20:1.03:3.12:1.75; the relative molecular weight is 3.24 × 10⁻⁶. 4 The structure of the polysaccharide from *Salvia splendens* is shown below: ； Where n is a natural number ≥ 1.

2. The application according to claim 1, characterized in that, The preparation method of the polysaccharide from *Sargassum fusiforme* includes the following steps: Step 1) Raw material pretreatment: Collect fresh sea sedges, wash them, air dry them, dry them at 55℃, and crush them to obtain dried sea sedge powder; Step 2) Treat the dried sea sedge powder with ethanol solution overnight. Extract the residue after drying with hot water at a material-to-water ratio of 1:

10. Extract the precipitate at 60°C for 4 hours. Repeat the extraction operation twice and combine the supernatants. Concentrate the extracted solution and precipitate it with 4 times the volume of anhydrous ethanol at 4°C to obtain the crude polysaccharide sample. Step 3) The crude polysaccharide sample was redissolved in water, deproteinized using the Sevag method, and dialyzed with water sequentially. The molecular weight of the dialyzed sample was 3000 Da. The sample was then concentrated and freeze-dried to obtain the crude polysaccharide. Step 4) After redissolving the crude polysaccharide, separate the crude polysaccharide using a 26×500 mm DEAE seplife FF cellulose column, sequentially with distilled water and NaCl solution at a rate of 1.5 mL / min. The concentration gradient of the NaCl solution is 0, 0.1, 0.2, and 0.3 mol / L; Step 5) The first eluted fraction from Step 4) was eluted with a 26×1000 mm dextran gel column (Sephacryl S-400 HR column) and distilled water at a flow rate of 1.0 mL / min to obtain the sea sedge polysaccharide.

3. The application according to claim 1, characterized in that, In the drug, the concentration of sea sedge polysaccharide is ≥100 μg / mL.

Images