A method for preparing brown algae polysaccharide sulfate oligomers

Abstract

This invention discloses a method for preparing alginate sulfate oligomers. By strictly controlling the addition amounts of TEMPO, sodium bromide, and sodium hypochlorite under specific conditions, highly efficient degradation of alginate sulfate is achieved. Specifically, the mass ratio of TEMPO to alginate sulfate is 0.5-5%, the mass ratio of sodium bromide to alginate sulfate is 5-40%, and the mass ratio of alginate sulfate to sodium hypochlorite is 1 g:0.1-10 mmol / L. No strong acid or enzyme preparations are required, and no microwave or ultrasonic auxiliary methods are relied upon. Oligomers with an average molecular weight below 80 kDa can be obtained within 1 hour, and oligomers with an average molecular weight of approximately 20 kDa can be obtained within 4 hours, while maintaining a sulfate content of approximately 25%. The method causes almost no damage to the functional groups of alginate polysaccharides, and the prepared oligomers exhibit significant effects in in vitro anticoagulant activity.

Description

Technical Field

[0001] This invention belongs to the field of deep processing of seaweed polysaccharides, and particularly relates to a method for preparing brown algae polysaccharide sulfate oligomers. Background Technology

[0002] Fucoidan sulfate is a water-soluble sulfated heteropolysaccharide that has attracted widespread attention in the food and pharmaceutical fields due to its various biological activities. However, fucoidan sulfate naturally extracted from the cell wall matrix of brown algae typically suffers from drawbacks such as high molecular weight, poor solubility, and high viscosity, limiting its practical applications. In contrast, fucoidan sulfate oligomers, due to their low viscosity, good bioabsorption, and strong bioactivity, are gradually becoming a research hotspot. Currently, the main methods for preparing fucoidan sulfate oligomers include acid hydrolysis, enzymatic degradation, photodegradation, and combined ultrasonic-microwave degradation.

[0003] Chinese invention patent application CN118515790A discloses "a method for preparing low molecular weight fucoidan and its application," specifically involving treating fucoidan with different types of acids (0.1 M hydrochloric acid, 0.15 M acetic acid, and 0.5 M citric acid) at 85-100℃ for 15-20 minutes to obtain products with a molecular weight below 5 kDa and a yield exceeding 59%. However, the strong acids used in this method place high demands on the corrosion resistance of industrial production equipment, and the high-temperature treatment may damage the sulfate groups in the fucoidan.

[0004] Chinese invention patent CN118638882A discloses "a method for preparing and applying low molecular weight fucoidan degradation products." This method combines cellulase and a complex plant hydrolase (total enzyme concentration of 0.8 g / L, enzymatic hydrolysis time of 2 h) with an acid hydrolysis process. Although the reaction conditions are relatively mild, the degradation efficiency is low and the production and recovery costs of the enzyme preparation are high. Furthermore, the enzyme's insufficient stability poses a challenge for its application in industrial production, and currently, there are no suitable commercially available enzyme preparations.

[0005] Chinese invention patent application CN118420795A discloses "a fucoidan with antibacterial activity and its preparation method and application." The preparation method specifically includes the following steps: dissolving fucoidan in water; mixing with H2O2 solution; subjecting it to radiation degradation under ultraviolet light; dialysis, evaporation concentration, and drying to obtain fucoidan with a molecular weight of 3-70 kDa and a sulfate content of 10-35 wt%. However, photodegradation may produce uneven degradation effects and byproducts, affecting the purity and activity of the target product.

[0006] Chinese invention patent CN107011454B discloses a "method for preparing low molecular weight, highly sulfated sea cucumber fucoidan sulfate." Specifically, it involves using sea cucumber fucoidan sulfate as a raw material, employing an ultrasonic-microwave combined degradation method to break glycosidic bonds and decompose the sea cucumber fucoidan sulfate to obtain low molecular weight sea cucumber fucoidan sulfate. Then, methyl methanesulfonate is used to react with the low molecular weight sea cucumber fucoidan sulfate in a sulfation reaction to obtain low molecular weight, highly sulfated sea cucumber fucoidan sulfate. However, while ultrasonic degradation can accelerate the reaction, excessive energy input can damage key active groups and increase energy consumption. Microwave degradation can improve efficiency, but it requires sophisticated equipment and may cause localized overheating, leading to side reactions. This method suffers from high equipment investment, complex operation, and difficulty in maintaining the bioactivity of fucoidan sulfate, limiting its application in the health field.

[0007] TEMPO (2,2,6,6-tetramethylpiperidine-1-oxo radical) is a stable radical compound widely used in organic chemistry and materials science. Its molecular structure contains a nitrogen atom with an unpaired electron, enabling it to function as a highly efficient oxidant or catalyst in chemical reactions. TEMPO exhibits excellent catalytic performance in selective oxidation reactions, particularly in the oxidation of sugars, alcohols, and other reducing compounds, demonstrating mildness, high selectivity, and lenient reaction conditions. However, to date, there have been no reports on using TEMPO to selectively oxidize specific sugar residue sites in fucoidan sulfate to induce the breaking of glycosidic bonds in the molecular chain, thereby achieving the targeted degradation of polysaccharides and obtaining low molecular weight products. Summary of the Invention

[0008] The present invention aims to solve the above-mentioned technical problems existing in the prior art by providing a method for preparing alginate sulfate oligomers.

[0009] The technical solution of this invention is: a method for preparing alginate sulfate oligomer, which is carried out in the following steps:

[0010] Step 1. Dissolve alginate sulfate in water to prepare solution I;

[0011] Step 2. Add TEMPO and sodium bromide to solution I and stir until homogeneous to obtain solution II;

[0012] Step 3. Add sodium hypochlorite to solution II and stir until homogeneous to obtain solution III;

[0013] Step 4. Add sodium hydroxide dropwise to solution III to adjust the pH to a weakly alkaline state, thus obtaining solution IV;

[0014] Step 5. After adding anhydrous ethanol to solution IV, the reaction is terminated, yielding solution V;

[0015] Step 6. Adjust the pH of solution V to neutral to obtain solution VI;

[0016] Step 7. Solution VI is dialyzed and freeze-dried to obtain alginate sulfate oligomer.

[0017] Preferably, step 1 involves mixing alginate sulfate with water at a mass-to-volume ratio of 1-50 mg: 1 ml and continuously stirring at 30-40°C until homogeneous.

[0018] Preferably, in step 2, the mass ratio of TEMPO to alginate sulfate in solution II is 0.5-5%, and the mass ratio of sodium bromide to alginate sulfate is 5-40%.

[0019] Preferably, step 3 involves adding alginate sulfate in a ratio of 1 g to 0.1-10 mmol / L, and continuously stirring at 30-50°C to obtain solution III.

[0020] Preferably, step 4 involves continuously adding 0.5 mol / mL sodium hydroxide solution to adjust the pH to 8-11, and continuously stirring for 0.5-5 h to obtain solution IV.

[0021] Preferably, the volume ratio of anhydrous ethanol added in step 5 to solution IV is 1-20%.

[0022] Preferably, step 6 involves adjusting the pH of solution V to 7 using a 1 mol / mL sodium hydroxide solution to obtain solution VI.

[0023] The preferred dialysis molecular weight cutoff for step 7 is 300 Da.

[0024] This invention utilizes a TEMPO radical catalytic system to selectively oxidize specific sugar residue sites in alginate sulfate, inducing glycosidic bond breakage in the molecular chain, thereby achieving the directed degradation of the polysaccharide and obtaining a low molecular weight product. Compared with existing technologies, it has the following advantages:

[0025] 1. High selectivity: TEMPO exhibits strong selectivity in the catalytic oxidation of alginate sulfate, oxidizing only specific glycosidic bonds in the polysaccharide without affecting other parts. This overcomes the problem of excessive damage to the polysaccharide structure in traditional degradation methods and effectively protects the active groups from damage.

[0026] 2. Mild reaction conditions: TEMPO can function under relatively mild reaction conditions, without the need for strong acids or enzymes, and without relying on auxiliary means such as microwaves or ultrasound, thus reducing the likelihood of side reactions or excessive oxidation. Oligomers with an average molecular weight below 80 kDa can be obtained within 1 hour, and oligomers with an average molecular weight of approximately 20 kDa can be obtained within 4 hours, while maintaining a sulfate group content of around 25%.

[0027] 3. Reusable: TEMPO exhibits good stability in the reaction and can be recycled multiple times, thereby reducing the preparation cost of alginate sulfate oligomers.

[0028] 4. High anticoagulant activity: The prepared alginate sulfate oligomer has high anticoagulant activity and significantly prolongs the clotting time of APTT. Attached Figure Description

[0029] Figure 1 This is a relative molecular mass distribution diagram of the alginate sulfate oligomers and alginate sulfate obtained in Examples 1-4 of this invention.

[0030] Figure 2 The image shows the infrared spectra of the alginate sulfate oligomer and alginate sulfate obtained in Example 1 of this invention.

[0031] Figure 3 This is a schematic diagram of the anticoagulant activity indicators of the alginate sulfate oligomer and alginate sulfate obtained in Example 1 of the present invention. Detailed Implementation

[0032] The raw material used in this invention embodiment—alglucan sulfate—was purchased from Qingdao Mingyue Algae Group Co., Ltd., and conforms to the industry standard SC / T 3404-2012. Example 1

[0033] Step 1. Dissolve alginate sulfate in water to a concentration of 10 mg / mL, and stir continuously at 35°C to obtain solution I;

[0034] Step 2. Take 100 mL of solution I, add 0.02 g TEMPO and 0.2 g sodium bromide, and stir continuously to obtain solution II;

[0035] Step 3. Add 0.4 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0036] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 4 h to obtain solution IV;

[0037] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0038] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0039] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0040] The raw material, fucoidan sulfate, before degradation, had a relative molecular weight of 336 kDa, a total sugar content of 52.02 ± 1.81%, and a sulfate content of 26.68 ± 0.40%. The fucoidan sulfate oligomer prepared in Example 1 was a white powder with a relative molecular weight of approximately 20 kDa, a total sugar content of 50.88 ± 0.74%, and a sulfate content of 25.09 ± 0.61%. Fourier transform infrared (FTIR) spectra (e.g.) Figure 2 As shown in the figure, compared with the raw material alginate sulfate, the sulfate characteristic peaks of the 20kDa oligomer did not change significantly. Specifically, there was a peak at 1245.32 cm⁻¹ (attributed to S=O stretching vibration), a peak at 851.12 cm⁻¹ (attributed to COS axial coordination stretching vibration), and a peak at 814.41 cm⁻¹ (attributed to COS equatorial coordination stretching vibration), indicating that the sulfate groups were not destroyed during the preparation process.

[0041] The in vitro anticoagulant activity of alginate sulfate oligomers was detected using a commercial kit, and the results are as follows: Figure 3 As shown. The results indicate that, at the same concentration as the raw material alginate sulfate and heparin, the alginate sulfate oligomer of the present invention significantly prolongs the activation time of partial thromboplastin. Therefore, the alginate sulfate oligomer prepared in this invention has certain anticoagulant activity. Example 2

[0042] Step 1. Dissolve alginate sulfate in water to a concentration of 10 mg / mL, and stir continuously at 35°C to obtain solution I;

[0043] Step 2. Take 100 mL of solution I, add 0.04 g TEMPO and 0.4 g sodium bromide, and stir continuously to obtain solution II;

[0044] Step 3. Add 0.4 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0045] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 1 h to obtain solution IV;

[0046] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0047] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0048] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0049] The relative molecular weight of the alginate sulfate oligomer prepared in Example 2 was determined to be approximately 41 kDa, with a total sugar content of 50.32 ± 0.52% and a sulfate group content of 24.56 ± 0.61%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 81.89 ± 2 s. Example 3

[0050] Step 1. Dissolve alginate sulfate in water to a concentration of 10 mg / mL, and stir continuously at 35°C to obtain solution I;

[0051] Step 2. Take 100 mL of solution I, add 0.04 g TEMPO and 0.4 g sodium bromide, and stir continuously to obtain solution II;

[0052] Step 3. Add 0.1 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0053] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 2 h to obtain solution IV;

[0054] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0055] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0056] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery alginate sulfate oligomer.

[0057] The relative molecular weight of the alginate sulfate oligomer prepared in Example 3 was determined to be approximately 61 kDa, with a total sugar content of 50.33 ± 0.22% and a sulfate group content of 24.92 ± 1.82%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 78.24 ± 2 s. Example 4

[0058] Step 1. Dissolve alginate sulfate in water to a concentration of 10 mg / mL, and stir continuously at 35°C to obtain solution I;

[0059] Step 2. Take 100 mL of solution I, add 0.02 g TEMPO and 0.2 g sodium bromide, and stir continuously to obtain solution II;

[0060] Step 3. Add 0.1 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0061] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 1 h to obtain solution IV;

[0062] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0063] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0064] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0065] The relative molecular weight of the alginate sulfate oligomer prepared in Example 4 was determined to be approximately 83 kDa, with a total sugar content of 50.12 ± 2.88% and a sulfate group content of 25.98 ± 0.24%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 75.24 ± 3 s.

[0066] The relative molecular mass distribution of the alginate sulfate oligomers and alginate sulfate prepared in Examples 1-4 are shown in the figure below. Figure 1 As shown. Example 5

[0067] Step 1. Dissolve alginate sulfate in water to a concentration of 5 mg / mL, and stir continuously at 35°C to obtain solution I;

[0068] Step 2. Take 100 mL of solution I, add 0.01 g TEMPO and 0.1 g sodium bromide, and stir continuously to obtain solution II;

[0069] Step 3. Add 0.4 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0070] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 2 h to obtain solution IV;

[0071] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0072] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0073] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0074] The relative molecular weight of the alginate sulfate oligomer prepared in Example 5 was determined to be approximately 26 kDa, the total sugar content of the oligomer was 50.91 ± 0.45%, and the sulfate group content was 25.57 ± 0.52%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 82.14 ± 1 s. Example 6

[0075] Step 1. Dissolve alginate sulfate in water to a concentration of 10 mg / mL, and stir continuously at 35°C to obtain solution I;

[0076] Step 2. Take 100 mL of solution I, add 0.03 g TEMPO and 0.3 g sodium bromide, and stir continuously to obtain solution II;

[0077] Step 3. Add 0.4 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0078] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 3 h to obtain solution IV;

[0079] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0080] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0081] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0082] The relative molecular weight of the alginate sulfate oligomer prepared in Example 6 was determined to be approximately 32 kDa, the total sugar content of the oligomer was 50.68 ± 0.41%, and the sulfate group content was 24.69 ± 0.59%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 80.10 ± 3 s. Example 7

[0083] Step 1. Dissolve alginate sulfate in water to a concentration of 20 mg / mL, and stir continuously at 35°C to obtain solution I;

[0084] Step 2. Take 100 mL of solution I, add 0.08 g TEMPO and 0.8 g sodium bromide, and stir continuously to obtain solution II;

[0085] Step 3. Add 5 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0086] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 0.5 h to obtain solution IV;

[0087] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0088] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0089] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0090] The relative molecular weight of the alginate sulfate oligomer prepared in Example 7 was determined to be approximately 48 kDa, the total sugar content of the oligomer was 50.69 ± 0.56%, and the sulfate group content was 24.55 ± 1.81%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 80.01 ± 2 s. Example 8

[0091] Step 1. Dissolve alginate sulfate in water to a concentration of 40 mg / mL, and stir continuously at 35°C to obtain solution I;

[0092] Step 2. Take 100 mL of solution I, add 0.05 g TEMPO and 0.5 g sodium bromide, and stir continuously to obtain solution II;

[0093] Step 3. Add 4 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0094] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 0.5 h to obtain solution IV;

[0095] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0096] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0097] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0098] The relative molecular weight of the alginate sulfate oligomer prepared in Example 8 was determined to be approximately 53 kDa, the total sugar content of the oligomer was 50.88 ± 0.24%, and the sulfate group content was 24.55 ± 1.88%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 79.89 ± 2 s. Example 9

[0099] Step 1. Dissolve alginate sulfate in water to a concentration of 45 mg / mL, and stir continuously at 35°C to obtain solution I;

[0100] Step 2. Take 100 mL of solution I, add 0.1 g TEMPO and 1 g sodium bromide, and stir continuously to obtain solution II;

[0101] Step 3. Add 3 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0102] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 0.5 h to obtain solution IV;

[0103] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0104] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0105] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0106] The relative molecular weight of the alginate sulfate oligomer prepared in Example 9 was determined to be approximately 68 kDa, with a total sugar content of 51.46 ± 0.45% and a sulfate group content of 24.85 ± 1.83%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 77.56 ± 1 s.

[0107] Example 10:

[0108] Step 1. Dissolve alginate sulfate in water to a concentration of 50 mg / mL, and stir continuously at 35°C to obtain solution I;

[0109] Step 2. Take 100 mL of solution I, add 0.2 g TEMPO and 2 g sodium bromide, and stir continuously to obtain solution II;

[0110] Step 3. Add 1 mmol of sodium hypochlorite to solution II and stir continuously to obtain solution III;

[0111] Step 4. Add 0.5 mol / L sodium hydroxide dropwise to solution III to adjust the pH of the solution to 10±0.5, and stir continuously for 1 h to obtain solution IV;

[0112] Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V;

[0113] Step 6. Adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI;

[0114] Step 7. Solution VI was dialyzed (with a cutoff of 300 Da) and freeze-dried to obtain a white powdery brown alginate sulfate oligomer.

[0115] The relative molecular weight of the alginate sulfate oligomer prepared in Example 10 was determined to be approximately 71 kDa, with a total sugar content of 52.77 ± 0.42% and a sulfate group content of 25.12 ± 1.89%. At a concentration of 200 μg / mL, the activation time of partial thromboplastin was 76.32 ± 3 s.

Claims

1. A method for preparing a brown algae polysaccharide sulfate oligomer, characterized in that... Follow these steps in sequence: Step 1. Dissolve alginate sulfate in water to prepare solution I; Step 2. Add TEMPO and sodium bromide to solution I and stir until homogeneous to obtain solution II; Step 3. Add sodium hypochlorite to solution II and stir until homogeneous to obtain solution III; Step 4. Add sodium hydroxide dropwise to solution III to adjust the pH to a weakly alkaline state, thus obtaining solution IV; Step 5. After adding anhydrous ethanol to solution IV, the reaction is terminated, yielding solution V; Step 6. Adjust the pH of solution V to neutral to obtain solution VI; Step 7. Solution VI is dialyzed and freeze-dried to obtain alginate sulfate oligomer.

2. The method for preparing the alginate sulfate oligomer according to claim 1, characterized in that... Step 1 involves mixing alginate sulfate with water at a mass-to-volume ratio of 1-50 mg: 1 ml and stirring continuously at 30-40°C until homogeneous.

3. The method for preparing the alginate sulfate oligomer according to claim 2, characterized in that... In step 2, the mass ratio of TEMPO to alginate sulfate in solution II is 0.5-5%, and the mass ratio of sodium bromide to alginate sulfate is 5-40%.

4. The method for preparing the alginate sulfate oligomer according to claim 3, characterized in that... In step 3, the ratio of alginate sulfate to sodium hypochlorite is 1g:0.1-10mmol, and the solution is continuously stirred at 30-50℃ to obtain solution III.

5. The method for preparing the alginate sulfate oligomer according to claim 4, characterized in that... Step 4 involves continuously adding 0.5 mol / mL sodium hydroxide solution to adjust the pH to 8-11, and continuously stirring for 0.5-5 h to obtain solution IV.

6. The method for preparing the brown alginate sulfate oligomer according to claim 5, characterized in that... The volume ratio of anhydrous ethanol added in step 5 to solution IV is 1-20%.

7. The method for preparing the alginate sulfate oligomer according to claim 6, characterized in that... Step 6 involves adjusting the pH of solution V to 7 using a 1 mol / mL sodium hydroxide solution to obtain solution VI.

8. The method for preparing the alginate sulfate oligomer according to claim 7, characterized in that... The dialysis molecular weight cutoff in step 7 is 300 Da.

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