Radix tetrastigmae polysaccharide, preparation method and application in gastric mucosa injury

Abstract

The application belongs to the technical field of biological medicine, and discloses a radix tetrastigme rhizome polysaccharide, a preparation method and application in gastric mucosa injury, and an acid heteropolysaccharide SYQ-P is obtained by separation and purification from the radix tetrastigme rhizome, the average molecular weight of the acid heteropolysaccharide SYQ-P is 399.7 kDa, the total sugar mass content reaches 91.75+ / -0.40%, and the acid heteropolysaccharide SYQ-P is composed of mannose, glucuronic acid, glucose, galactose, arabinose and rhamnose; the polysaccharide is prepared by hot water extraction, ethanol precipitation, deproteinization and decolorization, anion exchange chromatography and gel filtration chromatography. The SYQ-P has good biological safety, can significantly reduce alcoholic gastric mucosa injury, promote alcohol metabolism of the gastric tissue, inhibit inflammatory reaction, maintain the integrity of a tight connection barrier, regulate a cell pyroptosis path to play a gastric protection role, and can be used for preparing medicines and health foods for preventing and treating alcoholic gastric mucosa injury, and improving the resource utilization value of the radix tetrastigme.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a polysaccharide from the rootlets of *Trifolium repens*, its preparation method, and its application in gastric mucosal injury. Background Technology

[0002] Alcoholic gastric mucosal injury is a common digestive system disease in clinical practice. Short-term heavy drinking can induce acute gastric mucosal congestion, edema, erosion, and even ulcers. Long-term drinking can progress to chronic gastritis and gastric ulcers, and in severe cases, it can lead to critical complications such as gastric bleeding and perforation. Currently, clinical treatment mainly relies on proton pump inhibitors and chemically synthesized gastric mucosal protectants. Although these can quickly relieve symptoms, long-term use carries the risk of adverse reactions, and there is still a lack of preventive and therapeutic drugs that are of natural origin and have multiple targets.

[0003] Three-leaf green ( Tetrastigma hemsleyanum *Diels et Gilg* is a perennial herb belonging to the genus *Diels* in the family Vitaceae. It is a traditional Chinese medicine with traditional effects such as clearing heat and detoxifying, reducing swelling and dissipating nodules, and strengthening the spleen and resolving phlegm. Modern pharmacological studies have confirmed that *Diels et Gilg* possesses anti-inflammatory, immunomodulatory, and anti-tumor activities, and its active substances include various types such as flavonoids, triterpenes, and polysaccharides. Existing research mainly focuses on the tuberous roots of *Diels et Gilg*, with insufficient systematic research on the active ingredients in non-medicinal parts such as the fibrous roots, resulting in a waste of plant resources.

[0004] Plant polysaccharides, as natural biological macromolecules, are characterized by low toxicity, multiple targets, and good biocompatibility, showing great potential for development in the field of digestive system mucosal protection. Currently, there are no reports of structurally well-defined and highly homogeneous acidic heteropolysaccharides isolated from the roots of *Trifolium repens*, nor are there any systematic studies on the protective effects and molecular mechanisms of such polysaccharides against alcoholic gastric mucosal damage. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a polysaccharide from the fibrous roots of *Tripterygium wilfordii*, its preparation method, and its application in gastric mucosal injury. The crude polysaccharide is obtained through a pretreatment process involving hot water extraction, ethanol precipitation, Sevag deproteinization, and macroporous resin decolorization. It is then purified stepwise by DEAE-dextran gel anion exchange chromatography and Smartdex G-100 gel filtration chromatography to obtain the highly homogeneous acidic heteropolysaccharide SYQ-P. This achieves a balance between high extraction rate and high purity, providing a material basis and technical support for developing naturally derived products for the prevention and treatment of gastric mucosal injury, while simultaneously enhancing the resource utilization value of non-medicinal parts of *Tripterygium wilfordii*.

[0006] To achieve the above objectives, the present invention provides a polysaccharide from the rootlets of *Trifolium repens*, which is an acidic heteropolysaccharide. The monosaccharide composition of the *Trifolium repens* rootlets polysaccharide includes mannose, glucuronic acid, glucose, galactose, arabinose, and rhamnose.

[0007] Preferably, the proportions of each monosaccharide in the monosaccharide composition, by molar percentage, are: galactose 43.42%, glucuronic acid 16.61%, mannose 29.83%, arabinose 8.05%, glucose 1.20%, and rhamnose 0.89%.

[0008] A method for preparing the polysaccharide from the rootlets of *Trifolium repens* is also provided, comprising the following steps: (1) After washing, drying and cutting the roots of *Trifolium repens*, extract them with distilled water, combine the extracts and concentrate them, add anhydrous ethanol, let them precipitate overnight, and collect the precipitate. (2) The precipitate obtained in step (1) is redissolved in water, and then subjected to deproteinization treatment and decolorization treatment to obtain crude polysaccharide; (3) The crude polysaccharide was freeze-dried and dissolved in distilled water, then loaded onto a DEAE-dextran gel FF anion exchange column for gradient elution. The target component was collected, dialyzed with distilled water, and then freeze-dried. (4) The target component obtained in step (3) is loaded onto a Smartdex G-100 gel filtration chromatography column for elution. The eluent is collected, dialyzed, and freeze-dried to obtain polysaccharide from the roots of *Trifolium repens*.

[0009] Preferably, in step (1), the temperature of distilled water extraction is 100℃, the number of distilled water extractions is 3, the extraction time for each extraction is 1.5h, the material-to-liquid ratio is 1:10 (w / v); the concentration is made to a density of 0.05g / mL; and the volume ratio of anhydrous ethanol to the extract is 1:1.

[0010] Preferably, in step (2), the protein removal treatment is performed by the Sevag method; the decolorization treatment is performed by AB-8 macroporous resin.

[0011] Preferably, in step (3), the gradient elution is performed by sequentially eluting with distilled water, 0.4 mol / L NaCl solution, 0.8 mol / L NaCl solution and 1.0 mol / L NaCl solution at a flow rate of 2.0 mL / min; the target component is 0.4 mol / L NaCl eluent.

[0012] Preferably, in step (4), the eluent is distilled water and the elution flow rate is 2.0 mL / min.

[0013] The use of the polysaccharide from the roots of *Trifolium repens* in the preparation of products for the prevention and / or treatment of gastric mucosal damage is also provided.

[0014] Preferably, the gastric mucosal injury is ethanol-induced alcoholic gastric mucosal injury.

[0015] Preferably, the product works through at least one of the following pathways: reducing gastric mucosal bleeding and epithelial defects, lowering the gastric ulcer index, improving gastric tissue pathological damage, promoting gastric mucus secretion, increasing the activity of gastric tissue alcohol metabolism enzymes, inhibiting the release of gastric tissue pro-inflammatory factors, maintaining the expression of gastric mucosal tight junction proteins, and inhibiting gastric epithelial cell pyroptosis.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention achieves efficient separation and purification of polysaccharides from the fibrous roots of *Trifolium repens*, obtaining a homogeneous polysaccharide component with a clear structure and high purity. Specifically, a purification process of hot water extraction-ethanol precipitation, protein removal and decolorization, and anion exchange chromatography combined with gel filtration chromatography was used to obtain homogeneous polysaccharide SYQ-P from the fibrous roots of *Trifolium repens*. The total sugar content reached 91.75±0.40%, and there were no impurities such as proteins and nucleic acids. It is a highly branched acidic heteropolysaccharide composed of 6 monosaccharides with a clear and complete structure, providing a material basis for the in-depth development of non-medicinal parts of *Trifolium repens* and significantly improving the comprehensive utilization value of plant resources.

[0017] (2) The SYQ-P of this invention has significant in vivo protective effects against alcohol-induced gastric mucosal damage, with comprehensive and dose-dependent effects. Animal experiments have confirmed that SYQ-P pretreatment can significantly improve the pathological manifestations of ethanol-induced gastric mucosal congestion, edema, and erosion in mice, reduce the gastric ulcer index and histological damage score, and the protective effect of the high-dose group is comparable to that of omeprazole, a commonly used gastric mucosal protective drug in clinical practice; at the same time, it can increase the activity of ADH and ALDH in gastric tissue, promote local alcohol metabolism in the stomach, and reduce the accumulation of toxic metabolites such as acetaldehyde; it can also dose-dependently inhibit the excessive release of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, reduce the mucosal inflammatory cascade reaction, and significantly increase the PAS-positive mucus area of ​​the gastric mucosa, repair the mucus barrier, and exert gastric mucosal protective effects from multiple dimensions such as damage repair, metabolic regulation, inflammation suppression, and mucus protection.

[0018] (3) The present invention SYQ-P has good biocompatibility. In the concentration range of 0.26~67μg / mL, SYQ-P has no obvious cytotoxicity to normal human gastric epithelial GES-1 cells and has good biocompatibility. In the ethanol-induced GES-1 cell injury model, SYQ-P can dose-dependently increase the viability of damaged cells, reduce lactate dehydrogenase release, and alleviate cell membrane integrity damage, which verifies its protective activity on gastric epithelial cells and provides cellular support for in vivo efficacy.

[0019] (4) This invention has deeply developed the medicinal value of the rootlets of Tripterygium wilfordii, and provides a new direction for the resource utilization of non-root parts of Tripterygium wilfordii and further development into a natural gastric mucosa protectant. It can improve the comprehensive utilization benefits of medicinal materials and has good economic value and development prospects.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 This is a flowchart of the extraction and purification of SYQ-P from the rootlets of *Trifolium repens* in Example 1; Figure 2 The stepwise NaCl gradient elution curve (absorbance at 490 nm) of the crude polysaccharide in Example 1 on a DEAE-dextran gel fast flow column is shown. Figure 3 The elution curve is shown on the Smartdex G-100 gel filter column in Example 1. Figure 4 The molecular weight distribution curve of SYQ-P in Example 2; Figure 5 The Fourier transform infrared (FTIR) spectrum of SYQ-P in Example 2; Figure 6 The HPLC chromatogram of the SYQ-P monosaccharide composition in Example 2 is shown in Figure 2. A is the standard solution and B is SYQ-P. Figure 7 The image shows the UV-Vis absorption spectrum of the SYQ-P aqueous solution in the wavelength range of 200~800nm ​​obtained by the UV-Vis spectrophotometer in Example 2. Figure 8 The image is a scanning electron microscope (SEM) image of SYQ-P in Example 2, with a scale bar of 50 μm; Figure 9 This is the GC-MS chromatogram of SYQ-P methylation analysis in Example 2; Figure 10 The NMR results for SYQ-P polysaccharide in Example 2 are shown in the figure. A is the H spectrum, B is the C spectrum, C is the HSQC spectrum, and D is the NMR spectrum. 1 H, 1 H-COSY plot, E is TOCSY plot, F is HMBC plot, G is NOESY plot; Figure 11This figure illustrates the protective effect of SYQ-P against ethanol-induced gastric injury in vivo, as shown in Example 3. Figure A shows observations of mouse gastric mucosa (n=10); B shows quantitative gastric ulcer scoring; C shows representative histological micrographs of gastric tissue stained with H&E and AB-PAS; D shows the histological damage score stained with H&E; E shows the quantitative analysis of PAS-positive mucus area; F shows the expression level of the alcohol-metabolizing enzyme ALDH in gastric tissue homogenate; G shows the expression level of the alcohol-metabolizing enzyme ADH in gastric tissue homogenate; H shows the IL-1β pro-inflammatory cytokine profile in gastric tissue; I shows the IL-6 pro-inflammatory cytokine profile in gastric tissue; and J shows the TNF-α pro-inflammatory cytokine profile in gastric tissue. Data are expressed as mean ± standard deviation. Compared with the control group, # represents P < 0.05, ## represents P < 0.01, and compared with the model group... This means P < 0.05. P < 0.01, SYQ-PH represents a concentration of 400 mg / kg for SYQ-PH treatment, SYQ-PM represents a concentration of 200 mg / kg for SYQ-PM treatment, and SYQ-PL represents a concentration of 100 mg / kg for SYQ-PL treatment; Figure 12 This figure illustrates the protective effect of SYQ-P against ethanol-induced gastric injury in vitro, as shown in Example 4. Figure A shows the viability of GES-1 cells exposed to gradient ethanol concentrations to optimize modeling conditions; B shows the biosafety assessment of SYQ-P on GES-1 cells under normal conditions; C shows the dose-dependent cytoprotective effect of SYQ-P on ethanol-challenged GES-1 cells; and D shows the quantitative assessment of cell membrane damage via lactate dehydrogenase (LDH) release. Data are expressed as mean ± standard deviation. Compared with the control group, # represents P < 0.05, ## represents P < 0.01, and compared with the model group, ... This means P < 0.05. P < 0.01, Control represents the control group, Model represents the model group, and SYQ-P represents the SYQ-PH treatment group; Figure 13To demonstrate how SYQ-P maintains the integrity of tight junctions of the gastric mucosa in vivo and in vitro in Example 5, Figure A shows representative protein blot bands of Occludin and Claudin-5 in mouse gastric tissue; B shows the in vivo optical density analysis of Occludin relative to β-actin protein expression; C shows the in vivo optical density analysis of Claudin-5 relative to β-actin protein expression; D shows representative protein blot bands of ZO-1, Occludin, and Claudin-5 in GES-1 cells; E shows the quantitative statistical analysis of Claudin-5 protein level relative to β-actin in vitro; F shows the quantitative statistical analysis of Occludin protein level relative to β-actin in vitro; G... The table shows the IHC staining and semi-quantitative analysis (percentage of positive cells) of Occludin in mouse gastric tissue, with a scale bar of 200 μm. H represents the IF staining of ZO-1 (green) and DAPI (blue) counterstaining in gastric tissue and the relative semi-quantitative analysis of the percentage of positive cells (n=6), with a scale bar of 200 μm. I represents the immunofluorescence micrographs and mean fluorescence intensity statistics of Claudin-5 in GES-1 cells, with a scale bar of 200 μm. J represents the immunofluorescence micrographs and mean fluorescence intensity statistics of ZO-1 in GES-1 cells, with a scale bar of 200 μm. Data are expressed as mean ± standard deviation. Compared with the control group, # represents P < 0.05, ## represents P < 0.01, and compared with the model group, ... This means P < 0.05. P < 0.01, SYQ-PH represents a concentration of 400 mg / kg for SYQ-PH treatment, SYQ-PM represents a concentration of 200 mg / kg for SYQ-PM treatment, and SYQ-PL represents a concentration of 100 mg / kg for SYQ-PL treatment; Figure 14To verify the inhibitory effect of SYQ-P on GSDME-mediated pyroptosis in vivo and in vitro in Example 6, Figure A shows representative Western blot images of GSDME, IL-1β, Caspase-3, and GSDME-N in mouse gastric tissue; BE represents the quantitative optical density analysis of GSDME, IL-1β, Caspase-3, and GSDME-N protein expression relative to β-actin in vivo; F shows representative Western blot images of corresponding pyroptosis-related proteins in GES-1 cells; GJ represents the quantitative optical density analysis of GSDME, IL-1β, Caspase-3, and GSDME-N protein expression relative to β-actin in vitro; K represents the concentration of IL-1β secreted in the culture supernatant of GES-1 cells measured by ELISA; and L represents the concentration of GES-1 cells and Hoechst cells. Representative fluorescence micrographs of 33342 cells (blue for nuclei) and PI (red for ruptured membrane cells) co-stained, scale bar at 200 μm; data are expressed as mean ± standard deviation. Compared with the control group, # represents P < 0.05, ## represents P < 0.01, and compared with the model group, This means P < 0.05. P < 0.01, SYQ-PH represents a concentration of 400 mg / kg for SYQ-PH treatment, SYQ-PM represents a concentration of 200 mg / kg for SYQ-PM treatment, and SYQ-PL represents a concentration of 100 mg / kg for SYQ-PL treatment. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0025] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0026] In the examples, AB-8 macroporous resin and DEAE-dextran gel FF were purchased from Solarbio Science & Technology Co., Ltd. Smartdex G-100 was purchased from Changzhou Tianshiren Biotechnology Co., Ltd. (Jiangsu, China). Mannitol, rhamnose, galacturonic acid, galactose, glucose, glucuronic acid, arabinose, xylose, fucose, glucosamine, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and galactosamine were all purchased from Sigma-Aldrich (Merck, USA). IL-1β, TNF-α, and IL-6 enzyme-linked immunosorbent assay kits were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. Rabbit anti-ZO-1, rabbit anti-Claudin-5, rabbit anti-Occludin, rabbit anti-GSDME, rabbit anti-GSDME-N, and rabbit anti-Caspase-3 antibodies, as well as NK cells and GES-1 cells, were all purchased from Wuhan Boster Biological Engineering Co., Ltd. FITC-Plus anti-human granzyme B (GZMB) rabbit recombinant antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd. Lipo6000 TM The transfection reagent was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0027] Example 1 Preparation and purification of polysaccharide SYQ-P from the roots of *Trifolium repens*.

[0028] 1. Materials and reagents: Trifolium repens rootlets (batch number: 20231121) were provided by Zhejiang Guangsheng Pharmaceutical Co., Ltd. (Zhejiang, China).

[0029] 2. The preparation steps of SYQ-P, a polysaccharide from the roots of *Trifolium repens*, are as follows: Figure 1 As shown, specifically: (1) Hot water extraction and ethanol precipitation: The roots of *Trifolium repens* were thoroughly washed, dried, and cut into small sections. The prepared sample was extracted three times with hot distilled water at 100℃ for 1.5 hours each time, with a material-to-liquid ratio of 1:10 (w / v). The filtrates were combined, concentrated to a density of 0.05 g / mL, and an equal volume of anhydrous ethanol (1:1, v / v) was added for precipitation overnight.

[0030] (2) Deproteinization and decolorization: The extract obtained in step (1) was deproteinized using the Sevag method, and then decolorized using AB-8 macroporous resin to obtain crude polysaccharide.

[0031] (3) DEAE-dextran gel FF anion exchange chromatography: The crude polysaccharide was freeze-dried, dissolved in distilled water, and loaded onto a DEAE-dextran gel fast flow (FF) anion exchange column. It was eluted sequentially with distilled water, followed by gradient elution with NaCl solutions of concentrations of 0.4, 0.8, and 1.0 mol / L at a flow rate of 2.0 mL / min. The carbohydrate content of the eluent was continuously monitored using the phenol-sulfuric acid method. The elution curve (e.g., [image missing]) was then analyzed. Figure 2 As shown in the figure, the corresponding components were combined, dialyzed with distilled water, and freeze-dried to obtain seven different components, namely: SYQ-PW-1, SYQ-PW-2, SYQ-PN-4-1, SYQ-PN-4-2, SYQ-PN-4-3, SYQ-PN-8 and SYQ-PN-10.

[0032] (4) Smartdex G-100 gel filtration chromatography purification: Among them, the fraction SYQ-PN-4-1, which eluted with 0.4 mol / L NaCl and yielded the highest amount, was selected as the target major polysaccharide fraction. SYQ-PN-4-1 was further purified by Smartdex G-100 gel filtration chromatography column, using distilled water as the eluent at a flow rate of 2.0 mL / min. Figure 3 As shown, a single, highly symmetrical elution peak was obtained, indicating that a homogeneous polysaccharide component was successfully separated. The eluent corresponding to the main symmetrical peak was collected, dialyzed, and freeze-dried to obtain the final purified polysaccharide, named SYQ-P, with an extraction rate of 36.646%.

[0033] Example 2 Structural characterization of SYQ-P, a polysaccharide from the roots of *Clerodendrum trifoli

[0034] 1. Molecular weight determination.

[0035] The molecular weight and homogeneity of purified polysaccharides were determined using a high-performance size exclusion chromatography-multi-angle laser light scattering-differential refractive index detection system (HPSEC-MALLS-RI). Samples were prepared to a concentration of 1 mg / mL with 0.1 M NaNO3 solution and filtered through a 0.45 μm filter. An UltiMate 3000 high-performance liquid chromatography system was used, coupled with a DAWN HELEOS II multi-angle laser light scattering detector and an Optilab T-rEX differential refractive index detector. Ohpak SB-805 HQ and SB-803HQ size exclusion columns (both 300 × 8 mm) were used in series at a column temperature of 45 °C and an injection volume of 100 μL. A 0.1 M NaNO3 solution containing 0.02% NaN3 was used as the mobile phase at a flow rate of 0.6 mL / min, with isocratic elution for 35 min. Data processing: The obtained chromatographic and light scattering data were acquired, analyzed, and processed using ASTRA 6.1 software (Wyatt Technology) to determine the molecular weight distribution.

[0036] The results are as follows Figure 4 As shown, a single, sharp, and narrow symmetrical peak is observed, confirming the absence of heterogeneous macromolecular impurities. The average molecular weight of SYQ-P was determined to be 399.7 kDa.

[0037] 2. Fourier Transform Infrared Spectroscopy Analysis: The organic functional groups and structural characteristics of SYQ-P were analyzed using a Fourier Transform Infrared (FT-IR) spectrometer (Nicolet IS5, ThermoFisher, USA). The dried SYQ-P polysaccharide sample was uniformly mixed with spectral-grade KBr powder at a concentration of approximately 2% (w / w). The mixture was ground and pressed into transparent sheets, which were then spectroscopy-spectrated at 4000–400 cm⁻¹. -1 Record infrared absorption spectra within the wavenumber range.

[0038] The results are as follows Figure 5 As shown, the spectrum is at 3224.75 cm⁻¹ -1 A broad and strong characteristic absorption band is observed at this point, attributed to the OH stretching vibrations of hydroxyl groups both intermolecularly and intramolecularly. 1596.92 cm⁻¹ -1 The significant absorption peak at 1413.46 cm⁻¹ is typically associated with the asymmetric stretching vibration of the carbonyl group (C=O), suggesting the possible presence of uronic acid in the polysaccharide chain. Furthermore, the peak at 1413.46 cm⁻¹... -1 The spectral band at this location corresponds to the CH bending vibration. Specifically, the band at 1018.58 cm⁻¹... -1 The strong absorption peak observed is a classic fingerprint region of carbohydrates, indicating the stretching vibration of the COC and COH glycosidic bonds in the pyran ring structure.

[0039] 3. Determination of total sugar content.

[0040] The total carbohydrate content of SYQ-P was determined using the classic phenol-sulfuric acid method. Anhydrous glucose was used as the standard, and the absorbance of the sample reaction mixture was measured at 490 nm. The carbohydrate content was calculated based on the generated glucose calibration curve. The results showed that the total polysaccharide content of SYQ-P was 91.75 ± 0.40 (w / w%), indicating high polysaccharide purity.

[0041] 4. Monosaccharide composition analysis.

[0042] The monosaccharide composition of SYQ-P was determined by high-performance liquid chromatography (HPLC) combined with pre-column derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP). The sample was hydrolyzed with 2M trifluoroacetic acid (TFA) at 110℃ for 4 h, neutralized with NaOH, and derivatized under alkaline conditions at 70℃ in the dark for 1 h after the addition of an internal standard. The reaction solution was neutralized with HCl, and excess PMP was removed by chloroform extraction four times. The aqueous phase was filtered through a 0.22 μm filter. A Shim-pack GIST column (5 μm, 4.6 × 150 mm) was used at 30℃; the mobile phase was phosphate buffer (A) and acetonitrile (B) in a ratio of 83:17, the flow rate was 1.0 mL / min, and the UV detection wavelength was 254 nm. The results are as follows: Figure 6 As shown, SYQ-P is a complex heteropolysaccharide composed of six different monosaccharide residues, including mannose (Man), glucuronic acid (Glc-UA), glucose (Glc), galactose (Gal), arabinose (Ara), and rhamnose (Rha).

[0043] Quantitative analysis revealed that Gal (43.42%) and Man (29.83%) were the main monosaccharide components, collectively forming the main structural framework of SYQ-P. Glc-UA (16.61%) and Ara (8.05%) were present in moderate proportions, while Glc (1.20%) and Rha (0.89%) were identified as minor components. Glc-UA (16.61%) definitively classifies SYQ-P as an acidic heteropolysaccharide. This confirms the previously observed 1596.92 cm⁻¹ observation by FTIR. -1 There is a strong asymmetric carbonyl stretching band.

[0044] 5. Ultraviolet spectroscopy analysis: To detect potential impurities, the ultraviolet-visible absorption spectrum of the SYQ-P aqueous solution was recorded in the wavelength range of 200~800nm ​​using an ultraviolet-visible spectrophotometer.

[0045] The results are as follows Figure 7 As shown, the spectrum of SYQ-P exhibits a smooth curve with no discernible absorption peaks at 260 nm or 280 nm. Since proteins and nucleic acids exhibit strong characteristic UV absorption at 280 nm and 260 nm, respectively, these results demonstrate that large molecular weight proteins and nucleic acid impurities were successfully removed during purification, verifying the high purity of SYQ-P.

[0046] 6. Scanning electron microscopy (SEM) analysis.

[0047] The microstructure and three-dimensional structure of purified SYQ-P were characterized using scanning electron microscopy (SEM). A suitable amount of lyophilized polysaccharide powder was fixed onto an aluminum sample stage using a double-sided conductive carbon ribbon. Since the polysaccharide itself is non-conductive, a gold sputtering coating was applied to the sample under vacuum conditions to avoid charge effects and improve electron emission. Subsequently, the microstructural features of SYQ-P were observed and photographed at an accelerating voltage of 10.0 kV; representative microimages were acquired at 1000x magnification.

[0048] The results are as follows Figure 8 As shown, SYQ-P exhibits an irregular, porous three-dimensional sheet-like or membrane-like network structure; the surface of the scales is relatively smooth, but it is scattered with dispersed circular pores and interconnected cavities of varying sizes, which significantly increases the specific surface area, contributing to the excellent water solubility of SYQ-P and promoting its binding and interaction with biological receptors.

[0049] 7. Methylation analysis.

[0050] The glycosidic linkage of SYQ-P was elucidated by gas chromatography-mass spectrometry (GC-MS) analysis of partially methylated sugar alcohol acetates (PMAAs). First, the uronic acid in the SYQ-P polysaccharide was reduced with NaBD4, followed by complete methylation with iodomethane in a DMSO / NaOH system. The fully methylated product was hydrolyzed with 2M trifluoroacetic acid (121 °C, 90 min), reduced with NaBD4 to open the sugar ring, and then acetylated with acetic anhydride (100 °C, 2.5 h). The resulting PMAA derivatives were extracted with dichloromethane and analyzed using an Agilent 6890A-5977B GC-MS system equipped with a TG-200MS capillary column, with a temperature programmed gradient of 150 °C to 240 °C under electron impact (EI) ionization.

[0051] The results are as follows Figure 9As shown, based on their characteristic retention time and mass fragmentation spectra, a total of 12 different linkage modes were identified. Specifically, the results can be classified as follows: Terminal residues: The non-reducing ends of the polysaccharide are mainly composed of t-Gal(p) (molar ratio 36.07%), accompanied by small amounts of t-Ara(f) (5.30%), t-Glc(p)-UA (1.01%), and t-Rha(p) (0.39%). The high proportion of t-Gal(p) indicates that galactose is the main end-capping residue at the branch ends of the polysaccharide. Linear (main chain) residues: The structural backbone and extended chain contain various monosubstituted linkages. Among them, 4-Glc(p)-UA (13.94%) is the most abundant linear linkage, indicating that 1→4 linked glucuronic acid constitutes the main structural component. Other linear fragments included 2-Man(p) (6.50%), 4-Gal(p) (1.13%), 3-Man(p) (0.87%), 3-Glc(p) (0.79%), and 6-Glc(p) (0.54%). Branched residues: SYQ-P exhibited a highly branched structural feature. The main branching site was identified as 2,3-Man(p), with a molar ratio as high as 30.32%, indicating that the mannose residues were extensively disubstituted at both the O-2 and O-3 positions. In addition, a small amount of 3,6-Gal(p) (3.12%) was detected as a branching point. These results suggest that SYQ-P is a highly branched acidic heteropolysaccharide. Its core structural backbone may consist of 1→4 linked glucuronic acid and highly branched 2,3-linked mannose residues, with its numerous short branches or side chains mainly terminated by galactopyranose.

[0052] 8. Nuclear magnetic resonance analysis.

[0053] After the SYQ-P polysaccharide sample was repeatedly exchanged with heavy water three times, NMR analysis was performed (instrument: 500MHz nuclear magnetic resonance spectrometer (purchased from Bruker GmbH, Germany, 500MHz model), equipped with cryogenic probes (5mm C / H dual resonance helium cryogenic probe; 5mm broadband room temperature probe)).

[0054] The results are as follows Figure 10 As shown, 1 H-NMR spectrum and 13C-NMR spectroscopy results showed that the sample contained multiple distinct anomeric proton signal peaks (5.19, 5.06, and 4.29 ppm) and anomeric carbons (102.6, 102.1, 100.9, and 98.6 ppm), indicating that the glycosidic bonds in the sample were α / β type. HSQC spectroscopy revealed three distinct anomeric signal peaks (5.19 / 98.6 ppm, 5.06 / 100.9 ppm, and 4.29 / 102.6 ppm) and three weak anomeric signal peaks (5.24 / 98.6 ppm, 5.15 / 102.1 ppm, and 4.29 / 101.3 ppm). The inferred results are as follows: 1 H, 1 H-COSY results revealed coupling between 5.19 ppm (H1) and 4.08 ppm (H2); 4.08 ppm (H2) and 3.81 ppm (H3); 3.81 ppm (H3) and 3.60 ppm (H4); and 3.60 ppm (H4) and 3.71 ppm (H5). TOCSY results indicated that 5.19, 4.08, 3.81, and 3.60 ppm are in the same spin system. HSQC results showed coupling between 4.08 ppm and 78.8 ppm; 3.81 ppm and 76.0 ppm; 3.60 ppm and 68.8 ppm; 3.71 ppm and 72.9 ppm; and 3.60 and 3.71 ppm and 59.6 ppm. The residue corresponding to the anodic signal peak (5.19 / 98.6 ppm) is defined as residue A, with the linkage configuration →2,3)α-D-Man. p -(1→).

[0055] pass 1 H, 1 H-COSY results revealed coupling between 5.06 ppm (H1) and 3.63 ppm (H2); 3.63 ppm (H2) and 3.73 ppm (H3); 3.73 ppm (H3) and 3.75 ppm (H4); 3.75 ppm (H4) and 4.08 ppm (H5); and 4.08 ppm (H5) and 3.58 ppm (H6). TOCSY results indicated that 5.06, 3.75, 3.73, and 3.63 ppm are in the same spin system. HSQC results showed coupling between 4.08 ppm and 71.2 ppm; 3.75 ppm and 69.5 ppm; 3.73 ppm and 69.3 ppm; 3.63 ppm and 68.8 ppm; and 3.58 ppm and 61.8 ppm. The residue corresponding to the anodic signal peak (5.06 / 100.9ppm) is defined as residue B, which is linked in the manner of α-D-Gal. p -(1→).

[0056] pass 1 H, 1 H-COSY results revealed coupling between 4.29 ppm (H1) and 3.19 ppm (H2); 3.19 ppm (H2) and 3.47 ppm (H3); 3.47 ppm (H3) and 3.63 ppm (H4); and 3.63 ppm (H4) and 3.58 ppm (H5). TOCSY results indicated that 4.29, 3.19, 3.47, 3.63, and 3.58 ppm are in the same spin system. HSQC results showed coupling between 3.19 ppm and 72.4 ppm; 3.47 ppm and 76.0 ppm; 3.63 ppm and 77.0 ppm; and 3.58 ppm and 76.0 ppm. The residue corresponding to the anodic signal peak (4.29 / 102.6 ppm) is defined as residue C, with the linkage →4)-β-D-Glc. p A-(1→).

[0057] We define the residue corresponding to the anodic signal peak (5.24 / 98.6ppm) as residue D, and then... 1 H, 1 H-COSY results revealed coupling between 5.24 ppm (H1) and 3.99 ppm (H2); coupling between 3.99 ppm (H2) and 3.66 ppm (H3); and coupling between 3.66 ppm (H3) and 3.60 ppm (H4). Due to the weak signal intensity, these couplings could not be definitively identified. TOCSY results also showed relatively weak signals, making it impossible to confirm which signals resided in the same spin system. HSQC results showed coupling between 3.99 ppm and 77.0 ppm; and coupling between 3.66 ppm and 69.3 ppm. Based on the methylation result (6.50%), the linkage is presumed to be →2)-α-D-Man. p -(1→).

[0058] The HMBC results show coupling at 5.19 ppm and 77.0 ppm, and coupling at 3.63 ppm and 98.6 ppm, indicating that residues A and C are linked by a bond of →2,3)-α-D-Manp-(1→4)-β-D-GlcpA-(1→). The NOESY results show coupling at 4.29 ppm and 4.08 ppm, indicating that residue C is linked to residue A at position 2. Furthermore, a weak cross-peak (5.06 / 76.0 ppm) in the HMBC, combined with a cross-peak (5.06 / 3.81 ppm) in the NOESY, indicates that residue B is linked to residue A at position 3. The cross-peak (5.19 / 3.99 ppm) in the NOESY indicates that residue A is linked to residue D at position 2.

[0059] Combined with the methylation results, it is speculated that the sample structure of SYQ-P polysaccharide has →2)α-D-Manp-(1→ and →4)-β-D-GlcpA-(1→ as the main chain, and there is a branch at the C3 position of →2)-α-D-Manp-(1→, and the branch is mainly α-D-Gal p -(1→ as the main component.

[0060] Example 3 In vivo protective effect of SYQ-P on alcoholic gastric mucosal injury.

[0061] 1. Experimental animals. Sixty male ICR mice (20±2 g) were purchased from Hangzhou Medical College (certificate No.: 20250625Abzz01000180112; license No.: SYXK (Zhejiang) 2024-0010). The animals were acclimated and housed in the Animal Center of Zhejiang Academy of Traditional Chinese Medicine. Throughout the study, mice were kept under controlled environmental conditions with a 12-hour light / dark cycle, a temperature of 22±2°C, and a relative humidity of 40%~60%. Standard rodent chow and free access to water were provided. All animal experimental procedures were formally approved by the Ethics Committee of Zhejiang Academy of Traditional Chinese Medicine (approval No.: Pro-2025-490).

[0062] 2. Modeling and administration: For the first 14 days, the control group (n=10) was intragastrically administered with normal saline (10 mL / kg), and the remaining mice (n=50) were intragastrically administered with an equal volume of 40% ethanol. Then the mice were divided into model group, SYQ-P low-, medium- and high-dose groups (100, 200, 400 mg / kg) and omeprazole group (20 mg / kg), with 10 mice in each group, and continuous intragastric administration for 7 days. On the 8th day, the control group was given 0.5% sodium carboxymethyl cellulose (CMC-Na), and the other groups were intragastrically administered with 100% ethanol (10 mL / kg). One hour later, the mice were euthanized under deep anesthesia with isoflurane, the stomach was taken for photography, and the area of mucosal injury was measured; part of the tissue was fixed with 4% paraformaldehyde, and the rest was stored at -80°C.

[0063] 3. Biochemical analysis and test results.

[0064] (1) Gastric ulcer index: Macroscopic evaluation and quantification of gastric mucosal injury were performed by calculating the ulcer index (UI). The excised stomach of the mouse was incised along the greater curvature and gently rinsed with cold normal saline. The number of visible ulcers was counted and classified according to their size.

[0065] Wherein, UI is calculated by the following formula: UI=A+2B+3C.

[0066] Wherein A, B and C represent the number of ulcers with diameter less than or equal to 1 mm, between 1 mm and 3 mm, and greater than or equal to 3 mm, respectively.

[0067] The results are shown in Figure 11 China A Figure 11 As shown in Figure B, administration of anhydrous ethanol induced severe hemorrhagic lesions, which corresponded to a significant increase in the gastric ulcer score in the model group (P<0.01). Treatment in the SYQ-P group alleviated these morphological lesions, demonstrating a protective effect comparable to that of the positive control omeprazole (P<0.01).

[0068] (2) Histological and PAS staining analysis: Gastric tissue was fixed in 4% formalin for more than 48 hours, dehydrated, embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin (H&E) and observed under a microscope. The severity of gastric mucosal injury was semi-quantitatively assessed based on a 4-point scoring system of injury depth and area. For each section, six randomly selected fields of view (200x magnification) were evaluated and the average score was calculated according to the following criteria: (1) bleeding (blue arrow): 0-4 points; (2) mucosal edema (red triangle): 0-4 points; (3) epithelial cell loss (black arrow): 0-3 points; (4) inflammatory cell infiltration (yellow arrow): 0-3 points.

[0069] In addition, to assess the consumption and preservation of gastric mucin, adjacent sections were stained with periodic acid-Schiff (PAS). PAS-positive areas indicated the presence of glycoproteins (mucin) in the mucosal layer, which were captured under a microscope and quantitatively analyzed using ImageJ software.

[0070] The results are as follows Figure 11 C, Figure 11 China D and Figure 11 As shown in Figure E, histological evaluation by H&E and PAS staining further confirmed this cytoprotective effect. The model group exhibited typical pathological features, including mucosal hemorrhage and edema, epithelial cell loss, inflammatory cell infiltration, and depletion of the mucus layer. In contrast, SYQ-P administration effectively alleviated mucosal structural damage, as evidenced by a decrease in H&E histological scores (P<0.01), and restored the protective mucus barrier, as evidenced by the quantitative recovery of PAS-positive areas (P<0.01).

[0071] (3) Alcohol Metabolizing Enzyme Activity Assay: The activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in gastric tissue were determined using a commercial assay kit according to the manufacturer's instructions. Each group of gastric tissue samples (approximately 80 mg) was homogenized in 9 volumes of ice-cold physiological saline and centrifuged at 12,000 rpm for 10 minutes to obtain the supernatant. The total protein concentration of the supernatant was quantified at 562 nm using a quinoline carboxylic acid (BCA) protein assay kit, with bovine serum albumin (BSA) as the standard. For ADH and ALDH activity assays, the reaction mixture was incubated at 37°C, and absorbance changes were monitored spectrophotometrically at 360 nm. Enzyme activity was normalized to total protein content, expressed as units per milligram of protein (U / mg protein).

[0072] The results are as follows Figure 11 China F, Figure 11 As shown in Figure G, SYQ-P treatment significantly reversed the ethanol-induced decrease in ALDH (P<0.05) and ADH (P<0.05) levels and promoted local alcohol metabolism in the stomach.

[0073] (4) ELISA assay for inflammatory cytokine levels: Following the manufacturer's instructions, the levels of pro-inflammatory cytokines in gastric tissue supernatant, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), were quantified using a commercial ELISA kit. Gastric tissue supernatant samples and standards were incubated in antibody-coated microplates and then reacted with horseradish peroxidase (HRP)-labeled detection antibodies. After color development with substrate solution and the addition of stop solution, the optical density (OD) of each well was measured at 450 nm using a microplate reader. The concentration of each cytokine was calculated based on its respective standard calibration curve.

[0074] The results are as follows Figure 11 H, Figure 11 I and Figure 11 As shown in Figure J, all three pro-inflammatory factors in the model group were significantly elevated; SYQ-P can dose-dependently inhibit the overexpression of inflammatory factors and reduce mucosal inflammatory response.

[0075] Example 4 In vitro protective effect of SYQ-P against ethanol-induced GES-1 cell damage.

[0076] 1. Cell Culture: The human gastric epithelial cell line (GES-1) was used. GES-1 cells were cultured in standard RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S). Cell culture was incubated at 37°C in a humidified environment with 5% CO2. Cells in the logarithmic growth phase were collected for subsequent experimental processing.

[0077] 2. Experimental testing and result analysis.

[0078] (1) CCK-8 assay: The viability of GES-1 cells was assessed using the Cell Count Kit-8 (CCK-8) assay.

[0079] Modeling concentration screening: GES-1 cells were treated with 1%–9% ethanol for 4 hours, and cell viability was detected by CCK-8 assay. Results are as follows: Figure 12 As shown in Figure A, 4% (v / v) ethanol was determined to be the optimal concentration for modeling.

[0080] Cell safety evaluation: Cells were treated with 0.26–67 μg / mL SYQ-P for 24 h. Results are as follows: Figure 12 As shown in Figure B, cell viability showed no significant change (P>0.05), indicating that SYQ-P had no significant cytotoxicity within this concentration range.

[0081] Subsequently, GES-1 cells were treated with 4% ethanol and SYQ-P at concentrations ranging from 0.52 to 67 μg / mL for 4 hours. Suspended NK-92 cells were cultured in 96-well plates and exposed to gradient concentrations of SYQ-P (1.04 to 67 μg / mL) for 24 hours. After each treatment, CCK-8 reagent was added to each well, and absorbance was measured at 450 nm using a microplate reader to calculate cell viability. Results are as follows: Figure 12 As shown in Figure C, SYQ-P treatment significantly restored cell viability under ethanol challenge in a dose-dependent manner (16.75~67 μg / mL) (P>0.05, 0.01).

[0082] (2) LDH Assay: Cytotoxicity was assessed using a commercial LDH assay kit. After treatment, a control was used to induce maximum LDH release by adding a release reagent. The supernatant was collected by centrifugation and incubated with LDH working solution. The absorbance was recorded at 490 nm (reference wavelength: 600 nm) using a microplate reader. Results are as follows: Figure 12 As shown in Figure D, SYQ-P (8.37~67 μg / mL) effectively alleviated ethanol-induced cytotoxicity and cell membrane damage, as evidenced by a significant reduction in LDH release. This indicates that SYQ-P possesses significant gastric protective properties against ethanol-induced damage both in vivo and in vitro.

[0083] Example 5 SYQ-P's protective effect on the tight junction barrier of the gastric mucosa.

[0084] Stomach tissues from mice in each group in Example 3 were collected, and Western blot was used to detect the expression of Occludin and Claudin-5 proteins; immunohistochemistry was used to detect the localization of Occludin; and immunofluorescence was used to detect the distribution of ZO-1.

[0085] 1. In vivo experiments: Paraffin-embedded sections underwent antigen retrieval and non-specific blocking, followed by overnight incubation at 4°C with anti-Occludin primary antibody (1:500). Subsequently, sections were treated with horseradish peroxidase (HRP)-labeled secondary antibody. Immunoreactive sites were stained with diaminobenzidine (DAB), and cell nuclei were counterstained with hematoxylin. Stained sections were observed under a light microscope, and Occludin-positive areas were quantitatively analyzed using ImageJ software.

[0086] 2. In vitro experiments: GES-1 cells were fixed, permeabilized, and blocked with normal serum. Samples were incubated overnight at 4°C with primary antibodies against ZO-1 (1:500) and Claudin-5 (1:500), followed by incubation with the corresponding fluorophore-labeled secondary antibodies. Finally, cell nuclei were counterstained with DAPI, and sections were mounted with anti-quenching mounting medium. Immunofluorescence images were captured and analyzed using a fluorescence microscope or digital section scanning system.

[0087] 3. Test results and analysis: such as Figure 13 As shown, ethanol treatment significantly disrupted the expression of tight junction (TJ) proteins in vivo and in vitro. Western blot results showed that the expression of Occludin and Claudin-5 in the gastric tissue of the model group was significantly reduced (P<0.05), and SYQ-P intervention, especially high-dose SYQ-PH, significantly restored their expression. Immunohistochemistry (IHC) showed that SYQ-P increased the proportion of Occludin-positive cells in the gastric epithelium in a dose-dependent manner (P<0.01); immunofluorescence (IF) showed that it maintained the continuity and abundance of ZO-1 (P<0.01). In GES-1 cells, SYQ-P also restored the expression of Occludin and Claudin-5 in a dose-dependent manner and alleviated the breakage and fluorescence attenuation of the Claudin-5 and ZO-1 cell boundary networks (P<0.01). The results indicate that SYQ-P can inhibit ethanol-induced tight junction damage and maintain the integrity of the gastric mucosal barrier.

[0088] Example 6 SYQ-P inhibits GSDME-mediated pyroptosis.

[0089] (1) Western blotting: Total protein was extracted using RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and the concentration was standardized using a BCA protein assay kit. Equal volumes of protein samples were separated by SDS-PAGE and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with primary antibodies against tight junction proteins (Occludin, 1:2000; Claudin5, 1:2000 and ZO-1, 1:1000) and pyroptosis markers (GSDME, 1:2000; GSDME-N, 1:2000 and IL-1β, 1:2000; Caspase-3, 1:1000). After incubation with appropriate HRP-labeled secondary antibodies, protein bands were visualized using enhanced chemiluminescence (ECL) substrates and captured on an ImageQuant LAS 500. Protein bands were quantified using image analysis software, with β-actin as an internal control.

[0090] The results are as follows Figure 14 China A~ Figure 14 As shown in Figure K, the protein levels of upstream executor Caspase-3, full-length GSDME and its active pore-forming fragment (GSDME-N), and the key pro-inflammatory cytokine IL-1β were sharply increased in the model group (P<0.05, 0.01). Conversely, SYQ-P administration effectively attenuated this activation cascade. SYQ-P treatment not only inhibited the upregulation of Caspase-3 and total GSDME, but more importantly, significantly inhibited the cleavage of GSDME into its functional N-terminal domain (GSDME-N) in both gastric tissue and GES-1 cells (P<0.05, 0.01). To further confirm these biochemical findings at the cellular level in terms of function and morphology, extracellular IL-1β secretion and membrane permeability were assessed in vitro. Consistent with the intracellular protein trend, the excessive release of ethanol-triggered IL-1β into the cell culture supernatant was significantly reduced after SYQ-P treatment (P<0.01).

[0091] (2) Hoechst / PI dual staining assay: To visualize pyroptosis characterized by membrane permeability, GES-1 cells were subjected to dual fluorescence assays using Hoechst 33342 and propidium iodide (PI). After the specific modeling and SYQ-P intervention described in Example 3, cells were co-stained with Hoechst 33342 (labeling all cell nuclei) and PI (selectively recognizing cells with membrane rupture) using a commercial kit. Fluorescence signals were immediately captured using a fluorescence microscope, and the percentage of PI-positive cells was calculated to determine the incidence of pyroptosis.

[0092] The results are as follows Figure 14As shown in Figure L, the model group exhibited a high proportion of PI-positive (red) cells, indicating that cell membrane damage is a characteristic of pyroptosis. SYQ-P intervention significantly reduced the number of PI-positive cells, confirming its efficacy in preventing pyroptotic cell death.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A polysaccharide from the rootlets of *Ilex trifoliata*, characterized in that, The polysaccharide from the roots of *Trifolium repens* is an acidic heteropolysaccharide; the monosaccharide composition of the polysaccharide from the roots of *Trifolium repens* includes mannose, glucuronic acid, galactose, arabinose, and rhamnose.

2. The polysaccharide from the rootlets of *Trifolium repens* according to claim 1, characterized in that, In molar percentage, the proportions of each monosaccharide in the monosaccharide composition are as follows: galactose 43.42%, glucuronic acid 16.61%, mannose 29.83%, arabinose 8.05%, glucose 1.20%, and rhamnose 0.89%.

3. A method for preparing the polysaccharide from the rootlets of *Trifolium repens* according to any one of claims 1-2, characterized in that, Includes the following steps: (1) After washing, drying and cutting the roots of *Trifolium repens*, extract them with distilled water, combine the extracts and concentrate them, add anhydrous ethanol, let them precipitate overnight, and collect the precipitate. (2) The precipitate obtained in step (1) is redissolved in water, and then subjected to deproteinization treatment and decolorization treatment to obtain crude polysaccharide; (3) The crude polysaccharide was freeze-dried and dissolved in distilled water, then loaded onto a DEAE-dextran gel FF anion exchange column for gradient elution. The target component was collected, dialyzed with distilled water, and then freeze-dried. (4) The target component obtained in step (3) is loaded onto a Smartdex G-100 gel filtration chromatography column for elution. The eluent is collected, dialyzed, and freeze-dried to obtain polysaccharide from the roots of *Trifolium repens*.

4. The method according to claim 3, characterized in that, In step (1), the temperature of the distilled water extraction is 100℃, the number of distilled water extractions is 3, the extraction time for each extraction is 1.5h, and the material-to-liquid ratio is 1:10 (w / v); the concentration is made to a density of 0.05g / mL; and the volume ratio of anhydrous ethanol to the extract is 1:

1.

5. The method according to claim 3, characterized in that, In step (2), the deproteinization treatment is performed by the Sevag method to remove protein; the decolorization treatment is performed by AB-8 macroporous resin.

6. The method according to claim 3, characterized in that, In step (3), the gradient elution specifically involves eluting with distilled water, 0.4 mol / L NaCl solution, 0.8 mol / L NaCl solution, and 1.0 mol / L NaCl solution in sequence at a flow rate of 2.0 mL / min; the target component is 0.4 mol / L NaCl eluent.

7. The method according to claim 3, characterized in that, In step (4), the eluent used for elution is distilled water at a flow rate of 2.0 mL / min.

8. The use of the polysaccharide from the rootlets of *Trifolium repens* as described in any one of claims 1 to 2 in the preparation of products for the prevention and / or treatment of gastric mucosal damage.

9. The application according to claim 8, characterized in that, The gastric mucosal injury mentioned is ethanol-induced alcoholic gastric mucosal injury.

10. The application according to claim 8, characterized in that, The product works through at least one of the following pathways: reducing gastric mucosal bleeding and epithelial defects, lowering the gastric ulcer index, improving gastric tissue pathological damage, promoting gastric mucus secretion, increasing the activity of gastric tissue alcohol metabolism enzymes, inhibiting the release of gastric tissue pro-inflammatory factors, maintaining the expression of gastric mucosal tight junction proteins, and inhibiting gastric epithelial cell pyroptosis.

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