Sargassum fusiforme polysaccharide, and preparation method and application thereof

Abstract

The application provides a saccharide of Sargassum fusiforme, a preparation method and application thereof, and belongs to the technical field of extraction and application of bioactive ingredients. 6 The saccharide of Sargassum fusiforme has a relative molecular weight of (6.0-6.5) * 10 The preparation method comprises the following steps: boiling water reflux extraction of Sargassum fusiforme raw materials to obtain an extraction liquid, decolorization of the extraction liquid through a macroporous resin, alcohol precipitation, water redissolution, dialysis to obtain a cut-off liquid, and deproteinization by adding a protease to obtain a crude saccharide of Sargassum fusiforme; and the crude saccharide of Sargassum fusiforme is separated through an anion exchange chromatography column and a gel permeation chromatography column in sequence. The application defines the structure of the saccharide, the saccharide has high affinity to blood coagulation factor IIa and antithrombin III, and can play an anticoagulant activity through double-target point action.

Description

Technical Field

[0001] This invention belongs to the field of bioactive ingredient extraction and application technology, and particularly relates to a Sargassum fusiforme polysaccharide, its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Thromboembolic diseases encompass both venous and arterial thromboembolic disorders, including venous thromboembolism, pulmonary embolism, acute coronary syndrome, and ischemic stroke, which can rapidly progress to life-threatening complications. Anticoagulation has become a key treatment approach throughout all clinical stages. Vitamin K antagonists such as warfarin and heparin were once the first-line anticoagulation therapies, but these drugs have many limitations, including individual variability in response and increased bleeding risk. Compared to warfarin, direct oral anticoagulants (DOACs), such as dabigatran (a factor IIa inhibitor), apixaban, and rivaroxaban (a factor Xa inhibitor), have shown significant advantages in terms of ease of administration (e.g., no need for routine monitoring) and patient compliance (e.g., more lenient dietary restrictions). However, the use of DOACs still requires careful balancing of efficacy and bleeding risk. Therefore, there remains a clear need to develop anticoagulant products that combine excellent efficacy, high safety, and convenience.

[0004] Sargassum fusiforme ( Sargassum fusiforme Sargassum (Harv.) Setch., belonging to the Sargassum family and the Sargassum genus, is widely distributed along the warm temperate coast of East Asia. It has a history of edible and medicinal use spanning over two thousand years, and is rich in protein, vitamins, and trace elements. As a traditional Chinese medicine, Sargassum fusiforme is used to treat thyroid diseases, edema, and cardiovascular diseases. Modern research further reveals that its polysaccharide components possess significant anticoagulant bioactivity and exhibit a lower bleeding risk compared to heparin, potentially achieving a better risk-benefit balance. However, there is currently a lack of research and reports on isolating structurally defined and homogeneously composed anticoagulant polysaccharides from Sargassum fusiforme, making the discovery of key substances responsible for its anticoagulant activity crucial. Summary of the Invention

[0005] In view of the current state of technology, the purpose of this invention is to provide a Sargassum fusiforme polysaccharide, its preparation method and application, which isolates an anticoagulant polysaccharide with a well-defined structure and uniform composition from Sargassum fusiforme. It has a high affinity for coagulation factor IIa and antithrombin III and exerts anticoagulant activity through dual-target action.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] Firstly, a Sargassum fusiforme polysaccharide with a relative molecular mass of (6.0~6.5) × 10⁻⁶. 6 Da, comprising glucose, galactose, xylose and fucose in a molar ratio of (3.8~4.8):(0.7~1.3):(0.8~1.4):(2.3~3.1), and containing 15~20% sulfate.

[0008] Secondly, the preparation method of the above-mentioned Sargassum polysaccharide includes the following steps: Sargassum fusiforme raw material was extracted by boiling water reflux to obtain an extract. The extract was decolorized by macroporous resin and then precipitated with alcohol to obtain a precipitate. The precipitate was redissolved in water and dialyzed to obtain a truncation. Protease was added to the truncation for enzymatic hydrolysis, and the protein was removed to obtain Sargassum fusiforme crude polysaccharide. The crude polysaccharide of Sargassum fusiforme was dissolved in water and then separated by anion exchange chromatography and gel permeation chromatography to obtain the Sargassum fusiforme polysaccharide.

[0009] Thirdly, an anticoagulant product comprising the aforementioned Sargassum polysaccharide.

[0010] Optionally, the pharmaceutical composition may include pharmaceutically acceptable excipients.

[0011] Optionally, the oral dosage form of the pharmaceutical composition includes capsules, lozenges, oral liquids, tablets, granules, or pills. Fourthly, the application of the above-mentioned Sargassum polysaccharide in at least one of the following (a1) to (a3); (a1) To prepare products for the prevention and / or treatment of thromboembolic diseases; (a2) Prepare products for the prevention and / or treatment of venous thromboembolic diseases.

[0012] (a3) Preparation of anticoagulant products.

[0013] The beneficial effects of this invention are as follows: 1. This invention provides a Sargassum fusiforme polysaccharide with a novel chemical structure. The polysaccharide structure was clarified through comprehensive chemical analysis. It exhibits high affinity for coagulation factor IIa and antithrombin III, and can exert anticoagulant activity through dual-target action.

[0014] 2. The preparation method provided by this invention, based on the traditional water extraction and alcohol precipitation process, further combines decolorization, deproteinization, ion exchange chromatography, gel permeation chromatography and other methods to prepare polysaccharides with high purity, uniform molecular weight distribution and significant anticoagulant activity. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 This is the elution curve of the anion exchange chromatography column in Example 1 of the present invention.

[0017] Figure 2 This is the elution curve of the gel permeation chromatography column in Example 1 of the present invention.

[0018] Figure 3 The results are the determination results of the molecular weight distribution in Example 1 of the present invention.

[0019] Figure 4 The results show the determination of monosaccharide composition in Example 1 of this invention.

[0020] Figure 5 The results are infrared spectral analysis results of Embodiment 1 of the present invention.

[0021] Figure 6 The one-dimensional nuclear magnetic resonance spectrum of Embodiment 1 of the present invention 1 H NMR spectrum.

[0022] Figure 7 The one-dimensional nuclear magnetic resonance spectrum of Embodiment 1 of the present invention 13 C10 NMR spectrum.

[0023] Figure 8 The DEPT135 spectrum is a one-dimensional nuclear magnetic resonance spectrum of Embodiment 1 of the present invention.

[0024] Figure 9 The image shows the HSQC spectrum of the two-dimensional nuclear magnetic resonance spectrum of Embodiment 1 of the present invention.

[0025] Figure 10 The image shows the HMBC spectrum of the two-dimensional nuclear magnetic resonance spectrum of Embodiment 1 of the present invention.

[0026] Figure 11 The COSY spectrum is the two-dimensional nuclear magnetic resonance spectrum of Embodiment 1 of the present invention.

[0027] Figure 12 The NOESY spectrum is the two-dimensional nuclear magnetic resonance spectrum of Embodiment 1 of the present invention.

[0028] Figure 13 The results of hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis in Example 1 of this invention are shown, where A is... m / z Secondary mass spectra of ion 259.0, B and C are... m / z Secondary mass spectrum of ion 389.1, where D is... m / zSecondary mass spectrum of ion 405.1, E is m / z Secondary mass spectrum of 535.1 ions.

[0029] Figure 14 The results of the anticoagulant activity evaluation in Example 2 of the present invention are shown in the figure. A is the thrombin time graph, B is the activated partial thromboplastin time graph, C is the prothrombin time graph, and D is the bleeding risk graph.

[0030] Figure 15 The results of the anticoagulant activity evaluation in Example 3 of the present invention are shown, where A is the thrombin time graph, B is the activated partial thromboplastin time graph, and C is the prothrombin time graph.

[0031] Figure 16 The results of the anticoagulant activity evaluation in Example 4 of the present invention are shown. In this example, A is the concentration curve of direct inhibition rate of coagulation factor IIa, and B is the concentration curve of inhibition rate of coagulation factor IIa mediated by antithrombin III.

[0032] Figure 17 The results of the anticoagulant activity evaluation in Example 5 of the present invention are shown. In this example, A is the concentration curve of direct inhibition rate of coagulation factor IIa, and B is the concentration curve of inhibition rate of coagulation factor IIa mediated by antithrombin III.

[0033] Figure 18 The results of molecular interactions in Example 6 of the present invention are shown below. In this example, A is an isothermal titration calorimetric curve of interaction with coagulation factor IIa, the upper part of A is an exothermic curve, and the lower part of A is a binding curve fitting graph. In this example, B is an isothermal titration calorimetric curve of interaction with antithrombin III, the upper part of B is an exothermic curve, and the lower part of B is a binding curve fitting graph. Detailed Implementation

[0034] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0036] One or more embodiments of the present invention provide a Sargassum fusiforme polysaccharide with a relative molecular mass of (6.0~6.5) × 10⁻⁶. 6Da, comprising glucose, galactose, xylose and fucose in a molar ratio of (3.8~4.8):(0.7~1.3):(0.8~1.4):(2.3~3.1), and containing 15~20% sulfate.

[0037] The above structural information was obtained through comprehensive chemical analysis techniques. Sargassum polysaccharide was isolated and purified from Sargassum fusiforme. It has a clear structure and uniform composition, and exerts anticoagulant activity through dual-target action.

[0038] Optionally, the anticoagulant polysaccharide backbone consists of →4)-α-Fuc p -(1→ Composition: Fucoidose exists in the α configuration as a long chain linked by glycosidic bonds, with the C1 position of each sugar ring connected to the C4 position of the next sugar ring; branching structures exist at the C2 position; the branching structures include β-Gal p 2S-(1→) is a β-configuration galactose with a sulfation substitution at C2; the branched structure also includes β-Xyl p -(1→6)-α-Glc p -(1→6)-α-Glc p -(1→6)-α-Glc p -(1→) is composed of three glucose molecules linked by (1→6) bonds, with xylose in the β configuration at the end of the entire branch.

[0039] Optionally, the main chain residues of the anticoagulant polysaccharide are in O The -3 position has a sulfation substitution, and some branch structures also have sulfation substitutions.

[0040] Optional, the total sugar content is 70-75%; the remainder includes sulfate groups and residual protein.

[0041] Optionally, the glycosidic bond linkage includes: T-Xyl p T-Gal p 1, 2, 4-Fuc p and 1,6-Glc p That is, terminal xylose, terminal galactose, 1,2,4-linked fucose and 1,6-linked glucose.

[0042] Optionally, the Sargassum polysaccharide comprises a plurality of structural units repeatedly linked together as shown in the following formula: .

[0043] One or more embodiments of the present invention provide a method for preparing the above-mentioned Sargassum fusiforme polysaccharide, comprising the steps of: S1. The raw material of Sargassum fusiforme is extracted by boiling water reflux to obtain an extract. The extract is decolorized by macroporous resin and then precipitated with alcohol to obtain a precipitate. The precipitate is redissolved in water and dialyzed to obtain a truncation. Protease is added to the truncation for enzymatic hydrolysis, and the protein is removed to obtain Sargassum fusiforme crude polysaccharide. S2. Dissolve the crude polysaccharide of Sargassum fusiforme in water, and then separate it sequentially using anion exchange chromatography and gel permeation chromatography to obtain the Sargassum fusiforme polysaccharide.

[0044] The above method combines water extraction and alcohol precipitation, ion exchange chromatography, and gel permeation chromatography to obtain Sargassum polysaccharide with high anticoagulant activity.

[0045] Optionally, in S1, the boiling water reflux extraction method includes: extracting for 1 to 3 hours at a material-to-liquid ratio of 1:(7~12) by mass-to-volume ratio; and using high temperature to fully dissolve and release the polysaccharide into the water to improve the polysaccharide yield.

[0046] Optionally, in S1, the macroporous resin decolorization treatment method includes: using one or more macroporous resins selected from AB-8, D101, HPD100, HPD450 and HP-20, with a macroporous resin to extract volume ratio of 1:(0.8~1.2), adsorbing at 40~60℃ for 3~5 hours; the macroporous resin is preferably AB-8; selectively removing pigment impurity molecules in the extract to improve polysaccharide purity.

[0047] Optionally, in S1, the alcohol precipitation method includes: mixing ethanol and extract in a volume ratio of (3~5):1, and letting it stand at 2~10℃ for 8~16 hours; taking advantage of the characteristic that polysaccharides have reduced solubility in ethanol, the polysaccharides are precipitated from the solution to achieve preliminary separation and enrichment.

[0048] Optionally, in S1, the dialysis method includes: dialysis using a dialysis bag with a molecular weight cutoff of 2-5 kDa; further removal of small molecule impurities to achieve preliminary purification of the polysaccharide.

[0049] Optionally, in S1, the protease is papain, and the enzymatic hydrolysis method includes: enzymatic hydrolysis for 2-4 hours at 40-60°C and pH 5.0-7.0; and degradation of proteins in the extract.

[0050] Optionally, in S1, the deproteinization method includes: using the Sevag method for deproteinization; specifically: chloroform and n-butanol are mixed at a volume ratio of (4~6):1 to obtain the Sevag reagent, the Sevag reagent is mixed with the enzymatic hydrolysate at a volume ratio of 1:(3~5), stirred, centrifuged and the aqueous phase is collected, and repeated 2~5 times; the protein is denatured and a gel layer is formed at the interface between the organic phase and the aqueous phase, thereby removing the denatured protein.

[0051] Optionally, in the anion exchange chromatography method in S2, the anion exchange chromatography column uses DEAE-52 packing material, and eluents are sequentially used as ultrapure water, various NaCl solutions of 0.1 M to 0.4 M, and 0.1 M NaOH solution, with each eluent volume being 150 to 250 mL. The eluent in the volume range of 450 to 750 mL is collected. Preferably, the starting point of the collection range is 450 to 550 mL, and the ending point of the collection range is 650 to 750 mL. Based on the difference in negative charge (sulfate groups) carried by polysaccharides, the separation of polysaccharides is achieved by gradient elution with different concentrations of NaCl solution. Preferably, the NaCl solution includes NaCl solutions with concentrations of 0.1 M, 0.2 M, 0.3 M, and 0.4 M.

[0052] Optionally, in S2, after separation by anion exchange chromatography column, the solution is rotary evaporated to a set volume and transferred to a 2.5~3.5 kDa dialysis bag for desalting.

[0053] Optionally, in S2, in the gel permeation chromatography column separation method, the gel permeation chromatography column packing material is Sephadex G100, ultrapure water is used as the eluent for elution, and the eluent volume from the 40th to the 86th mL is collected; preferably, the starting point of the collection interval is the 40th to the 52nd mL, and the ending point of the collection interval is the 74th to the 86th mL; based on the difference in the molecular weight of polysaccharides, separation is performed through the molecular sieve effect to obtain SF4B polysaccharides with uniform molecular weight.

[0054] One or more embodiments of the present invention provide an anticoagulant product comprising the above-mentioned Sargassum fusiforme polysaccharide.

[0055] The aforementioned Sargassum fusiforme polysaccharide exhibits dual-target anticoagulant activity. Its direct inhibitory effect on coagulation factor IIa has an IC50 value. 50 The value was 11.09 μg / mL. Under antithrombin III-mediated regulation, its IC50 value was... 50 The value further decreased to 7.89 μg / mL. The Sargassum fusiforme anticoagulant polysaccharide SF4B can bind to coagulation factor IIa and antithrombin III molecules. K d The values ​​are 6.10 × 10 –9 M and 5.75×10 –10 M.

[0056] Optionally, the pharmaceutical composition may include pharmaceutically acceptable excipients.

[0057] Optionally, the oral dosage form of the pharmaceutical composition includes capsules, lozenges, oral liquids, tablets, granules, or pills. One or more embodiments of the present invention provide the application of the above-mentioned Sargassum polysaccharide in at least one of the following (a1) to (a3); (a1) To prepare products for the prevention and / or treatment of thromboembolic diseases; (a2) Prepare products for the prevention and / or treatment of venous thromboembolic diseases.

[0058] (a3) Preparation of anticoagulant products.

[0059] The present invention will be further described below with reference to specific embodiments.

[0060] Example 1 A polysaccharide from Sargassum fusiforme, named Sargassum fusiforme anticoagulant polysaccharide SF4B, abbreviated as SF4B; the preparation method includes the following steps: S1. After pulverizing dried Sargassum fusiforme, add pure water at a material-to-liquid ratio of 1:10 (g:mL) and extract by boiling water reflux for 2 hours. After extraction, separate the solid and liquid components and collect the extract. Rotate the extract to an appropriate volume and add AB-8 macroporous resin at a macroporous resin-to-extract volume ratio of 1:1. Adsorb and decolorize in a 50℃ water bath for 4 hours. After decolorization, add 95% ethanol to the decolorized filtrate at an ethanol-to-extract volume ratio of 4:1 (mL:mL) and stir. Let stand at 4℃ for 12 hours and collect the precipitate. Evaporate the ethanol from the precipitate, redissolve it with ultrapure water, and transfer it to a 3.5 kDa dialysis bag for dialysis. After complete dialysis, collect the retentate in the bag and dialyze it at 50℃ and pH. At 6.0°C, papain was added and hydrolyzed for 3 hours. After hydrolysis, Sevag reagent (chloroform: n-butanol = 5:1, mL:mL) was added and stirred, with the volume ratio of Sevag reagent to hydrolysate being 1:4 (mL:mL). The hydrolysate was centrifuged and collected after deproteinization. The deproteinization process was repeated 3 times. The deproteinized extract was collected, rotary evaporated to an appropriate volume, and then freeze-dried to obtain Sargassum fusiforme crude polysaccharide sample.

[0061] S2. Dissolve the crude polysaccharide sample of Sargassum fusiforme in ultrapure water and load it onto a DEAE-52 packed anion exchange chromatography column for separation. Elute sequentially with ultrapure water, 0.1 M, 0.2 M, 0.3 M, 0.4 M NaCl solution and 0.1 M NaOH solution, with each concentration gradient having a volume of 200 mL. Collect the eluted fractions according to the peaks and combine them. After rotary evaporation to the set volume, transfer them to a 3.5 kDa dialysis bag for desalting to obtain the Sargassum fusiforme polysaccharide fraction.

[0062] S3. Then, the Sargassum fusiforme polysaccharide fraction was loaded onto a Sephadex G100 packed gel permeation chromatography column for separation and elution with ultrapure water. The eluted fractions were collected and combined according to the peak conditions, rotary evaporated to the set volume, and then freeze-dried to obtain Sargassum fusiforme anticoagulant active polysaccharide SF4B.

[0063] Among them, the elution curve of the anion exchange chromatography column is as follows: Figure 1 As shown, seven elution peaks were observed. The Sargassum polysaccharide components represented by these peaks were named Sargassum polysaccharide components SF1 to SF7 in order of elution. The Sargassum polysaccharide component obtained from the eluent in the range of 500 mL to 695 mL was named polysaccharide component SF4. The elution curve of the gel permeation chromatography column is shown below. Figure 2 As shown, two elution peaks were observed. The eluent from the 45 mL to the 80 mL interval was collected to obtain Sargassum fusiforme anticoagulant polysaccharide SF4B.

[0064] The obtained SF4B was tested and structurally characterized, including the following:

[0065] (1) The total sugar content was determined by the phenol-sulfuric acid method: 1 mL of the test solution was taken into a stoppered test tube, 1 mL of 5% phenol solution was added, and after mixing, 5 mL of concentrated sulfuric acid was added. The mixture was boiled in a water bath for 30 minutes, then removed and cooled to room temperature in an ice bath. The absorbance was measured at 490 nm. A standard curve was plotted using fucose as a reference.

[0066] Calculations show that the total sugar content of Sargassum fusiforme anticoagulant polysaccharide SF4B is 73.38%, which is a percentage by mass.

[0067] (2) Determination of sulfate content: The sulfate content was determined by ion chromatography. The sample concentration was prepared as 10 mg / mL and hydrolyzed with 3 M trifluoroacetic acid at 105 °C for 8 hours. After hydrolysis, the trifluoroacetic acid was removed by vacuum drying and then dissolved in ultrapure water. The sample was filtered through a 0.22 μm microporous membrane and analyzed by ion chromatography. The chromatographic columns used were IonPac AS11-HC (4 × 50 mm, 4 μm) and IonPac AS11-HC (4 × 250 mm, 4 μm). The injection volume was 25 μL, the flow rate was 1 mL / min, the mobile phase was KOH solution, and the elution conditions were 0-5 min, 5 mM; 5-20 min, 5-30 mM; 20-23 min, 30 mM; 23-31 min, 30-5 mM.

[0068] Calculations show that the sulfate group content of Sargassum fusiforme anticoagulant polysaccharide SF4B is 17.69%, which is a percentage by mass.

[0069] (3) Determination of molecular weight distribution: The molecular weight was determined by gel permeation chromatography. The test sample was dissolved in ultrapure water and centrifuged at 13,000 rpm for 10 minutes. The supernatant was collected and injected into HPLC-ELSD for analysis. The chromatographic column used was a TSKgelG5000PWXL column (7.8 × 300 mm, 10 μm). The mobile phase was ultrapure water. The injection volume was 20 μL, the flow rate was 1 mL / min, the drift tube temperature was 90℃, the nebulizer temperature was 80℃, and the carrier gas flow rate was 1.5 L / min.

[0070] Test results are as follows Figure 3 As shown, the molecular weight of Sargassum fusiforme anticoagulant polysaccharide SF4B is 6.37 × 10⁻⁶. 6 Da.

[0071] (4) Determination of monosaccharide composition: The monosaccharide composition was determined by PMP pre-column derivatization high-performance liquid chromatography. The sample concentration was prepared at 10 mg / mL and hydrolyzed with 3 M trifluoroacetic acid at 105℃ for 2 hours. After hydrolysis, the trifluoroacetic acid was removed by vacuum drying and dissolved in ultrapure water. 200 μL of the hydrolyzed sample was mixed with 200 μL of 0.5 M PMP methanol solution and 200 μL of 0.3 M NaOH solution and incubated in a water bath at 70℃ for 60 minutes. After cooling to room temperature, 200 μL of 0.3 M HCl solution was added for neutralization. The derivative was extracted with chloroform, repeated three times. The aqueous phase was collected, centrifuged at 13000 rpm for 10 minutes, and the supernatant was collected for HPLC analysis. The chromatographic column used was a Kromasil 100-5-C18 column (4.6 × 250 mm, 5 μm), and the mobile phase was 0.1 M phosphate buffer (pH 5.5). 6.80): pure acetonitrile = 83:17 (mL:mL), injection volume was 10 μL, flow rate was 1 mL / min, temperature was 32℃, and detection wavelength was 249 nm.

[0072] Test results as follows Figure 4 As shown, the anticoagulant polysaccharide SF4B from Sargassum fusiforme is composed of glucose, galactose, xylose, and fucose in a molar ratio of 4.3:1.0:1.1:2.7.

[0073] (5) Infrared spectroscopy analysis: Weigh an appropriate amount of dried test sample and grind it with potassium chloride at a mass ratio of 1:100 (g:g), then compress it into tablets; analyze it using an infrared spectrometer with a wavenumber of 4000~400 cm⁻¹. –1 The resolution is 4 cm. –1 The number of scans was 32.

[0074] The infrared spectrum of Sargassum fusiforme anticoagulant polysaccharide SF4B is shown below. Figure 5 As shown. 3440 cm-1 The broad peak is attributed to O-H stretching vibration; 2926 cm -1 and 1417 cm -1 The absorption peak at 1630 cm⁻¹ corresponds to the C-H stretching and bending vibrations; -1 The absorption peak at 1250 cm⁻¹ originates from the bending (shear) vibration of the water of crystallization. -1 The absorption peak at 1038 cm⁻¹ indicates the S=O stretching vibration; while the peak at 1038 cm⁻¹ indicates the S=O stretching vibration. -1 The absorption peak at that point confirms the existence of the pyranose ring structure.

[0075] (6) Methylation analysis: Take 20 mg of polysaccharide sample, treat it with 732 cation exchange resin, neutralize the eluent with pyridine, transfer it to a 3.5 kDa dialysis bag for dialyzing, and freeze-dry to obtain polysaccharide pyridine salt. Take 10 mg of polysaccharide pyridine salt, dissolve it in 1.5 mL of anhydrous DMSO and 0.25 mL of pyridine, and add 13.5 mg of benzotetracarboxylic acid, 12.5 mg of antimony trioxide and 0.25 mL of pyridine in sequence; stir magnetically at 120℃ for 3 hours under nitrogen protection. Add 1 mL of 3% sodium bicarbonate to terminate the reaction. Dialyze the reaction solution thoroughly through a 3.5 kDa dialysis bag, freeze-dry, and obtain desulfurized polysaccharide sample. Take 4 mg of dried desulfurized polysaccharide sample, dissolve it in 4 mL of anhydrous DMSO, and sonicate it for 8 hours under nitrogen protection until it is completely dissolved; add 20 mg of ground and dried NaOH powder, and sonicate it for 4 hours under nitrogen protection. Under light-protected, magnetically stirred conditions and at 20°C, 1 mL of iodomethane was slowly added dropwise to the reaction system, and the reaction was carried out for 1 hour. The above methylation process was repeated once. After the reaction was completed, 2 mL of ultrapure water was added to quench the reaction. The reaction solution was extracted five times with 4 mL of dichloromethane, and the dichloromethane phases were combined and washed five times with 4 mL of ultrapure water. The obtained organic phase was dehydrated with 5 g of Na2SO4 for 24 hours, filtered, and rotary evaporated to dryness to obtain the methylated polysaccharide sample. Take the fully methylated sample, add 2 mL of 3 M trifluoroacetic acid and hydrolyze at 105 °C for 4 hours; after hydrolysis, dry under reduced pressure to remove trifluoroacetic acid; add 20% ammonia solution to the hydrolysis product to adjust the pH to alkaline, then add 0.5 mL of 20 mg / mL sodium borodeuteride solution for reduction, and react overnight at room temperature; slowly add 20% glacial acetic acid until the solution is weakly acidic, and dry under reduced pressure; add 0.5 mL of anhydrous pyridine and 0.5 mL of acetic anhydride to the reduction product, vortex mix, react at 105 °C for 2 hours, cool, and add 1 mL of ultrapure water to quench the reaction; extract with dichloromethane, collect the organic phase, wash three times with ultrapure water, collect the dichloromethane phase, and use it for GC-MS analysis. The chromatographic column used was an HP-5MS column (30 m × 250 μm × 0.25 μm), the carrier gas was helium, the injection volume was 1 μL, the flow rate was 1 mL / min, the injection port temperature was 280℃, and the detector temperature was 280℃. The temperature program was as follows: initial temperature 80℃, hold for 3 minutes, increase to 200℃ at a rate of 3℃ / min, hold for 5 minutes, then increase to 240℃ at a rate of 5℃ / min and hold for 5 minutes.

[0076] Based on GC-MS analysis of partially methylated sugar alcohol acetylation products, combined with relevant mass spectrometry databases and relative retention times of chromatographic peaks, four major glycosidic bond linkages were identified in desulfurized SF4B. As shown in Table 1, the four linkages correspond to the four major monosaccharides detected in the monosaccharide composition analysis.

[0077] Table 1 shows the main glycosidic bond linkages in desulfurized SF4B.

[0078] (7) Nuclear Magnetic Resonance Analysis: Take 30 mg of polysaccharide sample, dissolve it in 1 mL of deuterium water, freeze-dry, re-dissolve in deuterium water and freeze-dry again, repeat twice, and finally dissolve the sample in 600 μL of deuterium water. Perform nuclear magnetic resonance testing at 50℃. 1 32 H NMR scans were performed. 13 8192 C NMR scans, 4096 DEPT135 scans, 32 COSY scans, 32 HSQC scans, 128 HMBC scans, and 48 NOESY scans.

[0079] One-dimensional nuclear magnetic resonance (NMR) spectra provide information on the chemical shifts of each carbon and hydrogen atom. 1 H NMR spectrum as shown Figure 6 As shown, 13 The C NMR spectrum is as follows Figure 7 As shown, the DEPT135 spectrum is as follows: Figure 8 As shown. Two-dimensional nuclear magnetic resonance (NMR) spectra further reveal the correlation coupling signals between carbon and hydrogen, and between hydrogen atoms; the HSQC spectra are shown below. Figure 9 As shown, the HMBC spectrum is as follows Figure 10 As shown, the COSY spectrum is as follows: Figure 11 As shown, the NOESY spectrum is as follows: Figure 12 As shown in the figure. The violet color in the figure indicates the origin of β-Gal. p 2 S -(1→(residue A) signal; orange indicates origin from β-Xyl p -(1→(residue B) signal; dark purple indicates origin from →2,4)-α-Fuc p -(1→(residue C) signal; magenta indicates origin from →6)-α-Glc p -(1→(residue D) signal; beige indicates origin from →2,4)-α- Fuc p 3 S -(1→(residue E) signal. Through systematic analysis and cross-validation of NMR signals, the assignment of each carbon and hydrogen signal in SF4B and the identification of sugar residue types were completed, as shown in Table 2.

[0080] Table 2 shows the NMR signal assignments of each sugar residue in SF4B (unit: ppm).

[0081] (8) Oxidative degradation and hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis: The polysaccharide sample was prepared into a 4 mg / mL solution, and 100 mM H2O2-25 mM ascorbic acid reagent was added. The solution was ultrasonically treated at 50℃ for 1 hour to obtain the oxidative degradation product, which was then analyzed by hydrophilic interaction liquid chromatography-tandem mass spectrometry. The chromatographic column used was a BEH Amide column (2.1 × 100 mm, 1.7 μm). The mobile phase A was 0.35% ammonia solution of 20 mmol / L ammonium formate, and the mobile phase B was pure acetonitrile. The injection volume was 3 μL, the flow rate was 0.3 mL / min, and the temperature was 45℃. The elution conditions were as follows: 0~2 min, 90% B phase; 2~20 min, 90~50% B phase; 20~24 min, 50% B phase; 24~25 min, 50~90% B phase. The mass spectrometry scanning range was... m / z 60~1500.

[0082] After oxidative degradation of SF4B, the degradation products were separated by hydrophilic interaction liquid chromatography and analyzed by tandem mass spectrometry. The results are as follows: Figure 13 As shown, where, Figure 13 A in the middle is m / z Secondary mass spectrum of ion 259.0. Figure 13 B and C are m / z Secondary mass spectrum of ion 389.1 Figure 13 D is m / z Secondary mass spectrum of ion 405.1 Figure 13 E is m / z The secondary mass spectrum of ion 535.1 was obtained; the structure of the degradation products was identified based on the characteristic fragment ions in the secondary mass spectrum. The rich structures of the degradation products provided key information for resolving the sugar residue linkage sequences in SF4B polysaccharide. The specific identification results are summarized in Table 3. Table 3 shows the identification of SF4B degradation products.

[0083] Through comprehensive analysis and systematic structural characterization, the preferred chemical structure of Sargassum fusiforme anticoagulant polysaccharide SF4B described in this invention was determined, comprising multiple structural units of formula (Ⅰ) that are repeatedly linked: (I).

[0084] The Sargassum fusiforme anticoagulant polysaccharide SF4B described in this invention has a novel chemical structure, characterized by its main chain consisting of only (1→4) fucose residues. O -3 sulfation site and unique branching structure.

[0085] Example 2 This embodiment provides an evaluation of the anticoagulant activity of Sargassum fusiforme crude polysaccharide sample prepared in step S1 of Example 1 as the experimental raw material, including the following steps: Healthy adult male SD rats, 8 weeks old and weighing 250–300 g, were selected as experimental subjects and housed in an SPF-grade environment. Their circadian rhythm was controlled at 12 / 12 hours, the room temperature was maintained at 22±1℃, and they had free access to food and water. Prior to the experiment, they underwent an acclimatization period of at least one week. Forty-four rats were randomly divided into six groups: a blank control group (n=7), a heparin sodium positive control group (n=8, 5 mg / kg body weight), an argatroban positive control group (n=8, 0.5 mg / kg body weight), a low-dose group of Sargassum fusiforme crude polysaccharide (L, n=7, 0.1 g / kg body weight), a medium-dose group of Sargassum fusiforme crude polysaccharide (M, n=7, 0.4 g / kg body weight), and a high-dose group of Sargassum fusiforme crude polysaccharide (H, n=7, 1 g / kg body weight).

[0086] Rats in the blank control and positive control groups were administered physiological saline by gavage daily, while rats in each dose group of Sargassum fusiforme crude polysaccharide were administered the corresponding dose of Sargassum fusiforme crude polysaccharide solution by gavage for 7 consecutive days. Blood samples were collected from each dose group of Sargassum fusiforme crude polysaccharide 2 hours after the last gavage administration. Blood samples were collected from the positive control groups of heparin sodium and argatroban 0.5 hours after the corresponding drugs were injected into the tail vein. Blood was collected from the retro-orbital venous plexus. The collected blood samples were mixed with 3.8% sodium citrate anticoagulant at a ratio of 9:1 (v:v) and then centrifuged at 4°C and 3000 rpm for 15 minutes to obtain anemic platelet plasma.

[0087] Platelet-rich plasma from anemic individuals was incubated at 37°C for 3 minutes, followed by the addition of activated partial thromboplastin time (APTT) reagent and incubation for another 3 minutes. Then, 0.025M calcium chloride solution pre-warmed to 37°C was added, and timing was immediately started to record the time required for clot formation, i.e., the APTT time. Plasma was incubated at 37°C for 3 minutes, followed by the addition of thrombin time (TT) reagent or prothrombin time (PT) reagent pre-warmed to 37°C, and timing was immediately started to record the clot formation time, i.e., the TT time or PT time. Experimental data were presented as the ratio of clot formation time in each group to the blank control group.

[0088] The results are as follows Figure 14 As shown, where, Figure 14 In the graph, A represents the thrombin time. Figure 14 B in the graph represents the activated partial thromboplastin time. Figure 14 C in the graph represents the prothrombin time. Figure 14In the diagram, D represents the bleeding risk, indicating the bleeding time after tail amputation. Compared with the blank control group, both heparin sodium and argatroban treatment groups significantly prolonged APTT, TT, and PT. Orally administered Sargassum fusiforme crude polysaccharide exhibited significant in vivo anticoagulant activity, significantly prolonging TT and APTT in a dose-dependent manner. At all experimental doses, Sargassum fusiforme crude polysaccharide did not significantly prolong tail bleeding time, indicating its good oral safety. Therefore, it is possible to screen and identify active polysaccharides from Sargassum fusiforme crude polysaccharide.

[0089] Example 3 This embodiment uses Sargassum fusiforme polysaccharide fractions obtained after anion exchange chromatography separation in step S2 of Example 1 as experimental materials, including Sargassum fusiforme polysaccharide fractions SF1, SF2, SF3, SF4, SF5, SF6, and SF7, to evaluate their anticoagulant activity. Following the method in Example 2, the plasma reagent was mixed with any one of the seven Sargassum polysaccharide components, and the in vitro coagulation time was evaluated. The results are as follows: Figure 15 As shown, where, Figure 15 In the graph, A represents the thrombin time. Figure 15 B in the graph represents the activated partial thromboplastin time. Figure 15 C in the figure represents the prothrombin time curve. The experimental results show that Sargassum polysaccharide components SF4~SF7 exhibited significant dose-dependent prolongation effects on both TT and APTT, while Sargassum polysaccharide components SF1~SF3 did not have a significant effect on TT, APTT, and PT at all tested concentrations.

[0090] Example 4 This embodiment uses Sargassum fusiforme polysaccharide fractions obtained after anion exchange chromatography separation in step S2 of Example 1 as experimental materials, including Sargassum fusiforme polysaccharide fractions SF1, SF2, SF3, SF4, SF5, SF6, and SF7, to evaluate their inhibitory effect on coagulation factor IIa. Factor IIa and antithrombin III were dissolved and diluted separately with PBS buffer. In a 96-well plate, 25 μL of sample solution (any one of Sargassum polysaccharide fractions SF1 to SF7) and 25 μL of factor IIa solution (120 U / mL) were mixed. After incubation at 37°C for 30 minutes, 50 μL of S-2238 substrate solution (1 mg / mL) was added to each well, and incubation continued at 37°C for 10 minutes. Immediately after the reaction was complete, the absorbance of each well was measured at 405 nm, and the direct inhibition rate of factor IIa was calculated.

[0091] Mix 25 μL of sample solution (any one of Sargassum polysaccharide fractions SF1 to SF7), 25 μL of coagulation factor IIa solution (120 U / mL), and 25 μL of antithrombin III solution (10 μg / mL). After incubating at 37°C for 30 minutes, add 50 μL of S-2238 substrate solution (1 mg / mL) to each well and continue incubating at 37°C for 10 minutes. Immediately after the reaction is complete, measure the absorbance of each well at 405 nm and calculate the antithrombin III-mediated inhibition rate of coagulation factor IIa.

[0092] The results are as follows Figure 16 As shown, where, Figure 16 In the figure, A represents the concentration curve of the direct inhibition rate of coagulation factor IIa. Figure 16 B in the figure represents the concentration curve of factor IIa inhibition rate mediated by antithrombin III; indicating that Sargassum polysaccharide components SF4 to SF7 all exhibit factor IIa inhibitory activity: they can directly inhibit factor IIa and further inhibit factor IIa activity under the mediation of antithrombin III, and both pathways show a dose-dependent relationship.

[0093] Example 5 This embodiment uses Sargassum fusiforme anticoagulant polysaccharide SF4B obtained after separation by gel permeation chromatography in step S3 of Example 1 as the experimental material to evaluate its inhibitory effect on coagulation factor IIa, and calculate the direct inhibition rate of coagulation factor IIa and the inhibition rate of coagulation factor IIa mediated by antithrombin III.

[0094] The results are as follows Figure 17 As shown, where, Figure 17 In the figure, A represents the concentration curve of the direct inhibition rate of coagulation factor IIa. Figure 17 Figure B in the graph represents the concentration-inhibition curve of factor IIa mediated by antithrombin III; this indicates that SF4B can inhibit factor IIa directly or by activating antithrombin III, thereby inhibiting factor IIa activity, and both mechanisms of action exhibit a dose-dependent effect. The IC50 of SF4B for direct inhibition of factor IIa was calculated. 50 The value was 11.09 μg / mL, while its IC50 value was lower in the presence of antithrombin III. 50 The value further decreased to 7.89 μg / mL.

[0095] Example 6 This embodiment provides the molecular binding effect of SF4B with coagulation factor IIa and antithrombin III. The molecular interaction between SF4B and the two target proteins, coagulation factor IIa and antithrombin III, was studied using isothermal titration calorimetry. All solutions were diluted with PBS buffer. Polysaccharide sample SF4B (1.9 μM or 2.8 μM) was loaded into a syringe and titrated into a sample cell containing coagulation factor IIa (27.8 μM) or antithrombin III (8.8 μM). The titration program was set as follows: an initial injection of 0.4 μL over 0.8 seconds, followed by 18 injections of 2 μL each over 4 seconds, with a 100-second interval between each injection. The experiment was conducted at a constant temperature of 25°C. The binding constant and thermodynamic parameters between the two were calculated by recording and fitting binding isotherms.

[0096] The results are as follows Figure 18 As shown, Figure 18 In the diagram, A represents the isothermal titration calorimetry curves of the interaction with coagulation factor IIa, including the exothermic curve in the upper figure and the isothermal titration calorimetry curves of the interaction with antithrombin III in the lower figure. Figure 18 Figure B shows the isothermal titration calorimetry curves of the interaction between SF4B and antithrombin III, including the exothermic curve in the upper figure and the binding curve fitting curve in the lower figure. During the titration of SF4B to coagulation factor IIa and antithrombin III, obvious exothermic peaks were observed, and the heat of exothermic reaction decreased with the titration process, indicating gradual saturation of the binding sites. Calculations showed that SF4B binds to coagulation factor IIa... K d The value is 6.10 × 10 –9 M ( H =–730kJ / mol G = -47.0 kJ / mol, T S = -684 kJ / mol), SF4B binds to antithrombin III K d The value is 5.75 × 10 –10 M ( H =–1190kJ / mol G = -52.8 kJ / mol, T S = –1140 kJ / mol).

[0097] In summary, this invention has prepared a novel polysaccharide SF4B from Sargassum fusiforme. SF4B can directly inhibit coagulation factor IIa and further inhibit the activity of coagulation factor IIa by activating antithrombin III. Furthermore, SF4B exhibits affinity for both coagulation factor IIa and antithrombin III, demonstrating dual-target anticoagulant activity and possessing the potential and value for further development into an anticoagulant drug.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A Sargassum fusiforme polysaccharide, characterized in that, The relative molecular mass is (6.0~6.5) × 10⁻⁶. 6 Da is composed of glucose, galactose, xylose and fucose in a molar ratio of (3.8~4.8):(0.7~1.3):(0.8~1.4):(2.3~3.1), and has a sulfate content of 15~20%.

2. The Sargassum polysaccharide as described in claim 1, characterized in that, The main chain consists of →4)-α-Fuc p -(1→Composition, branching structure includes β-Gal p 2S-(1→ and β-Xyl) p -(1→6)-α-Glc p -(1→6)-α-Glc p -(1→6)-α-Glc p -(1→).

3. The Sargassum polysaccharide as described in claim 1, characterized in that, The total sugar content is 70-75%.

4. The Sargassum polysaccharide as described in claim 1, characterized in that, Glycosidic bond linkage modes include: T-Xyl p T-Gal p 1, 2, 4-Fuc p and 1,6-Glc p ; Alternatively, the Sargassum polysaccharide comprises repeatedly linked structural units as shown in the following formula: 。 5. A method for preparing Sargassum polysaccharide as described in any one of claims 1 to 4, characterized in that, Including the following steps: Sargassum fusiforme raw material was extracted by boiling water reflux to obtain an extract. The extract was decolorized by macroporous resin and then precipitated with alcohol to obtain a precipitate. The precipitate was redissolved in water and dialyzed to obtain a truncation. Protease was added to the truncation for enzymatic hydrolysis, and the protein was removed to obtain Sargassum fusiforme crude polysaccharide. The crude polysaccharide of Sargassum fusiforme was dissolved in water and then separated sequentially by anion exchange chromatography and gel permeation chromatography to obtain the Sargassum fusiforme polysaccharide.

6. The method for preparing Sargassum polysaccharide as described in claim 5, characterized in that, The boiling water reflux extraction method includes: extracting for 1 to 3 hours at a material-to-liquid ratio of 1:(7~12) by mass-to-volume ratio; Alternatively, the macroporous resin decolorization treatment method includes: using one or more macroporous resins selected from AB-8, D101, HPD100, HPD450 and HP-20, with a macroporous resin to extract volume ratio of 1:(0.8~1.2), adsorbing at 40~60℃ for 3~5 hours; the preferred macroporous resin is AB-8; Alternatively, the alcohol precipitation method includes mixing ethanol and extract in a volume ratio of (3~5):1 and letting it stand at 2~10℃ for 8~16 hours.

7. The method for preparing Sargassum polysaccharide as described in claim 5, characterized in that... Dialysis methods include: using dialysis bags with a molecular weight cutoff of 2-5 kDa for dialysis; Alternatively, the protease is papain, and the enzymatic hydrolysis method includes: enzymatic hydrolysis for 2-4 hours at 40-60°C and pH 5.0-7.0; Alternatively, in S1, the deproteinization method includes: using the Sevag method for deproteinization; specifically: chloroform and n-butanol are mixed at a volume ratio of (4~6):1 to obtain the Sevag reagent, and the Sevag reagent is mixed with the enzymatic hydrolysate at a volume ratio of 1:(3~5) and stirred; centrifuged and the aqueous phase is collected, and repeated 2~5 times.

8. The method for preparing Sargassum polysaccharide as described in claim 5, characterized in that, In the anion exchange chromatography column separation method, the anion exchange chromatography column uses DEAE-52 packing material. Elution is performed sequentially with ultrapure water, various NaCl solutions (0.1-0.4 M), and 0.1 M NaOH solution, with each eluent volume being 150-250 mL. The eluent from volume 450 to 750 mL is collected. Preferably, the starting point of the collection interval is 450-550 mL, and the ending point is 650-750 mL. Preferably, the NaCl solution includes NaCl solutions with concentrations of 0.1 M, 0.2 M, 0.3 M, and 0.4 M. Alternatively, in the gel permeation chromatography column separation method, the gel permeation chromatography column packing material is Sephadex G100, ultrapure water is used as the eluent for elution, and the eluent in the volume range of 40 to 86 mL is collected; preferably, the starting point of the collection range is 40 to 52 mL, and the ending point of the collection range is 74 to 86 mL.

9. An anticoagulant product comprising Sargassum polysaccharide as described in any one of claims 1 to 4.

10. The use of Sargassum polysaccharide as described in any one of claims 1 to 4 in at least one of the following (a1) to (a3); (a1) To prepare products for the prevention and / or treatment of thromboembolic diseases; (a2) Prepare products for the prevention and / or treatment of venous thromboembolic diseases. (a3) Preparation of anticoagulant products.

Images