Preparation method of fucoidan sulfate oligomer

Abstract

The invention discloses a preparation method of a fucoidan oligomer, which realizes efficient degradation of fucoidan under specific conditions by strictly controlling the addition amount of TEMPO, sodium bromide and sodium hypochlorite. Specifically, the ratio of the addition amount of TEMPO to the mass of fucoidan sulfate is 0.5-5%, the ratio of sodium bromide to fucoidan sulfate is 5-40%, the use amount ratio of fucoidan sulfate to sodium hypochlorite is 1 g: (0.1-10) mmoL, strong acid or enzyme preparations do not need to be added, and auxiliary means such as microwaves or ultrasound are not depended on, so that the preparation process is simple, and the preparation cost is low. The oligomer with the average molecular weight lower than 80 kDa can be obtained within one hour, the oligomer with the average molecular weight about 20 kDa can be obtained within four hours, and meanwhile the content of sulfate groups is kept to be about 25%. The functional groups of the fucoidan are hardly damaged, and the prepared oligomer has a remarkable effect in the aspect of in-vitro anticoagulant activity.

Description

Technical Field

[0001] The invention belongs to the field of deep processing of seaweed polysaccharides, and in particular relates to a method for preparing fucoidan sulfate oligomers. Background Art

[0002] Fucoidan sulfate is a water-soluble sulfated heteropolysaccharide that has attracted widespread attention in the food and pharmaceutical fields due to its multiple biological activities. However, fucoidan sulfate naturally extracted from the cell wall matrix of brown algae usually has disadvantages such as high molecular weight, poor solubility and high viscosity, which limits its practical application. In contrast, fucoidan sulfate oligomers have gradually become a research hotspot due to their low viscosity, good bioabsorbability and strong biological activity. At present, the preparation methods of fucoidan sulfate oligomers mainly include acid hydrolysis, enzyme degradation, photodegradation, ultrasonic-microwave combined degradation, etc.

[0003] The Chinese invention patent application with the patent publication number CN118515790A discloses "a method for preparing low molecular weight fucoidan and its application". Specifically, different types of acids (0.1 M hydrochloric acid, 0.15 M acetic acid and 0.5 M citric acid) are used to treat brown algae polysaccharide at 85-100°C for 15-20 min to obtain products with a molecular weight of less than 5 kDa and a yield of more than 59%. However, the strong acid used in this method places high corrosion resistance requirements on industrial production equipment, and high temperature treatment may cause the sulfate groups in brown algae polysaccharide to be destroyed.

[0004] The Chinese invention patent with the patent publication number CN118638882A discloses "a method for preparing and applying a low molecular weight fucoidan degradation product", which combines cellulase and composite 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, its degradation efficiency is low and the production and recovery costs of enzyme preparations are high. In addition, the enzyme's insufficient stability makes its application in industrial production challenging, and there are currently no suitable commercial enzyme preparations available.

[0005] The Chinese invention patent application with the patent publication number 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 H 2 O 2 The solutions are mixed; placed under ultraviolet light for radiation degradation; dialyzed, evaporated and concentrated, and dried 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 by-products, affecting the purity and activity of the target product.

[0006] The Chinese invention patent with patent announcement number CN107011454B discloses "a method for preparing low molecular weight and highly sulfated sea cucumber fucoidan sulfate". Specifically, sea cucumber fucoidan sulfate is first used as raw material, and ultrasonic-microwave combined degradation method is used to break the glycosidic bond, decompose sea cucumber fucoidan sulfate to obtain low molecular weight sea cucumber fucoidan sulfate, and then methyl methanesulfonate is used to carry out sulfation reaction with low molecular weight sea cucumber fucoidan sulfate to obtain low molecular weight and highly sulfated sea cucumber fucoidan sulfate. However, although ultrasonic degradation can accelerate the reaction, excessive energy input can easily destroy key active groups and increase energy consumption; microwave degradation can improve efficiency, but the equipment requirements are high and may cause local overheating and induce side reactions. There are problems such as high equipment investment, complex operation and difficulty in maintaining the biological activity of fucoidan sulfate, which limit its application in the health field.

[0007] TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl radical) is a stable free radical compound widely used in organic chemistry and materials science. Its molecular structure contains a nitrogen atom with an unpaired electron, which enables TEMPO to act as an efficient oxidant or catalyst in chemical reactions. TEMPO exhibits excellent catalytic performance in selective oxidation reactions, especially in the oxidation of sugars, alcohols and other reducing compounds, with the remarkable characteristics of mildness, strong selectivity and loose reaction conditions. However, to date, there have been no reports on the use of TEMPO to selectively oxidize specific sugar residue sites in fucoidan sulfate, induce the cleavage of molecular chain glycosidic bonds, thereby achieving directional 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 and provides a method for preparing fucoidan sulfate oligomers.

[0009] The technical solution of the present invention is: a method for preparing fucoidan sulfate oligomers, which is carried out in the following steps: Step 1. Dissolve fucoidan sulfate in water to prepare solution I; Step 2. Add TEMPO and sodium bromide to solution I and stir to obtain solution II; Step 3. Add sodium hypochlorite to solution II and stir evenly to obtain solution III; Step 4. Add sodium hydroxide dropwise to solution III to adjust the pH value to be weakly alkaline, thereby obtaining solution IV; Step 5. After adding anhydrous ethanol to solution IV, the reaction is terminated to obtain solution V; Step 6. Adjusting the pH value of solution V to neutral to obtain solution VI; Step 7. Solution VI is dialyzed and freeze-dried to obtain fucoidan sulfate oligomers.

[0010] Preferably, the step 1 is to mix the fucoidan sulfate with water in a mass volume ratio of 1-50 mg:1 ml and continue stirring at 30-40°C until uniform.

[0011] Preferably, in the solution II of step 2, the mass ratio of TEMPO to fucoidan sulfate is 0.5-5%, and the mass ratio of sodium bromide to fucoidan sulfate is 5-40%.

[0012] Preferably, in step 3, the ratio of added fucoidan sulfate to sodium hypochlorite is 1 g:0.1-10 mmoL, and stirring is continued at 30-50° C. to obtain solution III.

[0013] Preferably, the step 4 is to continuously drop 0.5 mol / mL sodium hydroxide solution to adjust the pH to 8-11, and continuously stir for 0.5-5 h to obtain solution IV.

[0014] Preferably, the volume ratio of anhydrous ethanol added in step 5 to solution IV is 1-20%.

[0015] Preferably, the step 6 is to adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI.

[0016] Preferably, the dialysis cut-off molecular weight in step 7 is 300 Da.

[0017] The present invention utilizes the TEMPO free radical catalytic system to selectively oxidize specific sugar residue sites of fucoidan sulfate, induce the cleavage of glycosidic bonds of the molecular chain, thereby achieving directional degradation of polysaccharides and obtaining low molecular weight products. Compared with the prior art, it has the following advantages: 1. High selectivity: TEMPO has strong selectivity in catalyzing the oxidation of fucoidan sulfate. It can only oxidize specific glycosidic bonds in polysaccharides without affecting other parts, overcoming the problem of excessive damage to the polysaccharide structure in traditional degradation methods, and can effectively protect the active groups from being destroyed.

[0018] 2. Mild reaction conditions: TEMPO can work under mild reaction conditions, without the need to add strong acids or enzymes, and does not rely on auxiliary means such as microwaves or ultrasound, and is not prone to side reactions or excessive oxidation. Oligomers with an average molecular weight of less than 80 kDa can be obtained within 1 hour, and oligomers with an average molecular weight of about 20 kDa can be obtained within 4 hours, while maintaining a sulfate content of about 25%.

[0019] 3. Reusable: TEMPO exhibits good stability in the reaction and can be recycled many times, thereby reducing the preparation cost of fucoidan sulfate oligomers.

[0020] 4. High anticoagulant activity: The prepared fucoidan sulfate oligomer has high anticoagulant activity and significantly prolongs the APTT coagulation time. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a relative molecular mass distribution diagram of the fucoidan sulfate oligomer and fucoidan sulfate prepared in Examples 1-4 of the present invention.

[0022] Figure 2 It is an infrared spectrum of the fucoidan sulfate oligomer and fucoidan sulfate prepared in Example 1 of the present invention.

[0023] Figure 3 It is a schematic diagram of the anticoagulant activity index of the fucoidan sulfate oligomer and fucoidan sulfate prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] The raw material used in the examples of the present invention, namely, fucoidan sulfate, was purchased from Qingdao Mingyue Seaweed Group Co., Ltd. and complies with the industry standard "SC / T 3404-2012". Example 1

[0025] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 10 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I, add 0.02 g TEMPO and 0.2 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 0.4 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 4 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0026] It was determined that the relative molecular weight of the raw material fucoidan sulfate before degradation was 336 kDa, the total sugar content was 52.02±1.81%, and the sulfate content was 26.68±0.40%. The fucoidan sulfate oligomer prepared in Example 1 was in the form of a white powder with a relative molecular weight of about 20 kDa, a total sugar content of 50.88±0.74%, and a sulfate content of 25.09±0.61%. The Fourier transform infrared spectroscopy (FTIR) spectrum (such as Figure 2 The results show that the characteristic peaks of sulfate in the 20 kDa oligomer did not change significantly compared with the raw material fucoidan sulfate, specifically the peaks at 1245.32 cm⁻¹ (attributed to S=O stretching vibration), 851.12 cm⁻¹ (attributed to COS axial coordination stretching vibration), and 814.41 cm⁻¹ (attributed to COS equatorial coordination stretching vibration), indicating that the sulfate group was not destroyed during the preparation process.

[0027] The in vitro anticoagulant activity of fucoidan sulfate oligomers was tested by a commercial kit. Figure 3 The results show that at the same concentration of the fucoidan sulfate oligomer of the present invention, the raw material fucoidan sulfate and heparin, the fucoidan sulfate oligomer significantly prolonged the activated partial prothrombin time, so the fucoidan sulfate oligomer prepared by the present invention has a certain anticoagulant activity. Example 2

[0028] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 10 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I, add 0.04 g TEMPO and 0.4 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 0.4 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 1 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0029] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 2 was about 41 kDa, the total sugar content of the oligomer was 50.32±0.52%, and the sulfate content was 24.56±0.61%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 81.89±2s. Example 3

[0030] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 10 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I, add 0.04 g TEMPO and 0.4 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 0.1 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 2 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0031] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 3 was about 61 kDa, the total sugar content of the oligomer was 50.33±0.22%, and the sulfate content was 24.92±1.82%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 78.24±2 s. Example 4

[0032] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 10 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I, add 0.02 g TEMPO and 0.2 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 0.1 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 1 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0033] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 4 was about 83 kDa, the total sugar content of the oligomer was 50.12±2.88%, and the sulfate content was 25.98±0.24%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 75.24±3s.

[0034] The relative molecular mass distribution diagram of the fucoidan sulfate oligomer and fucoidan sulfate prepared in Example 1-4 is as follows: Figure 1 shown. Example 5

[0035] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 5 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I, add 0.01 g TEMPO and 0.1 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 0.4 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 2 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0036] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 5 was about 26 kDa, the total sugar content of the oligomer was 50.91±0.45%, and the sulfate content was 25.57±0.52%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 82.14±1s. Example 6

[0037] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 10 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I, add 0.03 g TEMPO and 0.3 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 0.4 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 3 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0038] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 6 was about 32 kDa, the total sugar content of the oligomer was 50.68±0.41%, and the sulfate content was 24.69±0.59%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 80.10±3s. Example 7

[0039] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 20 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I and add 0.08 g TEMPO and 0.8 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 5 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 0.5 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0040] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 7 was about 48 kDa, the total sugar content of the oligomer was 50.69±0.56%, and the sulfate content was 24.55±1.81%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 80.01±2s. Example 8

[0041] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 40 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I and add 0.05 g TEMPO and 0.5 g sodium bromide, stirring continuously to obtain solution II; Step 3. Add 4 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 0.5 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0042] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 8 was about 53 kDa, the total sugar content of the oligomer was 50.88±0.24%, and the sulfate content was 24.55±1.88%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 79.89±2s. Example 9

[0043] Step 1. Dissolve the fucoidan sulfate in water at a concentration of 45 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I, add 0.1 g TEMPO and 1 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 3 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 0.5 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0044] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 9 was about 68 kDa, the total sugar content of the oligomer was 51.46±0.45%, and the sulfate content was 24.85±1.83%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 77.56±1s.

[0045] Embodiment 10: Step 1. Dissolve the fucoidan sulfate in water at a concentration of 50 mg / mL, and continue stirring and dissolving at 35°C to obtain solution I; Step 2. Take 100 mL of solution I, add 0.2 g TEMPO and 2 g sodium bromide, and continue stirring to obtain solution II; Step 3. Add 1 mmol sodium hypochlorite to solution II and continue stirring to obtain solution III; Step 4. Add 0.5 mol / L sodium hydroxide to solution III to adjust the pH of the solution to 10±0.5, and continue stirring for 1 h to obtain solution IV; Step 5. Add 1 mL of anhydrous ethanol to solution IV to terminate the reaction and obtain solution V; Step 6. Using 1 mol / mL sodium hydroxide solution, adjust the pH of solution V to 7 to obtain solution VI; Step 7. Solution VI was dialyzed (cutoff 300Da) and freeze-dried to obtain white powdery fucoidan sulfate oligomers.

[0046] It was determined that the relative molecular weight of the fucoidan sulfate oligomer prepared in Example 10 was about 71 kDa, the total sugar content of the oligomer was 52.77±0.42%, and the sulfate content was 25.12±1.89%. At a concentration of 200 μg / mL of the oligomer, the activated partial prothrombin time was 76.32±3 s.

Claims

1. A method for preparing fucoidan sulfate oligomers, characterized in that Follow these steps in order: Step 1. Dissolve fucoidan sulfate in water to prepare solution I; Step 2. Add TEMPO and sodium bromide to solution I and stir to obtain solution II; Step 3. Add sodium hypochlorite to solution II and stir evenly to obtain solution III; Step 4. Add sodium hydroxide dropwise to solution III to adjust the pH value to be weakly alkaline, thereby obtaining solution IV; Step 5. After adding anhydrous ethanol to solution IV, the reaction is terminated to obtain solution V; Step 6. Adjusting the pH value of solution V to neutral to obtain solution VI; Step 7. Solution VI is dialyzed and freeze-dried to obtain fucoidan sulfate oligomers.

2. The method for preparing fucoidan sulfate oligomer according to claim 1, characterized in that The step 1 is to mix the fucoidan sulfate and water in a mass volume ratio of 1-50 mg:1 ml and continue to stir evenly at 30-40°C.

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

4. The method for preparing fucoidan sulfate oligomer according to claim 3, characterized in that In step 3, the ratio of added fucoidan sulfate to sodium hypochlorite is 1 g:0.1-10 mmoL, and stirring is continued at 30-50° C. to obtain solution III.

5. The method for preparing fucoidan sulfate oligomer according to claim 4, characterized in that The step 4 is to continuously drop 0.5 mol / mL sodium hydroxide solution to adjust the pH to 8-11, and continuously stir for 0.5-5 h to obtain solution IV.

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

7. The method for preparing fucoidan sulfate oligomer according to claim 6, characterized in that The step 6 is to adjust the pH of solution V to 7 using 1 mol / mL sodium hydroxide solution to obtain solution VI.

8. The method for preparing fucoidan sulfate oligomer according to claim 7, characterized in that The dialysis cut-off molecular weight in step 7 is 300 Da.

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