Radix tetrastigme polysaccharide, preparation method thereof and application of radix tetrastigme polysaccharide in preparation of antithrombotic drugs

Abstract

The invention relates to the field of traditional Chinese medicines, and discloses radix tetrastigme polysaccharide, a preparation method thereof and application of the radix tetrastigme polysaccharide in preparation of antithrombotic drugs. The novel acid glucuronic acid galactomannan (THP5) is extracted from the overground part of the radix tetrastigme polysaccharide for the first time, it is found that the novel acid glucuronic acid galactomannan (THP5) can be used for preventing and treating thrombus diseases including arterial thrombosis, venous thrombosis and the like in a multi-target mode, the application range of the radix tetrastigme polysaccharide is widened, and a new way is provided for treating the thrombus diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine, and particularly relates to a Tripterospermum japonicum Hance polysaccharide, a preparation method thereof and application thereof in preparation of an anti-thrombus drug. BACKGROUND

[0002] Thrombosis is the formation of pathological clots in blood vessels under abnormal hemodynamic or inflammatory conditions and is the main cause of cardiovascular events, including myocardial infarction and ischemic stroke. Thrombosis is composed of fibrin, platelets, leukocytes, red blood cells and neutrophil extracellular traps (NETs). Although thrombosis is a physiological process that is strictly regulated in vascular injury or inflammatory response, its disorder can lead to life-threatening complications. In addition to cardiovascular diseases, thrombosis is increasingly recognized in autoimmune diseases, cancer and infectious diseases.

[0003] Tripterospermum japonicum Hance polysaccharide (THP) is the main physiological active component of Tripterospermum japonicum Hance and can significantly inhibit the overexpression of IFN-γ, TNF-α and IL-6, thereby playing an antipyretic and anti-inflammatory role. Tripterospermum japonicum Hance polysaccharide is a mixture of polysaccharides, and different types of polysaccharides have different effects. For example, the applicant isolated a pectin polysaccharide STHP4 highly rich in homogalacturonan (HG) from the aboveground part of Tripterospermum japonicum Hance in patent application CN202510297516.5, which has good immune activation effect and is safe and non-toxic, and can be used for product development of immune activators. THP5 is a new type of acidic glucuronoglycomannan separated from THP by the applicant, has a molecular weight of 3.225 x 10 5 kDa and is mainly composed of galactose, mannose and glucuronic acid. Our previous studies have confirmed that THP5 can inhibit the development of severe pneumonia through the TLR4 / NF-κB signaling pathway. However, there is no report on the related research of THP5 on thrombotic diseases, and the specific mechanism and target points thereof need to be further verified. SUMMARY

[0004] The present application provides a Tripterospermum japonicum Hance polysaccharide, a preparation method thereof and application thereof in preparation of an anti-thrombus drug. The present application first extracts a new type of acidic glucuronoglycomannan (THP5) from the aboveground part of Tripterospermum japonicum Hance polysaccharide and finds that it can prevent and treat thrombotic diseases through multiple targets, thereby not only widening the application range of Tripterospermum japonicum Hance polysaccharide but also providing a new way for treating thrombotic diseases.

[0005] The specific technical scheme of the present application is as follows: In a first aspect, the present application provides a Radix Tetrastigae polysaccharide, named THP5, which comprises 12 partially methylated sugar alcohol acetyl ester derivatives: T-Araf, 1,2-Rhap, T-GlcpA, T-Galp, 1,2-Manp, 1,4-GlcpA, 1,4-Galp, 1,2,3-Manp, 1,2,3,4-Manp, 1,4,6-Glcp, 1,4,6-Galp, 1,3,6-Galp; and monosaccharide composition comprises Rha, Ara, Gal, Glc, Man and GlcA.

[0006] The present application first extracts and separates the Radix Tetrastigae polysaccharide THP5 highly rich in glucuronic acid, galactose and mannose from the aerial part of Radix Tetrastigae. The present application further finds that the Radix Tetrastigae polysaccharide has the efficacy of treating thrombosis diseases including arterial thrombosis and venous thrombosis, and can broaden the application field of the Radix Tetrastigae polysaccharide.

[0007] Further, the Radix Tetrastigae polysaccharide has a molecular weight of 3.225*10 5 kDa; and the molar ratio of Rha, Ara, Gal, Glc, Man and GlcA is 2.95:11.89:35.77:1.53:20.90:26.95.

[0008] In a second aspect, the present application provides a preparation method of the Radix Tetrastigae polysaccharide, which comprises the following steps: 1) extracting the Radix Tetrastigae crude polysaccharide from the aerial part of Radix Tetrastigae; 2) dialyzing the Radix Tetrastigae crude polysaccharide, gradient eluting through a cellulose column to obtain an eluate; 3) water eluting through a gel column to obtain the Radix Tetrastigae polysaccharide.

[0009] Through the above preparation method, the Radix Tetrastigae polysaccharide THP5 highly rich in glucuronic acid, galactose and mannose can be extracted and separated from the aerial part of Radix Tetrastigae.

[0010] As a preferred, the preparation method specifically comprises: extracting the dried and crushed aerial part of Radix Tetrastigae with ethanol aqueous solution A, and filtering; drying the residue, extracting with water, centrifuging, taking the supernatant, filtering, and concentrating to a viscous state; adding ethanol aqueous solution B, stirring and extracting; sealing and standing, centrifuging, redissolving the precipitate with water, evaporating ethanol, and placing the concentrated solution in an environment of-90 to-70℃ for more than 12 hours, and freeze-drying to obtain the Radix Tetrastigae crude polysaccharide. Further dialyzing, using DEAE-52 cellulose as the filler, gradient eluting with different concentrations of NaCl solution, and obtaining the preliminary Radix Tetrastigae purified polysaccharide. Freeze-drying the purified polysaccharide, and eluting through a gel column Sephadex 200 to obtain the Radix Tetrastigae polysaccharide.

[0011] In a third aspect, the present application provides the application of the Radix Tetrastigae polysaccharide THP5 in preparing an anti-thrombosis drug.

[0012] Preferably, the thrombus is a thrombotic disease associated with the αⅡbβ3-MAPK signaling pathway. More preferably, the MAPK signaling pathway involves one or more of ERK, JNK, and p38.

[0013] Furthermore, the thrombotic diseases are arterial thrombosis and venous thrombosis. Even further, the arterial thrombosis includes myocardial infarction, cerebral infarction, and peripheral artery thrombosis; the venous thrombosis includes deep vein thrombosis (DVT), septic thrombosis, and tumor thrombosis.

[0014] This invention, through in vivo experiments, revealed that *Trifolium repens* polysaccharide THP5 can inhibit carrageenan-induced thrombus formation, suppressing thrombus formation in the tail, liver, and lung tissues of mice. Further analysis showed that THP5 exerts a therapeutic effect on thrombosis by downregulating the levels of leukocytes, neutrophils, and monocytes in thrombotic mice, inhibiting the increase of IL-6, TNF-α, and other inflammatory cytokines, as well as NO levels, prolonging the acute platelet-to-thrombotic time (APTT) in mice, and simultaneously regulating the MAPK pathway to reduce platelet activation. This indicates that THP5 can alleviate the formation of venous thrombosis.

[0015] This invention, through in vivo experiments, found that *Trifolium repens* polysaccharide THP5 can alleviate FeCl3-induced carotid artery thrombosis, prolong blood flow occlusion time, shorten thrombus length, and reduce thrombus weight. This indicates that THP5 can inhibit arterial thrombus formation.

[0016] This invention, through in vitro experiments, revealed that the polysaccharide THP5 from *Trifolium repens* can inhibit platelet aggregation induced by thrombin, collagen, and arachidonic acid, while also inhibiting the number and spreading area of ​​platelets adhering to fibrinogen. Furthermore, THP5 can inhibit the increase in thrombin-induced thrombus contraction speed, platelet ATP release level, and ROS level, while reducing thrombin-induced platelet CD62P and JON / A expression levels. Further analysis showed that THP5 has a strong affinity for platelet surface αⅡbβ3 and can reduce thrombin-induced phosphorylation of PKCα, ERK, JNK, and p38. This suggests that THP5 may inhibit platelet activation by targeting and binding to platelet surface αⅡbβ3 to regulate the MAPK pathway.

[0017] Fourthly, this invention provides the application of *Trifolium repens* polysaccharide THP5 in the preparation of MAPK pathway inhibitors.

[0018] Preferably, the MAPK pathway involves one or more of ERK, JNK, and P38. More preferably, the MAPK pathway is the platelet MAPK pathway.

[0019] Fifthly, the present invention provides a method for inhibiting the activation of the MAPK pathway in in vitro cells using the polysaccharide THP5 from *Trifolium repens*.

[0020] Preferably, the in vitro cells are in vitro platelets.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention is the first to extract a novel acidic glucuronic acid galactomannan (THP5) from the aerial parts of *Trifolium repens* polysaccharide, and further discovers that *Trifolium repens* polysaccharide THP5 can target the αIIbβ3 and MAPK pathways to inhibit platelet activation, thereby having a therapeutic effect on thrombosis-related diseases. This not only broadens the application field of *Trifolium repens* polysaccharide, but also provides new ideas and approaches for the treatment of thrombosis diseases. Attached Figure Description

[0022] Figure 1 This study focuses on the isolation, purification, monosaccharide composition, molecular weight analysis, and antithrombotic mechanism of THP5. a) Elution curve of THP5 eluted with 0.4 mol / L NaCl on a Superdex 200 gel chromatography column (1.5 × 100 cm); b) Light scattering (LS) and refractive index (RI) signals of THP5; c) Weight-average molecular weight (Mw) of THP5; d) Determination by Congo red assay; e) Relationship between root mean square radius and molecular weight on a logarithmic scale and the fitted line; f) Schematic diagram of the antithrombotic mechanism of THP5 polysaccharide.

[0023] Figure 2 This is a monosaccharide composition analysis of THP5. a) THP5; b) Standard.

[0024] Figure 3 These are the NMR spectra of THP5. a) 1H; b) 13C.

[0025] Figure 4 This is the NMR spectrum of THP5. c) HSQC.

[0026] Figure 5 These are the NMR spectra of THP5. d) COSY; e) TOCSY.

[0027] Figure 6 This is the NMR spectrum of THP5. f) NOESY; g) HMBC.

[0028] Figure 7 This is a hypothetical chemical structure diagram of THP5.

[0029] Figure 8This study investigated the therapeutic effect of *Trifolium repens* polysaccharide THP5 on κ-carrageenan-induced thrombosis in mice. (A) Images and black tail rate of thrombosis 48 hours after intraperitoneal injection of κ-carrageenan (100 mg / kg). Black tail rate (%) = (black tail length / total tail length) × 100%. (B) Laser speckle imaging of tail blood perfusion at 0-3 cm from the tip and tail blood perfusion volume. (C) Spleen index = spleen weight / body weight × 100%. (D) Plasma APTT level. (E) Peripheral blood cell count. (F) Serum IL-6, TNF-α, and NO levels. #### P<0.0001 ### P<0.001 ## P<0.01 # P<0.05 vs. controlgroup; **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05vs.Modelgroup; ns: P>0.05.

[0030] Figure 9 The polysaccharide THP5 from *Trifolium repens* inhibits κ-carrageenan-induced platelet thrombosis in the tail and liver / lung tissues. (A) HE staining of the tail and liver / lung tissues. (B) CD42b expression levels in the tail and liver / lung tissues. #### P<0.0001vs. control group; **** P<0.0001 vs. Model group.

[0031] Figure 10 This study investigated the inhibitory effect of *Trifolium repens* polysaccharide THP5 on platelet activation in vitro. (A) Platelet aggregation induced by thrombin, AA, or collagen after pre-incubation with THP5 or aspirin. (B) Platelet adhesion levels on fibrinogen. The study also examined the number of adherent platelets and the spreading area (μm). 2 (C) Quantification of platelet count. (D) Changes in thrombin-induced thrombus contraction within 15 min at 37°C. (E) Quantification of changes in thrombus contraction area. (F) ATP release level of platelets induced by thrombin after pre-incubation with THP5 or aspirin. (G) CD62P level of platelets induced by thrombin after pre-incubation with THP5 or aspirin. (H) JON / A level of platelets induced by thrombin after pre-incubation with THP5 or aspirin. #### P<0.0001 ### P<0.001 ##P<0.01 # P<0.05 vs. control group; **** P<0.0001 *** P<0.001 ** P<0.01 * P<0.05 vs. Model group.

[0032] Figure 11 This study explores potential targets for the interaction between Bio-THP5 polysaccharide and platelets. (A) Silver-stained bands in the Bio-THP5-platelet protein interaction pull-down experiment. (B) MS / MS spectrum of integrin β3 (m / z 1027.44518, z=2). (C) Western blot verification of integrin β3 in the THP5-human and mouse platelet protein pull-down experiment. (DE) Mass spectrometry enrichment analysis of GO and KEGG pathways identified by silver-stained bands in the pull-down experiment. (F) SPR sensing map of dose- and time-dependent binding of THP5 to GPIIb / IIIa. (G) Competitive inhibition of ADP-induced platelet aggregation by THP5 in the presence of tirofiban or abciximab.

[0033] Figure 12 This study assesses the in vivo antithrombotic efficacy and bleeding risk of *Trifolium repens* polysaccharide THP5 compared to tirofiban. (A) Images of black tail formation and black tail rate 48 h after intraperitoneal injection of κ-carrageenan (100 mg / kg). Black tail rate (%) = (black tail length / total tail length) × 100%. (B) Laser speckle imaging of tail blood perfusion at 0-3 cm from the tip and tail blood flow perfusion volume. (C) Spleen index = spleen weight / body weight × 100%. (D) Plasma fibrinogen (FIB) level. (E) Peripheral blood cell count. (F) Laser speckle imaging of carotid artery blood flow in FeCl3-induced carotid artery thrombosis mice. (G) Time to complete blood flow occlusion, thrombus length, and thrombus weight. (H) Organ indices of heart, liver, spleen, lung, and kidney in mice after administration of aspirin, tirofiban, or THP5. (I) Tail bleeding time after administration of aspirin, tirofiban, or THP5. (J) Platelet count and hematocrit. #### P<0.0001 ### P<0.001 ## P<0.01 # P<0.05 vs. control group; **** P<0.0001 *** P<0.001 **P<0.01, *P<0.05 vs. Model group; ns, P>0.05.

[0034] Figure 13 This is the platelet proteomics result of *Trifolium repens* polysaccharide THP5 treatment in κ-carrageenan-induced thrombotic mice. (A) Principal component analysis (PCA) illustrates the different platelet proteomics characteristics between the MOD and THP5 treatment groups. (B) Protein correlation heatmap. (C) KEGG pathway enrichment analysis of differentially expressed proteins. (D) Top 20 differentially expressed proteins.

[0035] Figure 14 This study investigated the regulatory role of THP5-mediated MAPK signaling in thrombosis and platelet activation. (A) Western blot analysis of phosphorylated and total PKCα, P38, ERK, and JNK proteins in platelets from κ-carrageenan-induced thrombotic mice. (B) Western blot analysis of phosphorylated and total PKCα, P38, ERK, and JNK proteins in thrombin-stimulated platelets pretreated with THP5 or tirofiban in vitro. #### P<0.0001 ### P<0.001 ## P<0.01 # P<0.05 vs. control group; **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05 vs. Model group. Detailed Implementation

[0036] Firstly, a *Trifolium repens* polysaccharide, named THP5, comprises 12 partially methylated sugar alcohol acetyl ester derivatives: T-Araf, 1,2-Rhap, T-GlcpA, T-Galp, 1,2-Manp, 1,4-GlcpA, 1,4-Galp, 1,2,3-Manp, 1,2,3,4-Manp, 1,4,6-Glcp, 1,4,6-Galp, and 1,3,6-Galp; the monosaccharide composition includes Rha, Ara, Gal, Glc, Man, and GlcA. Further, the molecular weight of the *Trifolium repens* polysaccharide is 3.225 × 10⁻⁶. 5 kDa; the molar ratio of Rha, Ara, Gal, Glc, Man and GlcA is 2.95:11.89:35.77:1.53:20.90:26.95.

[0037] Secondly, a method for preparing *Trifolium repens* polysaccharide includes the following steps: 1) Extraction of crude polysaccharides from the aerial parts of *Tripterygium wilfordii*. Preferably, step 1) specifically includes: extracting the dried and pulverized aerial parts of *Tripterygium wilfordii* with an ethanol-water solution A, followed by filtration; drying the filter residue and extracting with water, centrifuging, filtration of the supernatant, and concentration to a viscous state; adding an ethanol-water solution B and stirring for extraction; sealing and allowing to stand, centrifuging, redissolving the precipitate in water, evaporating the ethanol, placing the concentrated solution in an environment of -90 to -70°C for at least 12 hours, and freeze-drying to obtain crude polysaccharides from *Tripterygium wilfordii*. More preferably, in step 1), the aerial parts of *Tripterygium wilfordii* are leaves and / or stems. The concentration of ethanol in the ethanol-water solution A is 90-98 vol%, and the temperature is 75-85°C; the solid-liquid ratio of the extraction is 1:10-20. The temperature of the water extraction is 90-98°C. The centrifugation speed is 2500-3500 g, and the time is 5-15 min. The temperature of the first concentration is 55-65°C; the temperature of the second concentration is 55-65°C. The ethanol concentration in the aqueous ethanol solution B is 90-98 vol%, and the solid-liquid ratio during extraction is ≤1:3. The temperature for sealing and settling is 1-6℃, and the time is 8-12 hours. The temperature for evaporating the ethanol is 55-65℃.

[0038] 2) The crude polysaccharide from *Trifolium repens* was dialyzed and eluted using a cellulose column gradient to obtain the eluent. Further preferably, in step 2), the dialysis bag used for dialysis has a molecular weight cutoff of 3.0 kDa, and the dialysis time is 72 h. The packing material for the cellulose column is DEAE-52 cellulose. Gradient elution was performed sequentially with water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions at a flow rate of 2.2 rpm (approximately 2 mL / min), and the final eluent was collected.

[0039] 3) Elute with water using a gel column to obtain *Trifolium repens* polysaccharide THP5. Further preferred embodiment, step 3) specifically includes: concentrating the eluent at 55-65℃, dialysis for desalting (pore size cutoff 3.0 kDa), and freeze-drying to obtain preliminarily purified *Trifolium repens* polysaccharide; preparing a 20 mg / mL aqueous solution of *Trifolium repens* polysaccharide, loading it onto a Sephadex 200 gel column at a flow rate of 2.2 rpm (approximately 2 mL / min), with each loading volume being 5-10% of the column bed volume; eluting with water at a flow rate of 2.2 rpm (approximately 2 mL / min), and freeze-drying the eluent to obtain purified *Trifolium repens* polysaccharide.

[0040] Thirdly, the application of *Trifolium repens* polysaccharide THP5 in the preparation of antithrombotic drugs. Preferably, the thrombosis is a thrombotic disease related to the αⅡbβ3-MAPK signaling pathway. More preferably, the MAPK signaling pathway involves one or more of ERK, JNK, and p38. More preferably, the thrombotic disease is arterial thrombosis or venous thrombosis. Further, the arterial thrombosis is myocardial infarction, cerebral infarction, or peripheral arterial thrombosis; the venous thrombosis is deep vein thrombosis (DVT), septic thrombosis, or tumor thrombosis. Preferably, the drug comprises: *Trifolium repens* polysaccharide THP5 as the main active ingredient, and pharmaceutically acceptable excipients.

[0041] Fourthly, the application of *Trifolium repens* polysaccharide THP5 in the preparation of MAPK pathway inhibitors. Preferably, the MAPK pathway involves one or more of ERK, JNK, and p38. More preferably, the MAPK pathway is the platelet MAPK pathway.

[0042] Fifthly, a method for inhibiting MAPK pathway activation in in vitro cells using *Trifolium repens* polysaccharide THP5. Preferably, the in vitro cells are in vitro platelets.

[0043] Example 1: Preparation of refined polysaccharide THP5 from the aerial parts of *Trifolium repens* 1.1 Experimental Reagents and Instruments: Chemical reagents: 95% ethanol, concentrated sulfuric acid (98%), phenol, carbazole, trifluoroacetic acid, NaBD4, DMSO, acetic acid, dichloromethane, iodomethane, Congo red, acetic anhydride, methanol, hydrochloric acid, chloroform, sodium hydroxide, tetramethylsilane (TMS), deuterated water (D2O), etc. DEAE-52 cellulose, Superdex 200 glucose-agar gel. Reagent kit: Brandford protein assay kit (Beyotime P0006). Experimental Apparatus: Constant flow peristaltic pump, 1.5×30cm glass chromatography column, 2.6×50cm glass chromatography column, HC-0210-10 medium-pressure chromatography column, BS-100N automatic fraction collector, rotary evaporator, LDZ5-2 low-speed balancing centrifuge, SHZ-D(III) circulating water vacuum pump, Freezone 4.5L lyophilizer, 3kDa dialysis bag, high-performance liquid chromatography system (equipped with OHpak SB-805HQ, SB-804HQ, SB-803HQ gel columns), Waters 2410 differential refractive index detector, Ultimate 3000 high-performance liquid chromatography system, multi-angle laser scattering detector, ICS 5000+ high-performance liquid chromatography system (equipped with Dionex). TM CarboPac TMPA-20 anion exchange column), Agilent 7890A-5977B gas chromatography system (equipped with an Agilent BPX70 column), and various pipettes. Laboratory equipment: water bath, round-bottom flask, Buchner funnel, filter paper, glass rod, and beakers.

[0044] 1.2 Experimental Methods 1.2.1 Extraction of crude polysaccharides from *Trifolium repens*: Dried aerial parts of *Trifolium repens*, including leaves and stems, were pulverized and weighed (100g). Extraction was performed twice with 80% (v / v) ethanol aqueous solution (80℃), followed by filtration. The residue was dried in a ventilated place at room temperature, then extracted twice with 95℃ pure water. The residue was centrifuged at 4000 rpm for 5 min, and the supernatant was collected and filtered. The mixture was concentrated to a viscous consistency using a rotary evaporator at 60℃. At least three times the volume of 95% ethanol was slowly added, and the mixture was rapidly stirred using a magnetic stirrer (slow stirring followed by fast stirring). The mixture was sealed and incubated overnight at 4℃. After centrifugation at 4000 rpm for 5 min, the precipitate was collected and reconstituted with pure water. The ethanol was removed by rotary evaporation at 40℃. The concentrated solution was incubated at -80℃ for at least 12 h, and then freeze-dried to obtain crude polysaccharides from *Trifolium repens*.

[0045] 1.2.2 Isolation and purification of crude polysaccharides from *Trifolium repens* 1.2.2.1 Dialysis: Dissolve the crude polysaccharide in pure water and dialyze with pure water for 72 hours (with a pore size of 3 kDa). Remove the liquid from the dialysis bag and store it in a refrigerator at 4°C.

[0046] 1.2.2.2 Anion Exchange Column Purification: DEAE-52 cellulose was used as the packing material for the anion exchange column. A constant flow pump was used to determine the flow rate. Four column volumes of deionized water were passed through the column to reach equilibrium (linear flow rate 1.5 cm / min). An appropriate amount of deionized water containing crude *Trifolium repens* polysaccharide was loaded (linear flow rate 0.25 cm / min), and eluted with a gradient of deionized water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions (linear flow rate 1 cm / min). The eluent was collected in test tubes using an automatic fraction collector. The eluent was analyzed using the sulfuric acid-phenol method. The 0.4 mol / L NaCl eluent was concentrated under reduced pressure by rotary evaporation (60℃), dialyzed for desalting (3 kDa pore cutoff), and freeze-dried to obtain preliminarily purified *Trifolium repens* polysaccharide THP5.

[0047] 1.2.2.3 Gel Chromatography Column Purification: Superdex 200 was used as the gel chromatography column packing material. A constant flow pump was used to determine the flow rate. 3–5 column volumes of deionized water were passed through the column to reach equilibrium (approximately 2 mL / min). A deionized aqueous solution of the pre-purified polysaccharide THP5 (concentration 20 mg / mL, approximately 2 mL / min) was loaded as a sample. To achieve good separation, the sample volume loaded each time was 5–10% of the column bed volume. Eluting was performed with deionized water (approximately 2 mL / min), and the fractions were collected in test tubes using an automatic fraction collector. The eluent was analyzed using the sulfuric acid-phenol method. The eluent was freeze-dried to obtain the purified polysaccharide THP5 from *Trifolium repens*.

[0048] 1.2.3 Molecular Weight Determination and Triple Helix Interpretation of *Trifolium repens* Polysaccharide THP5 THP5 was dissolved in 0.1 mol / L NaNO3 (containing 0.02% NaN3, w / w), filtered through a 0.45 μm syringe-type microporous membrane filter, and then transferred to a chromatographic injection vial. The sample was analyzed using a U3000 high-performance liquid chromatograph equipped with three tandem columns: Ohpak SB-805HQ (300×8 mm), SB-804HQ (300×8 mm), and SB-803HQ (300×8 mm). The eluent was 0.1 mol / L NaNO3 (containing 0.02% NaN3, w / w). Instrument parameters were set as follows: injection volume 100 μL, column temperature 45℃, flow rate 0.4 mL / min. Response signals were recorded using a differential refractive index detector and a multi-angle laser scattering detector.

[0049] A 0.5 mg / mL THP5 aqueous solution was completely mixed with Congo red solution (final concentration 20 μM), and different concentrations of NaOH solution (final concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mol / L) were added at room temperature. Pure water was used as a blank control. The spectrum of the solution was scanned in the range of 450-550 nm using an ultra-micro UV-Vis spectrophotometer, and the trend of the wavelength of the maximum absorption peak as a function of NaOH concentration was calculated.

[0050] 1.2.4 Monosaccharide Composition Determination of *Trifolium repens* Polysaccharide THP5: The monosaccharide composition was analyzed using the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization method. 5 mg of *Trifolium repens* polysaccharide was hydrolyzed in 1 mL of 2 mol / L trifluoroacetic acid (TFA) at 121 °C for 2 h. The solution was then dried under nitrogen and washed with methanol to remove residual TFA. This process was repeated three times. 0.2 mL of monosaccharide standard or sample solution was added to 0.5 mL of 0.5 mol / L PMP methanol solution and 0.2 mL of 0.5 mol / L sodium hydroxide solution. The mixture was incubated at 70 °C for 1 h. Finally, 0.2 mL of 0.5 mol / L hydrochloric acid was added to neutralize the sodium hydroxide, followed by 1 mL of chloroform to remove excess PMP. The aqueous phase was carefully separated, and water was added to bring the volume to 1 mL. The organic phase was discarded. The sample was filtered through a 0.22 μm filter and transferred to a chromatographic vial. Samples and standards were analyzed using high-performance anion exchange chromatography (HPAEC) equipped with an anion exchange column (3 × 150 mm, 10 μm). Instrument parameters were set as follows: injection volume 5 μL, elution flow rate 0.5 mL / min. Mobile phase settings: mobile phase A: ddH₂O, mobile phase B: 0.1 mol / L NaOH, mobile phase C: 0.1 mol / L NaOH and 0.2 mol / L NaAc. Elution program: volume ratio of mobile phases A, B, and C: 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. Response signals were recorded using a pulsed amperometric electrochemical detector (PAD). Data were processed using Chromeleon 7.2 CDS software. The retention time of the sample is compared with that of the standard to determine the monosaccharide composition of the sample. A linear regression equation is plotted with the concentration of the monosaccharide standard on the x-axis and the peak area on the y-axis to form a quantitative calibration curve (standard curve). The molar ratio of the monosaccharide composition of the sample is calculated based on the standard curve.

[0051] 1.2.5 Methylation analysis of *Trifolium repens* polysaccharide THP5: 10 mg of sample was dissolved in 1 mL of distilled water, and 1 mL of carbodiimide solution (100 mg / mL) was added. The reaction was allowed to proceed for 2 h, followed by the addition of 1 mL of imidazole solution (2 mol / L) and 1 mL of NaBD4 solution (30 mg / mL), and the reaction was continued for another 3 h. After the reaction was terminated, 100 μL of glacial acetic acid was added, followed by dialysis and freeze-drying for 48 h. Next, the sample was dissolved in 500 μL of dimethyl sulfoxide (DMSO), and 1 mg of sodium hydroxide was added for methylation reaction for 30 min. Then, 50 μL of iodomethane (CH3I) was added and the reaction was allowed to proceed for 1 h. Subsequently, 1 mL of water and 2 mL of dichloromethane were added, mixed thoroughly, centrifuged, and the aqueous phase was discarded. The mixture was washed with water three times. The dichloromethane phase was collected and evaporated to dryness. 100 μL of trifluoroacetic acid (TFA, 2 mol / L) was added and the reaction was carried out at 121 °C for 90 min, followed by evaporation to dryness. Add 50 μL of ammonia (2 mol / L) and 50 μL of NaBD4 solution (1 mol / L), mix, and react at room temperature for 2.5 h. After the reaction is terminated, add 20 μL of acetic acid and dry under nitrogen. Wash with 250 μL of methanol and dry again under nitrogen. Add 250 μL of acetic anhydride, mix, and react at 100 °C for 2.5 h. Add 1 mL of water and let stand for 10 min. Finally, add 500 μL of dichloromethane, mix, centrifuge, discard the aqueous phase, and repeat the washing with water 3 times. Take the lower dichloromethane phase and detect it using gas chromatography-mass spectrometry (GC-MS, 7890A-5977B) with an HP-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm). The temperature program was as follows: the initial column temperature was 50℃ and held for 1 min, then increased from 50℃ to 130℃ (heating rate of 3℃ / min), and finally held at 230℃ for 2 min. Mass spectrometry parameters: full scan mode, scan mass range of 30-600 m / z.

[0052] 1.2.6 Nuclear Magnetic Resonance Spectroscopy Analysis of *Trifolium repens* Polysaccharide THP5: Samples (20-30 mg) were dissolved in 0.5 mL of D₂O. To accelerate dissolution, a 60°C water bath was used, with occasional vortexing, until complete dissolution. After the solution returned to room temperature, it was injected into a 5 mm dedicated sample testing glass tube. One-dimensional nuclear magnetic resonance spectroscopy was used to record the results. 1 H-NMR, 13 C-NMR and two-dimensional NMR spectra (COSY, HSQC, HMBC, NOESY, and TOCSY). Measurements were performed at 600 MHz and 22 °C. Data processing and analysis were performed using MestReNova 12.0 software (Mestrelab Research SL).

[0053] 1.3 Experimental Results 1.3.1 Yield of crude polysaccharide from *Trifolium repens*: The polysaccharide was extracted and found to be pale yellow in appearance, with a yield of 13% (crude polysaccharide / powder of *Trifolium repens*, dry weight ratio).

[0054] 1.3.2 Preparation of *Trifolium repens* purified polysaccharide THP5: The yield of *Trifolium repens* polysaccharide THP after preliminary purification by anion exchange chromatography was 20.31% (preliminary purified *Trifolium repens* polysaccharide / crude *Trifolium repens* polysaccharide, dry weight ratio), and its appearance was pale yellow flocculent. The yield of *Trifolium repens* polysaccharide THP5 after gel column purification was 64.70%, and its appearance was white flocculent.

[0055] 1.3.3 Molecular weight of THP5: The average molecular weight of THP5 was measured to be 3.225 × 10⁻⁶. 5 The kDa THP-5 does not have a triple-helix structure; it has a random coil conformation and may have a compact coiled structure in some areas. Figure 1 ).

[0056] 1.3.4 Monosaccharide Composition of THP5: The monosaccharide composition of THP5 consists of Rha, Ara, Gal, Glc, Man, and GlcA. The molar ratio is 2.95:11.89:35.77:1.53:20.90:26.95. See details ( Figure 2 ).

[0057] 1.3.5 Chemical composition identification of THP5: According to the standard curves of phenol-sulfuric acid method, sulfuric acid-carbazole method and Coomassie brilliant blue method, the sugar content of THP5 is 83.5%, the uronic acid content is 24.73%, and the protein content is <1%, indicating that THP5 is an acidic polysaccharide with almost no protein impurities (Table 1).

[0058] 1.3.6 Methylation analysis of THP5: THP5 has 12 partially methylated sugar alcohol acetyl ester derivatives (PMAAs): T-Araf, 1,2-Rhap, T-GlcpA, T-Galp, 1,2-Manp, 1,4-GlcpA, 1,4-Galp, 1,2,3-Manp, 1,2,3,4-Manp, 1,4,6-Glcp, 1,4,6-Galp, 1,3,6-Galp (Table 2).

[0059] 1.3.7 Nuclear Magnetic Resonance Analysis of THP5: For details of the nuclear magnetic resonance analysis of THP5, please refer to... Figures 3-6 See Table 3. For details of the inferred possible structure of THP5, please refer to [Table 3]. Figure 7 .

[0060] Table 1: Standard Curve for Calculating the Chemical Composition Content of THP5 Method Standard curve R2 Phenol-sulfuric acid method y = 4.2091x + 0.102 0.9910 Sulfuric acid-carbazole method y = 3.3874x + 0.1561 0.9913 Coomassie brilliant blue method y = 0.6423x + 0.680 0.9900 Table 2: Methylation analysis of THP5 Note: T stands for Terminal, indicating that a sugar residue is attached to the end of the sugar chain. p represents the pyran conformation of the sugar ring, and f represents the furan conformation. The A in GlcpA stands for Acid, referring to a uronic acid.

[0061] Table 3: NMR chemical shifts of THP5 in D2O (unit: ppm) Note: Me is an abbreviation for Methyl, which stands for methyl (-CH3).

[0062] Example 2: Pharmacodynamic test of THP5 polysaccharide in inhibiting κ-carrageenan-induced thrombosis 2.1 Experimental materials 2.1.1 Experimental Animals and Drugs: Thirty male BALB / c mice, weighing 20±2g, were purchased from the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. The animal experiments were approved by Zhejiang University of Traditional Chinese Medicine (ethics approval number: IACUC-20240325-25). The experimental animals were housed in separate cages, with bedding changed every 3 days. An acclimatization period of 2 days was provided, during which time the animals had free access to water and food. The ambient temperature was 20±2℃, and the relative humidity was 60%, with a 12-hour light and 12-hour dark cycle daily. The mice were randomly divided into four groups: control group, model group, ASA group, THP5-L group (low-dose group), THP5-M group (medium-dose group), and THP5-H group (high-dose group), with 5 mice in each group. (THP5 is mentioned in Example 1.)

[0063] 2.1.2 Experimental Reagents and Instruments: Chemical reagents: κ-carrageenan (Sigma, 11114-20-8), ASA (aspirin, CSPC Pharmaceutical Group Co., Ltd.); Reagent kit: BD TM CBA mouse inflammation detection kit (BD Biosciences, 552364), NO detection kit (Beyotime, S0021). Experimental instruments: FA2204 electronic balance, Allegra 64R benchtop high-speed refrigerated centrifuge, 5427R high-speed refrigerated centrifuge, laser speckle blood flow monitor, ELISA reader, complete blood count (CBC). Coagulation function test, pipettes. Experimental equipment: curved forceps, surgical scissors, hemostatic forceps, beakers, graduated cylinders, EP tubes, centrifuge tubes, 1mL syringe.

[0064] 2.1.3 Drug Preparation 2.1.3.1 *Trifolium repens* polysaccharide solution: Weigh 1.25, 2.5, and 5 mg of *Trifolium repens* polysaccharide THP5 powder using an electronic balance and pour them into 10 mL centrifuge tubes. Add sterile physiological saline, sonicate to dissolve, and prepare working solutions with final concentrations of 0.125, 0.25, and 0.5 mg / mL. Filter through a 0.22 μM filter membrane to obtain sterile solutions. Prepare solutions immediately before use.

[0065] 2.1.3.2 Preparation of aspirin suspension: Weigh 100 mg of aspirin powder using an electronic balance, pour it into a 10 mL centrifuge tube, add sterile physiological saline, and sonicate to dissolve it to prepare an aspirin suspension with a final concentration of 10 mg / mL. Prepare and use immediately.

[0066] 2.1.3.3 Preparation of κ-carrageenan solution: Weigh 100 mg of carrageenan using an electronic balance and add it to a 10 mL centrifuge tube. Add sterile physiological saline and heat to 80 °C to dissolve, preparing a solution with a final concentration of 10 mg / mL.

[0067] 2.2 Experimental Methods 2.2.1 Animal Grouping and Model Establishment: Mice were randomly divided into groups of 5. A thrombosis model was established by intraperitoneal injection of κ-carrageenan (100 mg / kg). At 4 and 24 h after model establishment, the control group and model group were administered sterile saline via tail vein, the ASA group was administered aspirin by gavage, and the THP5 groups (1.25, 2.5, and 5 mg / kg) were administered THP5 via tail vein. Mice were sacrificed at 48 h after model establishment.

[0068] 2.2.2 Experimental Procedure: After anesthetizing mice, the blood perfusion volume in the mouse tail was measured and photographs were taken. Blood was collected from the mouse through the eyeball into EDTA or sodium citrate anticoagulant tubes. Simultaneously, mouse tail, liver, and lung tissues were collected and fixed in 4% formaldehyde solution. Plasma Processing: The plasma was centrifuged at 3500 rpm for 15 min at 4°C, and the supernatant was collected for APTT and cytokine level determination.

[0069] 2.2.3 Plasma cytokine level assay: Take a new 1.5 mL centrifuge tube, add 10 μL of cytokine mixture + 10 μL of cell supernatant sample / serially diluted standard solution + 10 μL of PE dye to each tube, and centrifuge to mix. Incubate at room temperature in the dark for 2 h, add 100 μL of water buffer to each sample, mix well, and load the sample for detection by flow cytometry.

[0070] 2.2.4 Determination of plasma NO level: Plasma sample was added to a 96-well plate at a rate of 50 μL / well. Then, room temperature Griess Reagent I and Griess Reagent II were added to each well in sequence. The absorbance was measured at 540 nm and the NO level was calculated by substituting the absorbance into the standard curve.

[0071] 2.2.5 HE staining of tissue: Fixed tissue sections were dehydrated and embedded in paraffin. HE staining: The sample was first hydrated, then stained with hematoxylin and eosin, and finally dehydrated before mounting and microscopic examination.

[0072] 2.2.6 Immunohistochemical determination of CD42b levels: The sections were dewaxed and hydrated, antigen was retrieved with EDTA, and non-specific interference was eliminated by blocking with 3% H2O2. The tissue sections were incubated with CD42b antibody at 4℃, and then incubated with secondary antibody-bound antibody in a 37℃ water bath for 30 min. The sections were observed and photographed under a microscope.

[0073] 2.2.7 Data Statistics: Measurement data were expressed as mean ± standard deviation (mean ± SD). Differences between groups were compared using analysis of variance (ANOVA), and pairwise comparisons were performed using one-way ANOVA. Statistical significance was defined as p < 0.05. # "" indicates that p < 0.05 compared to the normal control group. ## "" indicates that p < 0.01 compared to the normal control group. ### "" indicates that p < 0.001 compared to the normal control group. #### "Compared with the normal control group, p<0.0001;" * "" indicates p < 0.05 compared to the model group. ** "" indicates that p < 0.01 compared to the model group. *** "Indicates p < 0.001 compared to the model group, " **** "" indicates p < 0.0001 compared to the model group; statistics were performed using GraphpadPrism 9.0.

[0074] 2.3 Experimental Results 2.3.1 Therapeutic effect of Trifoliate Orange Polysaccharide THP5 on κ-carrageenan-induced thrombosis in mice: The results are as follows Figure 8 As shown: After modeling, the mouse tails gradually turned black, indicating peripheral thrombosis. Compared with the model group, the medium-dose THP5 group significantly reduced the level of black tail (…). Figure 8 A). Laser speckle contrast imaging showed that the blood perfusion in the tail (0-3 cm) of the model group was significantly reduced, while low- and medium-dose THP5 increased the blood perfusion in the tail. Figure 8 (B) The spleen index was significantly elevated in the model group and decreased after THP5 treatment. Figure 8 C). The APTT in the model group was significantly shortened, while the APTT in the THP5 group was prolonged. Figure 8:D). Hematological analysis showed that the model group had elevated white blood cell, neutrophil, and monocyte counts, and decreased hemoglobin and red blood cell counts. THP5 decreased white blood cell, neutrophil, and monocyte counts, while medium and high doses of THP5 significantly restored HGB or RBC levels. Platelet counts remained unchanged in all groups, indicating no thrombocytopenia or platelet toxicity. Figure 8 E). Serum IL-6 and TNF-α concentrations were elevated in the model group, and THP5 significantly reduced these two inflammatory cytokines. Following thrombosis induction, plasma nitric oxide (NO) levels decreased, and THP5 somewhat mitigated this effect. Figure 8 :F).

[0075] 2.3.2 *Trifolium repens* polysaccharide THP5 inhibits κ-carrageenan-induced platelet thrombus formation in tail and liver / lung tissues: Results are as follows... Figure 9 As shown: In the model group, vessels at 2 cm and 4 cm from the tail tip were almost completely blocked by thrombi, and approximately 70% of vessels at 6 cm were affected. THP5 significantly reduced thrombus formation; no thrombus formation was observed at 4 cm in the medium-dose THP5 group, and only a small number of thrombi were detected at 6 cm in both the low-dose and high-dose THP5 groups. In the lung tissue, the model group showed extensive platelet-rich thrombi, alveolar capillary hemorrhage, and inflammatory infiltration; THP5 significantly alleviated the symptoms in all cases. In the liver, the model group exhibited hepatic vein thrombosis, hepatocellular edema, and perivascular inflammation; THP5 significantly reduced all of these symptoms. Figure 9 A). Immunohistochemistry of CD42b confirmed that THP5 significantly reduced platelet deposition and CD42b expression in tail and liver / lung thrombi. Figure 9 :A).

[0076] Example 3: Study on the antiplatelet activation function of Trifolium repens polysaccharide THP5 in vitro 3.1 Experimental Materials 3.1.1 Experimental Animals and Drugs: Twenty male BALB / c mice, weighing 20±2g, were purchased from the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. The animal experiments were approved by Zhejiang University of Traditional Chinese Medicine (ethics approval number: IACUC-20240325-25). The experimental animals were housed in separate cages, with bedding changed every 3 days. An acclimatization period of 2 days was provided, during which the animals had free access to water and food. The ambient temperature was 20±2℃, the relative humidity was 60%, and the daily light and dark cycle was 12 hours.

[0077] 3.1.2 Experimental Reagents and Instruments: Chemical Reagents: Thrombin (Shanghai Yuanye Biotechnology Co., Ltd., 9002-04-4), Arachidonic Acid (MCE, HY-159590), Collagen (CHRONO-LOG, P / N385), CD62P Antibody (BioLegend, 148305), JON / A Antibody (Emfret, M023-2), PGE1 (Prostaglandin E1, Sigma, 900100P), Apyrase (Adenosine Triphosphatase; Sigma, 9000-95-7), EDTA (Aladdin, E196386), Phalloidin (YESEN, 40734ES75), Triton X-100 (Beyotime, P0096), Tyrode Buffer (Aladdin, M301990). Reagent Kits: ROS Detection Kit (Beyotime, S0033S), ATP Detection Kit (Beyotime, S0026) Experimental Instruments: FA2204 electronic balance, Allegra 64R benchtop high-speed refrigerated centrifuge, 5427R high-speed refrigerated centrifuge, Accuri TM C6 flow cytometer, ELISA reader, pipettes. Laboratory equipment: curved forceps, surgical scissors, hemostats, beakers, graduated cylinders, EP tubes, centrifuge tubes, 2mL syringes.

[0078] 3.1.3 Drug Preparation 3.1.3.1 THP5 solution of Trifolium repens polysaccharide: Weigh 30 mg of THP5 powder of Trifolium repens polysaccharide using an electronic balance and pour it into a 2 mL EP tube. Add sterile physiological saline, sonicate to dissolve, and prepare a working solution with a final concentration of 30 mg / mL. Filter through a 0.22 μM filter membrane to obtain a sterile solution, and aliquot and store at -20℃.

[0079] 3.1.3.2 Preparation of aspirin solution: Weigh 1 mg of aspirin powder using an electronic balance, pour it into a 2 mL centrifuge tube, add sterile physiological saline, and sonicate to dissolve it to prepare an aspirin solution with a final concentration of 1 mg / mL. Prepare and use immediately.

[0080] 3.2 Experimental Methods 3.2.1 Preparation of washed platelets: Blood was collected from mice via the abdominal aorta into a syringe containing sodium citrate anticoagulant. Whole blood was collected into centrifuge tubes and diluted 1:1 with 0.9% physiological saline. To prevent platelet activation, PGE1 (0.1 μg / mL) and Apyrase (1 U / mL) were added. After gentle inversion and mixing, the mixture was centrifuged at 230g for 10 min, and the supernatant was collected as platelet-rich plasma (PRP). Then, Apyrase (1 U / mL) and EDTA (5 mM) were added to the PRP, and the mixture was centrifuged at 800g for 10 min to obtain platelets as a precipitate. The platelet precipitate was resuspended in Tyrode buffer to prepare washed platelets for subsequent use.

[0081] 3.2.2 Platelet aggregation assay: Washed platelets were transferred to 96-well plates, and the OD value at 405 nm was adjusted to approximately 1.0. After incubation at 37°C for 10 min with different concentrations of THP5 (0.1, 0.5, 1 mg / mL) or aspirin (5 μg / mL), thrombin (0.5 U / mL), AA (1 mM), or collagen (0.1 μg / mL) were added. The plates were continuously stirred, and the OD at 405 nm was recorded at 1-min intervals using a microplate reader to assess aggregation kinetics.

[0082] 3.2.3 Thrombus Retraction Assay: Washed platelet pellet was resuspended in platelet-poor plasma (PPP) to a final concentration of 3 × 10⁷ / mL and pre-incubated with THP5 (0.1, 0.5, 1 mg / mL) at 37°C for 15 min. 100 μL of platelet suspension was mixed with 300 μL of physiological saline, and thrombin (0.5 U / mL) was added to induce thrombus formation. Samples were incubated at 37°C, and images were taken every 5 min to monitor thrombus retraction. Clot size was quantified using ImageJ, and the retraction rate was calculated based on the change in clot area over time.

[0083] 3.2.4 Platelet adhesion and spreading test: Wash platelets (5×10⁻⁶) 7 Platelets (0.1, 0.5, 1 mg / mL) were incubated with THP5 (0.1, 0.5, 1 mg / mL) at 37°C for 15 min. 100 μL of washed platelets (3 × 10⁻⁶) were then added. 7 Platelets were seeded onto fibrin-coated cell slides (50 μg / mL) and incubated at 37°C for 2 h. After washing with Tyrode buffer to remove unattached platelets, the slides were fixed with 4% paraformaldehyde and infiltrated with 0.2% Triton X-100. Platelet morphology and diffusion were observed under a fluorescence microscope after staining with phalloidin.

[0084] 3.2.5 Platelet ATP Release Assay: Washed platelets (5×10⁻⁶) 7Platelets were pre-incubated with THP5 (0.1, 0.5, 1 mg / mL) at 37°C for 15 min, and then activated with thrombin (0.5 U / mL) at 37°C for 5 min. The samples were centrifuged at 800 g for 10 min to precipitate platelets, and the supernatant was collected. ATP concentration was measured using an ATP assay kit according to the manufacturer's instructions.

[0085] 3.2.6 Platelet ROS Level Detection: Washed platelets (3×10⁻⁶) 7 Platelets were loaded with H2DCF-DA (50 μM) at 37 °C for 20 min, and incubated with THP5 (0.1, 0.5, 1 mg / mL) at 37 °C for 15 min, followed by stimulation with thrombin (0.5 U / mL) for 5 min. The reaction was terminated by adding 10 volumes of Tyrode buffer, and platelet ROS levels were detected by flow cytometry.

[0086] 3.2.7 Platelet CD62P and JON / A Level Detection: Washed platelets (5×10⁻⁶) 7 Platelet suspensions were incubated with THP5 (0.1, 0.5, 1 mg / mL) at 37°C for 15 min, followed by stimulation with thrombin (0.5 U / mL) at 37°C for 5 min. 50 μL of platelet suspension was added to either CD62P antibody or JON / A antibody and incubated in the dark for 30 min. The reaction was terminated by adding 450 μL of Tyrode buffer, and CD62P and JON / A levels were detected by flow cytometry.

[0087] 3.2.8 Data Statistics: Measurement data were expressed as mean ± standard deviation (mean ± SD). Differences between groups were compared using analysis of variance (ANOVA), and pairwise comparisons were performed using one-way ANOVA. Statistical significance was defined as p < 0.05. # "" indicates that p < 0.05 compared to the normal control group. ## "" indicates that p < 0.01 compared to the normal control group. ### "" indicates that p < 0.001 compared to the normal control group. #### "Indicates p < 0.0001 compared to the normal control group;" * "" indicates p < 0.05 compared to the model group. ** "" indicates that p < 0.01 compared to the model group. *** "Indicates p < 0.001 compared to the model group, " **** "" indicates p < 0.0001 compared to the model group; statistics were performed using GraphpadPrism 9.0.

[0088] 3.3 Experimental Results 3.3.1 THP5 inhibits platelet activation in vitro: such asFigure 10 As shown, pre-incubation with THP5 significantly inhibited platelet aggregation induced by thrombin, AA, or collagen, with effects superior to aspirin. Figure 10 A). THP5 inhibited platelet adhesion and diffusion on the fibrinogen-coated surface in a dose-dependent manner, reducing the number of platelets adhering and the platelet spreading area. Figure 10 B). In the thrombus contraction experiment, THP5 inhibited the thrombin-induced thrombus contraction rate ( Figure 10 :CD). THP5 dose-dependently inhibited thrombin-induced ATP release from dense particles ( Figure 10 :E), and significantly reduced ROS levels ( Figure 10 Following THP5 pre-incubation and thrombin induction, platelet surface CD62P expression was significantly reduced, indicating a decrease in α-granule release levels. Figure 10 Furthermore, THP5 inhibits thrombin-induced integrin αIIbβ activation in a dose-dependent manner, as indicated by JON / A binding (G). Figure 10 :H).

[0089] Example 4: Investigation of the interaction target between Trifoliate orange polysaccharide THP5 and platelets 4.1 Experimental Materials 4.1.1 Experimental Ethics 4.1.1.1 Blood Collection from Mice: Ten male BALB / c mice, weighing 20±2g, were purchased from the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. The animal experiment was approved by Zhejiang University of Traditional Chinese Medicine (ethics approval number: IACUC-20240325-25). The experimental animals were housed in separate cages, with bedding changed every 3 days. An acclimatization period of 2 days was provided, during which time the animals had free access to water and food. The ambient temperature was 20±2℃, the relative humidity was 60%, and the daily light and dark cycle was 12h.

[0090] 4.1.1.2 Human Blood Collection: Human studies were conducted in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of Zhejiang University of Traditional Chinese Medicine (Approval No.: 20250425-3). All participants provided written informed consent before sample collection, agreeing to the use and publication of data. Peripheral blood samples were collected from five healthy volunteers (2 males, 3 females; age range: 25-30 years).

[0091] 4.1.2 Experimental Reagents and Instruments: Chemical Reagents: MES buffer (Shanghai Yuanye Biotechnology Co., Ltd., No. 71119-23-8), acylhydrazide-biotin (MCE, HY-100215), EDC (Aladdin, E106172), HBS buffer (Aladdin, H301529), Tyrode buffer (Aladdin, M301990), streptavidin magnetic beads (Beyotime, P2151), tirofiban (MCE, HY-17369), abciximab (MCE; HY-P9934), ADP (MCE, HY-W010918), GPIIb / IIIa protein (MCE, HY-P77716), CD61 antibody (CST, 13166). Reagent kits: Biotin quantitative reagent kit (Beyotime, P0373S), rapid silver staining kit (Beyotime, P0017S). Experimental instruments: Biacore 8K instrument, CM5 sensor chip, microplate reader, pipettes. Experimental equipment: Curved forceps, surgical scissors, hemostats, beakers, graduated cylinders, EP tubes, centrifuge tubes, 2mL syringes.

[0092] 4.2 Experimental Methods 4.2.1 Preparation of washed platelets: Blood was collected from mice via the abdominal aorta into a syringe containing sodium citrate anticoagulant. Whole blood was collected into centrifuge tubes and diluted 1:1 with 0.9% physiological saline. To prevent platelet activation, PGE1 (0.1 μg / mL) and Apyrase (1 U / mL) were added. After gentle inversion and mixing, the mixture was centrifuged at 230g for 10 min, and the supernatant was collected as platelet-rich plasma (PRP). Then, Apyrase (1 U / mL) and EDTA (5 mM) were added to the PRP, and the mixture was centrifuged at 800g for 10 min to obtain platelets as a precipitate. The platelet precipitate was resuspended in Tyrode buffer to prepare washed platelets for subsequent use.

[0093] 4.2.2 Biotinylation of THP5: THP5 was dissolved in MES buffer, and then acylhydrazide-biotin was added at a 1:1 molar ratio. EDC was added to a final concentration of 5 mM to initiate coupling, and the mixture was shaken at 37 °C for 12 h. After heating and centrifugation, the supernatant was collected and dialyzed against 3 kDa. The concentration and labeling efficiency of biotinylated THP5 (Bio-THP5) were determined using a biotin quantitative kit.

[0094] 4.2.3 Bio-THP5 and Platelet Pull-Down Assay: Bio-THP5 and streptavidin magnetic beads were incubated with shaking at 4°C for 12 h. The beads were washed with TBS to remove unbound proteins, and then incubated with platelet protein lysates at 4°C for another 12 h. After washing with TBS, proteins bound to the beads were eluted with 1× loading buffer, separated by SDS-PAGE, and differential bands were observed by rapid silver staining. Selected differential bands were excised for LC-MS / MS analysis.

[0095] 4.2.4 SPR affinity analysis of *Trifolium repens* polysaccharide THP5 and ITGA2B3: Activation was performed using 400 mM MEDC and 100 mM NHS at a flow rate of 10 μL / min for 600 s. ITGA2B3 was diluted with 10 mM sodium acetate (pH 4.0) to 10 μg / mL / 20 μg / mL and diluted at a flow rate of 5 μL / min for 600 s. The CM5 chip was blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 600 s. THP5 was diluted using running buffer, serially diluted 2-fold from 10 nM to 0.078 nM. Each THP5 concentration was considered a cycle, with binding and dissociation performed at a flow rate of 30 μL / min for 180 s and 180 s respectively. For analysis, the effective concentration was selected and kinetics was performed using a 1:1 model.

[0096] 4.2.5 Competitive Platelet Aggregation Inhibition Assay: Washed platelets were transferred to 96-well plates, and the OD was adjusted to 1.0 at 405 nm. THP5 (1 mg / mL), tirofiban (2 μM), and abciximab (20 μg / mL) were added individually or together, and the plates were incubated at 37°C for 10 min. ADP (10 μM) was added to induce aggregation. The plates were continuously shaken, and the OD at 405 nm was recorded at 1-min intervals using a microplate reader to assess aggregation kinetics.

[0097] 4.3 Experimental Results 4.3.1 Integrin β3 has been identified as a potential molecular target mediating the interaction between THP5 and platelets: such as Figure 11 As shown, silver staining in the pull-down experiment revealed a distinct 100-150 kDa protein band enriched in the THP5 group. Figure 11 A). Mass spectrometry identification of integrin β3 is a prominent candidate. Figure 11 B), Western blot analysis using the Pulldown experiment verified this. Figure 11 :C). KEGG pathway analysis of differentially expressed protein bands showed enrichment during platelet aggregation, activation, and coagulation. Figure 11:D). GO analysis showed significant enrichment of platelet activation, focal adhesion, and complement / coagulation cascade. Figure 11 :E).

[0098] 4.3.2 GPIIb / IIIa exhibits high affinity for THP5: To clarify the interaction between THP5 and GPIIb / IIIa, SPR analysis showed that THP5 binds to purified αIIbβ3 integrin with high affinity (KD = 0.6 nM). Figure 11 :F).

[0099] 4.3.3 THP5 shares the same site of action as the GPIIb / IIIa targeted drugs tirofiban / abbciximab: In competitive platelet aggregation assays, the combination of THP5 with tirofiban or abciximab did not enhance the inhibitory effect compared to THP5 alone. Figure 11 The results suggest that THP5 may be similar to tirofiban or abciximab in that it exerts its antiplatelet activation effect by targeting the ligand-binding head fragment of αIIbβ3, thereby disrupting the RGD recognition site and the MIDAS metal ion coordination site.

[0100] Example 5: Comparison of the antithrombotic efficacy and drug-induced bleeding safety of THP5-tirofiban 5.1 Experimental Materials 5.1.1 Experimental Animals and Drugs: Sixty-five male BALB / c mice, weighing 20±2g, were purchased from the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. The animal experiments were approved by Zhejiang University of Traditional Chinese Medicine (ethics approval number: IACUC-20220530-10). The experimental animals were housed in separate cages, with bedding changed every 3 days. An acclimatization period of 2 days was provided, during which time the animals had free access to water and food. The ambient temperature was 20±2℃, the relative humidity was 60%, and the daily light and dark cycle was 12 hours.

[0101] 5.1.2 Experimental Reagents and Instruments: Chemical Reagents: κ-carrageenan (Sigma, 11114-20-8), TIF (tirofiban; MCE, HY-17369), FeCl3 (Aladdin, I112064). Experimental Instruments: FA2204 electronic balance, Allegra 64R benchtop high-speed refrigerated centrifuge, 5427R high-speed refrigerated centrifuge, laser speckle blood flow monitor, complete blood count, coagulation function test, pipettes. Experimental Apparatus: Curved forceps, surgical scissors, hemostatic forceps, beakers, graduated cylinders, EP tubes, centrifuge tubes, 1mL syringes.

[0102] 5.1.3 Drug Preparation 5.1.3.1 *Trifolium repens* polysaccharide THP5 solution: Weigh 2.5g of *Trifolium repens* polysaccharide THP5 powder using an electronic balance and pour it into a 10mL centrifuge tube. Add 10mL of sterile physiological saline, sonicate to dissolve, and prepare a working solution with a final concentration of 0.25mg / mL. Filter through a 0.22μM filter membrane to obtain a sterile solution.

[0103] 5.1.3.2 Preparation of tirofiban solution: Pipette 113.5 μL of tirofiban stock solution (10 mM) dissolved in DMSO, add it to 20% SBE-β-CD physiological saline solution, make up to 10 mL and mix well.

[0104] 5.1.3.3 Preparation of κ-carrageenan solution: Weigh 100 mg of carrageenan using an electronic balance and add it to a 10 mL centrifuge tube. Add 10 mL of sterile physiological saline and heat to 80 °C to dissolve, preparing a solution with a final concentration of 10 mg / mL.

[0105] 5.1.3.4 Preparation of 20% FeCl3 solution: Weigh 2 mg of carrageenan using an electronic balance and add it to a 10 mL centrifuge tube. Add 10 mL of sterile physiological saline to prepare a 20% FeCl3 solution.

[0106] 5.2 Experimental Methods 5.2.1 Experimental Grouping and Model Establishment: κ-carrageenan thrombosis model: Mice were randomly divided into four groups: control group, model group, TIF (0.5 mg / kg) group, and THP5 (2.5 mg / kg) group, with 5 mice in each group. A thrombosis model was established by intraperitoneal injection of κ-carrageenan (100 mg / kg). At 4 and 24 hours post-modeling, the control and model groups were administered sterile saline via tail vein, the TIF group was administered tirofiban via tail vein, and the THP5 group was administered THP5 via tail vein. Mice were sacrificed at 48 hours post-modeling.

[0107] FeCl3 carotid artery thrombosis model: Mice were randomly divided into four groups: control group, model group, TIF group (0.5 mg / kg), and THP5 group (2.5 mg / kg), with 5 mice in each group. Ten minutes after intravenous injection of saline, tirofiban, or THP5, the mice were anesthetized, and the carotid artery was dissected. A filter paper disc (1×1 mm) saturated with 20% FeCl3 was applied to the surface of the blood vessel to induce carotid artery thrombosis.

[0108] Drug-induced bleeding safety experiment: Mice were randomly divided into four groups: control group, ASA group (100 mg / kg), TIF group (0.5 mg / kg), and THP5 group (1.25, 2.5, and 5 mg / kg), with five mice in each group. At 0 and 24 hours, the control group was intravenously injected with saline, the aspirin group was given oral aspirin, and the tirofiban and THP5 groups were intravenously injected with tirofiban and THP5, respectively. The mice were euthanized 48 hours later.

[0109] 5.2.2 Experimental Procedure κ-carrageenan thrombosis model: After anesthetizing mice, the blood perfusion volume in the tail was measured and the tail was photographed. Blood was collected from the eyeballs into EDTA or sodium citrate anticoagulant tubes, and the spleen was weighed simultaneously. Plasma processing: The plasma was centrifuged at 3500 rpm for 15 min at 4°C, and the supernatant was collected for FIB assay.

[0110] FeCl3 carotid artery thrombosis model: Filter paper discs (1×1 mm) saturated with 20% FeCl3 were applied to the surface of the blood vessel, and blood flow was continuously monitored using a laser speckle flow imaging system. After euthanizing the mice, the carotid artery was excised and photographed. The thrombus was carefully dissected from the arterial specimen, weighed, and its length was measured for quantitative analysis.

[0111] Drug-induced hemorrhage safety experiment: At 48 hours, after anesthetizing mice, a 5mm section of the mouse tail was cut, and the tail was immersed in 37°C physiological saline to measure the duration of bleeding. After the mice were sacrificed, blood and major organs (heart, liver, spleen, lungs, and kidneys) were collected for analysis.

[0112] 5.3 Experimental Results 5.3.1 Both THP5 and tirofiban can significantly inhibit κ-carrageenan-induced thrombus formation: such as Figure 12 As shown, both THP5 and tirofiban significantly reduced κ-carrageenan-induced thrombosis, which can be demonstrated by improved tail perfusion and reduced black tail. Figure 12 :AB). THP5 has a greater impact on the increase in spleen index caused by thrombosis, indicating a broader regulatory scope for inflammation or hematopoietic responses. Figure 12 :C). Meanwhile, THP5 is more effective than tirofiban in reducing circulating fibrinogen levels ( Figure 12 :D), and more effectively alleviated the decrease in erythrocyte count and hemoglobin concentration induced by κ-carrageenan ( Figure 12 :E).

[0113] 5.3.2 Both THP5 and tirofiban significantly inhibited FeCl3-induced carotid artery thrombosis: In the FeCl3-induced carotid artery thrombosis model, both THP5 and tirofiban significantly prolonged occlusion time, reduced thrombus weight, and inhibited thrombus length.Figure 12 :F).

[0114] 5.3.3 THP5 drug bleeding safety test: such as Figure 12 As shown, organ indices (heart, liver, spleen, lung, and kidney) in mice treated with THP5 did not change, indicating good systemic safety. Figure 12 Aspirin significantly prolonged tail bleeding time, while intravenous tirofiban or THP5 had no significant effect on hemostasis. Figure 12 :I). Platelet counts remained stable after treatment with tirofiban or THP5, while aspirin caused a significant decrease in platelet count ( Figure 12 :J).

[0115] Example 6: Investigation on the effect of Trifoliate orange polysaccharide on platelet protein levels in thrombotic mice.

[0116] 6.1 Experimental Materials 6.1.1 Experimental Animals and Drugs: Ten male BALB / c mice, weighing 20±2g, were purchased from the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. The animal experiment was approved by Zhejiang University of Traditional Chinese Medicine (ethics approval number: IACUC-20220530-10). The experimental animals were housed in separate cages, with bedding changed every 3 days. An acclimatization period of 2 days was provided, during which the animals had free access to water and food. The ambient temperature was 20±2℃, the relative humidity was 60%, and the daily light and dark cycle was 12 hours.

[0117] 6.1.2 Experimental Reagents and Instruments: Chemical Reagents: κ-carrageenan (Sigma, 11114-20-8), TIF (tirofiban; MCE, HY-17369). Experimental Instruments: FA2204 electronic balance, Allegra 64R benchtop high-speed refrigerated centrifuge, 5427R high-speed refrigerated centrifuge, pipettes. Experimental Apparatus: curved forceps, surgical scissors, hemostats, beakers, graduated cylinders, EP tubes, centrifuge tubes, 1mL syringes.

[0118] 6.1.3 Drug Preparation 6.1.3.1 THP5 solution of Trifolium repens polysaccharide: Weigh 2.5g of THP5 powder of Trifolium repens polysaccharide using an electronic balance and pour it into a 10mL centrifuge tube. Add 10mL of sterile physiological saline, sonicate to dissolve, and prepare a working solution with a final concentration of 0.25mg / mL. Filter through a 0.22μM filter membrane to obtain a sterile solution.

[0119] 6.1.3.2 Preparation of κ-carrageenan solution: Weigh 100 mg of carrageenan using an electronic balance and add it to a 10 mL centrifuge tube. Add 10 mL of sterile physiological saline and heat to 80 °C to dissolve, preparing a solution with a final concentration of 10 mg / mL. 6.2 Experimental methods 6.2.1 Experimental grouping and model establishment: κ-carrageenan thrombosis model: Mice were randomly divided into two groups: a model group and a THP5 (2.5 mg / kg) group, with 5 mice in each group. A thrombosis model was established by intraperitoneal injection of κ-carrageenan (100 mg / kg). At 4 and 24 h after modeling, the model group received sterile physiological saline via the tail vein, and the THP5 group received THP5 via the tail vein. Mice were sacrificed at 48 h after modeling.

[0120] 6.2.2 Experimental procedure: Blood was collected from mice via the abdominal aorta. Platelets were extracted, washed, and the platelet precipitate was collected. An equal amount of platelet protein samples were transported to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for subsequent proteomics analysis.

[0121] 6.3 Experimental Results 6.3.1 THP5 affects platelet activation-related proteins and signaling pathways: such as Figure 13 As shown, PCA analysis revealed different proteomic characteristics among the groups, indicating that THP5 significantly induced changes in the platelet proteome. Figure 13 A). The sample correlation analysis confirmed a high degree of intragroup consistency, supporting the reliability of the data. Figure 13 KEGG pathway enrichment analysis identified significant regulation of thrombosis-related pathways by THP5, including complement and coagulation cascades, PI3K-Akt, MAPK, insulin signaling, and adipokines pathways. Figure 13 :C). Proteomics analysis further identified several thrombosis-related proteins that were significantly downregulated by THP5 (C). Figure 13 :D), including integrin α1, plasma serine protease inhibitor (Serpin), type VI collagen and high mobility group 2 (HMGB2).

[0122] Example 7: Investigation of the in vitro and in vivo THP5 antiplatelet activation signaling pathway 7.1 Experimental Materials 7.1.1 Experimental Animals and Drugs: Twelve male BALB / c mice, weighing 20±2g, were purchased from the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. The animal experiment was approved by Zhejiang University of Traditional Chinese Medicine (ethics approval number: IACUC-20220530-10). The experimental animals were housed in separate cages, with bedding changed every 3 days. An acclimatization period of 2 days was provided, during which time the animals had free access to water and food. The ambient temperature was 20±2℃, the relative humidity was 60%, and the daily light and dark cycle was 12 hours.

[0123] 7.1.2 Experimental Reagents and Instruments: Chemical Reagents: κ-carrageenan (Sigma, 11114-20-8), RIPA lysis buffer (Beyotime, P0013B), PKCα antibody (CST, 2056), p38 antibody (CST, 8690), ERK antibody (CST, 8690), JNK antibody (CST, 9252), p-PKCα antibody (Selleck, F2482), p-P38 antibody (CST, 4511), p-ERK antibody (CST, 4370), p-JNK antibody (CST, 9251), β-tubulin antibody (HUABIO, M1305-2). Experimental Instruments: FA2204 electronic balance, Direct-Q8 ultrapure water system, pipettes, XW-80A vortex mixer. Experimental equipment: curved forceps, surgical scissors, hemostats, beakers, graduated cylinders, EP tubes, centrifuge tubes, 1mL syringes.

[0124] 7.1.3 Drug Preparation 7.1.3.1 *Trifolium repens* polysaccharide THP5 solution: Weigh 2.5 mg of *Trifolium repens* polysaccharide THP5 powder using an electronic balance and pour it into a 10 mL centrifuge tube. Add 10 mL of sterile physiological saline, sonicate to dissolve, and prepare a working solution with a final concentration of 0.25 mg / mL. Filter through a 0.22 μM filter membrane to obtain a sterile solution. Weigh 30 mg of *Trifolium repens* polysaccharide THP5 powder using an electronic balance and pour it into a 2 mL EP tube. Add sterile physiological saline, sonicate to dissolve, and prepare a working solution with a final concentration of 30 mg / mL. Filter through a 0.22 μM filter membrane to obtain a sterile solution, and aliquot and store at -20℃.

[0125] 7.1.3.2 Preparation of κ-carrageenan solution: Weigh 100 mg of carrageenan using an electronic balance and add it to a 10 mL centrifuge tube. Add 10 mL of sterile physiological saline and heat to 80 °C to dissolve, preparing a solution with a final concentration of 10 mg / mL.

[0126] 7.1.3.3 Preparation of tirofiban solution: Pipette 113.5 μL of tirofiban stock solution (10 mM) dissolved in DMSO, add it to 20% SBE-β-CD physiological saline solution, make up to 10 mL and mix well to obtain a tirofiban solution with a final concentration of 0.05 mg / mL.

[0127] 7.2 Experimental Methods 7.2.1 Experimental Grouping and Model Establishment: Mice were randomly divided into four groups: control group, model group, TIF (0.5 mg / kg) group, and THP5 (2.5 mg / kg) group, with 5 mice in each group. A thrombosis model was established by intraperitoneal injection of κ-carrageenan (100 mg / kg). At 4 and 24 hours post-modeling, the control and model groups were administered sterile saline via tail vein, the TIF group was administered tirofiban via tail vein, and the THP5 group was administered THP5 via tail vein. Mice were sacrificed at 48 hours post-modeling.

[0128] 7.2.2 Experimental Procedure: Blood was collected from the abdominal aorta of mice in each treatment group. Platelets were extracted and washed, and the platelet precipitate was collected. RIPA was added and the platelets were lysed on ice for 30 min. After centrifugation at 12,000 rpm for 20 min at 4℃, the supernatant was collected, 5× loading buffer was added, and the mixture was incubated in a 100℃ metal bath for 10 min. The mixture was then stored at -80℃.

[0129] 7.2.3 Preparation of in vitro platelet activation samples: Blood was collected from the abdominal aorta of normal mice. Platelets were extracted, washed, and resuspended in Tyrode buffer. THP5 (0.1, 0.5, or 1 mg / mL) or tirofiban (2 μM) was added, and the mixture was incubated at 37°C for 15 min. Thrombin (0.5 U / mL) was then added and incubated for 5 min. The mixture was centrifuged at 800g for 10 min, and the precipitate was lysed on ice with RIPA for 30 min. After centrifugation at 12000 rpm for 20 min at 4°C, the supernatant was collected, added to 5× loading buffer, and incubated in a 100°C metal bath for 10 min. The mixture was then stored at -80°C.

[0130] 7.2.4 MAPK Pathway Protein Assay: Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) from gel wetting to nitrocellulose membrane. After sealing (5% BSA), primary antibodies (p-PKCα, p-ERK, p-JNK, p-P38, PKCα, ERK, JNK, P38, β-tubulin) and secondary antibodies (rabbit antibody, mouse antibody) were detected. Protein expression was detected by ECL and blot analysis was performed using a Tanon 5200CL imaging analysis system (Tanon, China). All protein levels were normalized to total protein and β-tubulin, and quantified using ImageJ software.

[0131] 7.3 Experimental Results 7.3.1 THP5 exerts its antithrombotic effect in vivo by inhibiting the MAPK pathway: In vivo, κ-carrageenan-treated mouse platelets showed increased phosphorylation of MAPK components, and increased p-PKCα / PKCα, p-P38 / P38, p-ERK / ERK, and p-JNK / JNK ratios, indicating pathway activation during thrombosis. Treatment with tirofiban or THP5 reduced these phosphorylation levels, with THP5 exhibiting a stronger inhibitory effect. Figure 14 :A).

[0132] 7.3.2 THP5 exerts its antiplatelet activation effect in vitro by inhibiting the MAPJ pathway: In vitro thrombin stimulation significantly increased the phosphorylation of MAPK proteins in platelets. Pre-incubation with THP5 and tirofiban attenuated thrombin-induced phosphorylation of PKCα, p38, ERK, and JNK. Figure 14 :B).

Claims

1. A polysaccharide of *Trifolium repens*, characterized in that: Named THP5, it comprises 12 partially methylated sugar alcohol acetyl ester derivatives: T-Ara f ,1,2-Rha p T-Glc p A, T-Gal p , 1,2-Man p 1,4-Glc p A ， 1,4-Gal p ,1,2,3-Man p , 1,2,3,4-Man p 1,4,6-Glc p ,1,4,6-Gal p ,1,3,6-Gal p ; The monosaccharide composition includes Rha, Ara, Gal, Glc, Man, and GlcA.

2. The *Trifolium repens* polysaccharide according to claim 1, characterized in that: The molecular weight of the *Trifolium repens* polysaccharide is 3.225 × 10⁻⁶. 5 kDa; The molar ratio of Rha, Ara, Gal, Glc, Man, and GlcA is 2.95:11.89:35.77:1.53:20.90:26.

95.

3. A method for preparing *Trifolium repens* polysaccharide, characterized in that... include: 1) Extract crude polysaccharides from the aerial parts of *Tripterygium wilfordii*; 2) The crude polysaccharide of *Trifolium repens* was dialyzed and eluted using a cellulose column gradient to obtain the eluent; 3) The polysaccharide was obtained by elution with water in a gel column.

4. The application of the *Trifolium repens* polysaccharide according to claim 1 or 2, or the *Trifolium repens* polysaccharide obtained by the preparation method according to claim 3, in the preparation of antithrombotic drugs.

5. The application as described in claim 4, characterized in that: The thrombosis is a thrombotic disease associated with the αⅡbβ3-MAPK signaling pathway.

6. The application as described in claim 5, characterized in that: The thrombotic diseases mentioned are arterial thrombosis and venous thrombosis.

7. The application as described in claim 6, characterized in that: The arterial thrombosis refers to myocardial infarction, cerebral infarction, and peripheral arterial thrombosis. The venous thrombosis refers to deep vein thrombosis (DVT), septic thrombosis, and tumor thrombosis.

8. The application of the *Trifolium repens* polysaccharide according to claim 1 or 2 or the *Trifolium repens* polysaccharide obtained by the preparation method according to claim 3 in the preparation of MAPK pathway inhibitors.

9. The application as described in claim 8, characterized in that: The MAPK pathway involves one or more of ERK, JNK, and P38.

10. A method for inhibiting MAPK pathway activation in in vitro cells using the polysaccharide of *Trifolium repens* according to claim 1 or 2 or the polysaccharide obtained by the preparation method according to claim 3.

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