Preparation method and application of G-type fucoidan

Abstract

The present invention discloses a preparation method and application of G-type fucoidan in the technical field of G-type fucoidan. S1: Prepare a sodium alginate solution with a concentration specific gravity of 1% to 4%, and homogenize the swollen sodium alginate solution with a high-pressure homogenizer to obtain Product 1; S2: Take an appropriate amount of Product 1 and an acid solution and mix them evenly to obtain a mixed solution with an acid concentration of 0.1 mol / L to 0.6 mol / L, and heat it to 90°C to 110°C for acid hydrolysis for 7 h to 24 h to obtain Product 2; S3: Centrifuge the obtained Product 2 for 10 min and collect the precipitate to obtain Precipitate 1; Adjust the pH of Precipitate 1 to neutral for re-dissolution, and then adjust the pH to 2.85 with acid, and perform secondary centrifugation under the same centrifugation conditions and collect the precipitate again to obtain Precipitate 2; Adjust the pH of Precipitate 2 to neutral and re-dissolve it to obtain Product 3; S4: After freeze-drying Product 3, G-type fucoidan is obtained. The G-type fucoidan obtained by the present invention requires a shorter time, has a lower degree of browning of the raw material, and a higher purity, and can be applied to medical care.

Description

Technical Field

[0001] The present invention relates to the technical field of G-type fucoidan, and specifically provides a preparation method and application of G-type fucoidan. Background Art

[0002] Sodium alginate, also known as alginate, is a natural polysaccharide formed by the alternating connection of β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G) through 1-4 glycosidic bonds.

[0003] According to the arrangement of M and G, there are three blocks in the sodium alginate molecule: polymannuronic acid fragment (M block, M-type fucoidan), polyguluronic acid fragment (G block, G-type fucoidan), and the alternating fragment of mannuronic acid and guluronic acid (MG block or GM block); the composition ratio of MG in sodium alginate is affected by factors such as algal species, region, season, age, and part; in M-type fucoidan, the glycosidic bond is bonded by an equatorial bond, forming a soft and extensible molecular conformation; in G-type fucoidan, the glycosidic bond is bonded by an axial bond, forming a rigid and compact molecular conformation. These structural differences result in different applications in the medical field.

[0004] Degrading sodium alginate can produce new biological activities in the resulting oligosaccharides. Research shows that G-type fucoidan has various excellent biological activities such as anti-inflammatory, antibacterial, and immunomodulatory effects. Acid hydrolysis of sodium alginate is a common method for preparing G-type fucoidan. Compared with enzymatic hydrolysis and oxidative degradation, the acid hydrolysis preparation method does not introduce new groups; during acid hydrolysis, the MG fragment in alginic acid is degraded into soluble components and enters the acidic solution; the insoluble part is centrifuged, the pH is adjusted to neutral for re-dissolution, and then adjusted to acidic pH for fractional precipitation, and crude products of G-type fucoidan and M-type fucoidan can be obtained respectively; by repeated pH fractional precipitation, the purity of G-type fucoidan can be improved to a certain extent, but the content of guluronic acid in G-type fucoidan can only reach 85%, and the β-D-mannuronic acid residues mixed in the G-type fucoidan molecule cannot be removed, which limits its application in the medical field. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of G-type fucoidan to solve the problem that the content of guluronic acid in G-type fucoidan can only reach 85% as mentioned in the above background art, and the mannuronic acid residues mixed in the G-type fucoidan molecule cannot be removed, which limits its application in the medical field.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A preparation method of G-type fucoidan, comprising the following steps:

[0008] S1: Prepare a sodium alginate solution with a concentration specific gravity of 1% - 4%. Treat the sodium alginate solution until it is fully swollen. After homogenizing the swollen sodium alginate solution with a high-pressure homogenizer, obtain Product 1.

[0009] S2: Take an appropriate amount of Product 1 and mix it evenly with an acid solution to obtain a mixed solution with an acid concentration of 0.1 mol / L - 0.6 mol / L, and heat it to 90°C - 110°C for acid hydrolysis for 7 h - 24 h to obtain Product 2.

[0010] S3: Centrifuge the obtained Product 2 for 10 min under a centrifugal force of 8000 g to collect the precipitate to obtain Precipitate 1. Adjust the pH of Precipitate 1 to neutral for re-dissolution, then adjust the pH to 2.85 with acid, and perform secondary centrifugation under the same centrifugation conditions and collect the precipitate again to obtain Precipitate 2. Adjust the pH of Precipitate 2 to neutral and re-dissolve it to obtain Product 3.

[0011] S4: After freeze-drying Product 3, obtain G-type fucoidan.

[0012] Preferably, the pressure of the high-pressure homogenizer is 400 bar - 860 bar, and the number of cycles is 3 - 6 times.

[0013] Preferably, the pressure of the high-pressure homogenizer is 520 bar - 860 bar, and the number of cycles is 3 - 6 times.

[0014] Preferably, the acid solution in S2 is any one of sulfuric acid, hydrochloric acid, trifluoroacetic acid, formic acid, and oxalic acid.

[0015] Preferably, the acid solution in S2 is a 0.3 mol / L hydrochloric acid solution.

[0016] Preferably, the hydrochloric acid solution is heated to 100°C.

[0017] An application of G-type fucoidan prepared by the above preparation method, applying the prepared G-type fucoidan to wound care and respiratory care.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] Compared with the existing acid hydrolysis technology, the present invention uses the high-pressure homogenization method for pretreatment and then acid hydrolysis, which not only effectively solves the related impurities of G-type fucoidan, but also can obtain G-type fucoidan with controllable molecular weight; compared with the traditional acid hydrolysis method, when preparing G-type fucoidan products with the same molecular weight, the required time is shorter, the degree of browning of the raw material is lower, and the purity is higher; and in the in vitro sputum rheology experiment, the high-purity G-type fucoidan prepared by the present invention can more effectively reduce the elastic modulus and viscous modulus of sputum. In wound care, the G-type fucoidan prepared by the present invention can promote the growth of granulation tissue and accelerate wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Data table of the effects of concentration and homogenization conditions on the viscosity of sodium alginate;

[0021] Figure 2 Data table of the effects of different acid hydrolysis conditions on the products of G-type fucoidan;

[0022] Figure 3 Data table of the effects of different temperatures and acid hydrolysis times on the products of G-type fucoidan;

[0023] Figure 4 Ultraviolet-visible light spectrum scanning diagram;

[0024] Figure 5 Data table of the molecular weight determination of different samples by GPC method;

[0025] Figure 6 G-type fucoidan obtained from Preparation Example 11 1 1H-NMR spectrum (25 °C);

[0026] Figure 7 G-type fucoidan obtained from Control Example 8 1 1H-NMR spectrum (25 °C);

[0027] Figure 8 G-type fucoidan obtained from Preparation Example 11 1 Partial 1H-NMR spectrum (80 °C);

[0028] Figure 9 G-type fucoidan obtained from Control Example 8 1 Partial 1H-NMR spectrum (80 °C);

[0029] Figure 10 Data table of the rheological property changes of sputum plus 5% physiological saline;

[0030] Figure 11 Data table of the rheological property changes of sputum plus 5% G-type fucoidan solution obtained from Preparation Example 10;

[0031] Figure 12 Data table of the rheological property changes of sputum plus 5% G-type fucoidan solution obtained from Control Example 7;

[0032] Figure 13 Graph of the rheological property changes of sputum plus 5% physiological saline;

[0033] Figure 14 Graph of the rheological property changes of sputum plus 5% G-type fucoidan solution obtained from Preparation Example 10;

[0034] Figure 15It is a graph showing the rheological property changes of the G-type fucoidan solution obtained by adding 5% sputum as a control example 7;

[0035] Figure 16 It is an effect diagram of the healing of second-degree scalds on the lower limbs;

[0036] Figure 17 It is the healing effect of the ulcerated wound of the local scar on the chest wall;

[0037] Figure 18 It is a data table showing the influence of the initial sodium alginate concentration on the viscosity of sodium alginate. Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the specific implementation manners; it should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention.

[0039] The present invention provides a technical solution:

[0040] A preparation method of G-type fucoidan, comprising the following steps:

[0041] S1: Prepare a sodium alginate solution with a concentration ratio of 1% to 4%, treat the sodium alginate solution until it is fully swollen, and homogenize the swollen sodium alginate solution with a high-pressure homogenizer to obtain product one;

[0042] S2: Take an appropriate amount of product one and an acid solution and mix them evenly to obtain a mixed solution with an acid concentration of 0.1 mol / L to 0.6 mol / L, and heat it to 90 °C to 110 °C and acidolyze it for 7 h to 24 h to obtain product two;

[0043] S3: Centrifuge the obtained product two for 10 min under a centrifugal force of 8000 g and collect the precipitate to obtain precipitate one; adjust the pH of precipitate one to neutral for re-dissolution, and then adjust the pH to 2.85 with acid, and perform secondary centrifugation under the same centrifugation conditions and collect the precipitate again to obtain precipitate two; adjust the pH of precipitate two to neutral and re-dissolve it to obtain product three;

[0044] S4: After freeze-drying product three, G-type fucoidan is obtained.

[0045] Experiment 1: To investigate the influence of different homogenization conditions on the viscosity of sodium alginate solution, the method is as follows:

[0046] The influence of the concentration of the sodium alginate solution on the viscosity decrease ratio of the sodium alginate solution before and after homogenization is negligible; three groups of Preparation Example 1, Preparation Example 1-1, and Preparation Example 1-2 are set for comparison:

[0047] Preparation Example 1: Weigh sodium alginate, add an appropriate amount of purified water, and stir until no visible patches are seen with the naked eye to obtain a sodium alginate solution with a weight ratio concentration of 4%. After sufficient swelling, its initial viscosity is measured; adjust the pressure of the high-pressure homogenizer to 400 bar and the number of cycles to 5 times. Under the above conditions, use the high-pressure homogenizer to treat the 4% weight ratio concentration sodium alginate solution after sufficient swelling to obtain a homogenized solution and measure its viscosity to obtain the viscosity after homogenization.

[0048] Preparation Example 1-1: The process is the same as that of Preparation Example 1, except that the weight ratio concentration of the sodium alginate solution is 3%.

[0049] Preparation Example 1-2: The process is the same as that of Preparation Example 1, except that the weight ratio concentration of the sodium alginate solution is 2%.

[0050] Conclusion: Refer to Figure 18 the experimental data. The solutions obtained in Preparation Example 1, Preparation Example 1-1, and Preparation Example 1-2 are clearer and more transparent in appearance than before homogenization; after homogenization, the viscosities of the solutions obtained in Preparation Example 1, Preparation Example 1-1, and Preparation Example 1-2 decreased by 62.6%, 61.9%, and 61.8% respectively; it can be obtained that through the treatment of high-pressure homogenization, the molecular spatial structure of sodium alginate changes, showing better flow performance, and the weight ratio concentration of the sodium alginate solution has no effect on the viscosity reduction ratio after homogenization.

[0051] Five groups of objects to be investigated are set for preparation and comparison, namely: Preparation Example 1, Preparation Example 2, Preparation Example 3, Preparation Example 4, and Preparation Example 5; the preparation methods of each group of objects to be investigated are as follows:

[0052] Preparation Example 2: Weigh sodium alginate, add an appropriate amount of purified water, and stir until no visible patches are seen with the naked eye to obtain a sodium alginate solution with a weight ratio concentration of 4%. After sufficient swelling, its initial viscosity is measured; adjust the pressure of the high-pressure homogenizer to 860 bar and the number of cycles to 3 times. Under the above conditions, use the high-pressure homogenizer to treat the 4% weight ratio concentration sodium alginate solution after sufficient swelling to obtain a homogenized solution and measure its viscosity to obtain the viscosity after homogenization.

[0053] Preparation Example 3: Weigh sodium alginate, add an appropriate amount of purified water, and stir until no visible patches are seen with the naked eye to obtain a sodium alginate solution with a weight ratio concentration of 3%. After sufficient swelling, its initial viscosity is measured; adjust the pressure of the high-pressure homogenizer to 800 bar and the number of cycles to 6 times. Under the above conditions, use the high-pressure homogenizer to treat the 3% weight ratio concentration sodium alginate solution after sufficient swelling to obtain a homogenized solution and measure its viscosity to obtain the viscosity after homogenization.

[0054] Preparation Example 4: Weigh sodium alginate, add an appropriate amount of purified water, and stir until no visible patches are present to obtain a 2% weight ratio concentration sodium alginate solution. After sufficient swelling, its initial viscosity is measured; adjust the pressure of the high-pressure homogenizer to 450 bar and the number of cycles to 5 times. Under the above conditions, use the high-pressure homogenizer to process the 2% weight ratio concentration sodium alginate solution after sufficient swelling to obtain a homogenized solution and measure its viscosity to obtain the viscosity after homogenization.

[0055] Preparation Example 5: Weigh sodium alginate, add an appropriate amount of purified water, and stir until no visible patches are present to obtain a 2% weight ratio concentration sodium alginate solution. After sufficient swelling, its initial viscosity is measured; adjust the pressure of the high-pressure homogenizer to 520 bar and the number of cycles to 4 times. Under the above conditions, use the high-pressure homogenizer to process the 2% weight ratio concentration sodium alginate solution after sufficient swelling to obtain a homogenized solution and measure its viscosity to obtain the viscosity after homogenization.

[0056] Conclusion: The data of Preparation Example 1, Preparation Example 2, Preparation Example 3, Preparation Example 4, and Preparation Example 5 are shown in Figure 1 ; among them, the solutions obtained in Preparation Example 2, Preparation Example 3, and Preparation Example 5 are clearer and more transparent in appearance than before homogenization; after homogenization, the viscosities of the solutions obtained in Preparation Example 1, Preparation Example 2, Preparation Example 3, Preparation Example 4, and Preparation Example 5 decreased by 62.6%, 86.4%, 84.7%, 67.3%, and 92.1% respectively; it can be seen that through the treatment of high-pressure homogenization, the molecular spatial structure of sodium alginate changes, showing better fluidity.

[0057] Preferably, the pressure of the high-pressure homogenizer is 400 bar to 860 bar, and the number of cycles is 3 to 6 times. From the experimental data, it can be obtained that the viscosities of the solutions obtained in Preparation Example 2, Preparation Example 3, and Preparation Example 5 decreased to 16% or less of the initial viscosity, and the obtained solutions showed excellent performance in subsequent obtaining of high-purity G-type fucoidan.

[0058] Preferably, the pressure of the high-pressure homogenizer is 520 bar to 860 bar, and the number of cycles is 3 to 6 times. From the experimental data, it can be obtained that the viscosity of the solution obtained in Preparation Example 5 decreased to 8% or less of the initial viscosity, and the obtained solution is extremely excellent in subsequent obtaining of high-purity G-type fucoidan.

[0059] Experiment 2: To investigate the influence of different conditions on the G-type fucoidan product, the method is as follows:

[0060] Set 8 groups of investigation objects for preparation and comparison, which are: Preparation Example 6, Preparation Example 7, Control Example 1, Preparation Example 8, Control Example 2, Preparation Example 9, Preparation Example 10, and Control Example 3; the preparation methods of each group of investigation objects are as follows:

[0061] Preparation Example 6: a1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 1 and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.6 mol / L. Under stirring conditions, carry out acid hydrolysis at 100 °C for 7 hours; a2. Take the product obtained in step a1, centrifuge the product at a centrifugal force of 8000 g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; a3. Freeze-dry the product in step a2 to obtain G-type fucoidan.

[0062] Preparation Example 7: b1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 2 and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.6 mol / L. Under stirring conditions, carry out acid hydrolysis at 100 °C for 7 hours; b2. Take the product obtained in step b1, centrifuge the product at a centrifugal force of 8000 g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; b3. Freeze-dry the product in step b2 to obtain G-type fucoidan.

[0063] Control Example 1: c1. Take an appropriate amount of untreated sodium alginate with a weight ratio concentration of 4% and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.6 mol / L. Under stirring conditions, carry out acid hydrolysis at 100 °C for 7 hours; c2. Take the product obtained in step c1, centrifuge the product at a centrifugal force of 8000 g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; c3. Freeze-dry the product in step c2 to obtain G-type fucoidan.

[0064] Preparation Example 8: d1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 3 and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.4 mol / L. Under stirring conditions, carry out acid hydrolysis at 100 °C for 7 hours; d2. Take the product obtained in step d1, centrifuge the product at a centrifugal force of 8000 g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; d3. Freeze-dry the product in step d2 to obtain G-type fucoidan.

[0065] Comparative Example 2: e1. Take an appropriate amount of untreated sodium alginate with a weight ratio concentration of 3% and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.4 mol / L. Under stirring conditions, carry out acid hydrolysis at 100°C for 7 hours; e2. Take the product obtained in step e1, centrifuge this product at a centrifugal force of 8000g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; e3. Lyophilize the product in step e2 to obtain G-type fucoidan.

[0066] Preparation Example 9: f1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 4 and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L. Under stirring conditions, carry out acid hydrolysis at 100°C for 7 hours; f2. Take the product obtained in step f1, centrifuge this product at a centrifugal force of 8000g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; f3. Lyophilize the product in step f2 to obtain G-type fucoidan.

[0067] Preparation Example 10: g1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 5 and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L. Under stirring conditions, carry out acid hydrolysis at 100°C for 7 hours; g2. Take the product obtained in step g1, centrifuge this product at a centrifugal force of 8000g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; g3. Lyophilize the product in step g2 to obtain G-type fucoidan.

[0068] Comparative Example 3: h1. Take an appropriate amount of untreated sodium alginate with a weight ratio concentration of 2% and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L. Under stirring conditions, carry out acid hydrolysis at 100°C for 7 hours; h2. Take the product obtained in step h1, centrifuge this product at a centrifugal force of 8000g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; h3. Lyophilize the product in step h2 to obtain G-type fucoidan.

[0069] Among them, the yield of G-type fucoidan: is the ratio of the weight of G-type fucoidan prepared under each condition to the weight of sodium alginate fed under the corresponding preparation conditions. The calculation formula is as follows:

[0070] Yield of G-type fucoidan = weight of G-type fucoidan / weight of sodium alginate × 100%;

[0071] Among them, the method for measuring molecular weight: The molecular weight and distribution of the sample are determined by gel permeation chromatography (GPC);

[0072] The following detection conditions are adopted: Instrument: Agilent 1260; Chromatographic conditions: Agilent infinity 1260; Chromatographic column: PL aquagel-OH mixed (8 μm); Detector: Differential refractive index detector and laser light scattering detector in combination; Mobile phase: 0.1 M sodium nitrate, 500 ppm aqueous solution of sodium azide; Column temperature: 30°C; Flow rate: 1.0 ml / min; Standard: PEO; Sampling concentration: 1 mg / ml; Injection volume: 40 μl.

[0073] Among them, the method for measuring the content of G-type fucoidan: It is determined by the ratio of the peak area related to guluronic acid to the peak area related to the prepared G-type fucoidan product measured by the 1H-NMR method;

[0074] Specifically, the hydrogen spectrum conditions are as follows: 1H-NMR at 25°C, using an Agilent nuclear magnetic resonance instrument (spectrometer frequency 600 MHz);

[0075] Before sampling, about 20 mg of G-type fucoidan prepared by different methods is taken and dissolved in 2 ml of 99% D2O respectively. After freeze-drying, they are respectively dissolved in 2 ml of 99.9% D2O again.

[0076] During the test, 0.7 ml of the G-type fucoidan sample to be measured is added to the NMR sample tube, and then 20 μl of TTHA (triethylenetetraminehexaacetic acid, 0.3 ml / L) is added.

[0077] Conclusion: The recorded data of Preparation Example 6, Preparation Example 7, Control Example 1, Preparation Example 8, Control Example 2, Preparation Example 9, Preparation Example 10 and Control Example 3 are referred to Figure 2 .

[0078] From Figure 2It can be seen that at each concentration level (4% concentration level: Preparation Example 6, Preparation Example 7, Control Example 1; 3% concentration level: Preparation Example 8, Control Example 2; 2% concentration level: Preparation Example 9, Preparation Example 10, Control Example 3), for the samples after high-pressure homogenization, after acid hydrolysis, the content and yield of G-fucoidan are higher than those of the corresponding samples without high-pressure homogenization, and the corresponding weight-average molecular weight is smaller; in addition, at the same concentration, after high-pressure homogenization treatment, the greater the degree of viscosity decrease, the higher the content and yield of G-fucoidan after acid hydrolysis and the smaller the weight-average molecular weight. Therefore, the mechanical forces such as high-speed shearing, high-frequency oscillation, cavitation phenomenon and convective impact generated during high-pressure homogenization and the corresponding thermal effects have caused physical, chemical and structural property changes to the biological macromolecule sodium alginate, reduced the crystallinity of each block site, made some degradation sites more easily exposed, thereby accelerating the degradation reaction and increasing the yield of G-fucoidan.

[0079] Experiment 3: To investigate the effects of different temperatures and acid hydrolysis times on G-fucoidan, the method is as follows: The acid solution is any one of sulfuric acid, hydrochloric acid, trifluoroacetic acid, formic acid, and oxalic acid.

[0080] 12 groups of investigation objects were set up for preparation and comparison, which were respectively: Preparation Example 12, Preparation Example 13, Preparation Example 14, Control Example 5, Control Example 6, Control Example 7, Preparation Example 11, Preparation Example 15, Preparation Example 16, Control Example 4, Control Example 8, and Control Example 9; the preparation methods of each group of investigation objects are as follows:

[0081] Preparation Example 12: i1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 5 and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 7 hours under stirring conditions at an acid hydrolysis temperature of 90°C; i2. Take the product obtained in step i1, centrifuge the product at a centrifugal force of 8000g for 10 minutes, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add NaOH to the precipitate to adjust the pH to neutral for re-dissolution; i3. Lyophilize the product in step i2 to obtain G-fucoidan.

[0082] Preparation Example 13: j1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 5 and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 15 hours under stirring conditions at an acid hydrolysis temperature of 90°C; j2. Take the product obtained in step j1, centrifuge the product at a centrifugal force of 8000g for 10 minutes, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add NaOH to the precipitate to adjust the pH to neutral for re-dissolution; j3. Lyophilize the product in step j2 to obtain G-fucoidan.

[0083] Preparation Example 14: k1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 5 and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 24 hours under stirring conditions at an acid hydrolysis temperature of 90°C; k2. Take the product obtained in step k1, centrifuge the product for 10 min under a centrifugal force of 8000 g, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then adjust the pH to 2.85 with hydrochloric acid, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; k3. Freeze-dry the product in step k2 to obtain G-type fucoidan.

[0084] Control Example 5: l1. Take an appropriate amount of untreated sodium alginate with a weight ratio concentration of 2% and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 7 hours under stirring conditions at an acid hydrolysis temperature of 90°C; l2. Take the product obtained in step l1, centrifuge the product for 10 min under a centrifugal force of 8000 g, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then adjust the pH to 2.85 with hydrochloric acid, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; l3. Freeze-dry the product in step l2 to obtain G-type fucoidan.

[0085] Control Example 6: m1. Take an appropriate amount of untreated sodium alginate with a weight ratio concentration of 2% and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 15 hours under stirring conditions at an acid hydrolysis temperature of 90°C; m2. Take the product obtained in step m1, centrifuge the product for 10 min under a centrifugal force of 8000 g, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then adjust the pH to 2.85 with hydrochloric acid, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; m3. Freeze-dry the product in step m2 to obtain G-type fucoidan.

[0086] Control Example 7: n1. Take an appropriate amount of untreated sodium alginate with a weight ratio concentration of 2% and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 24 hours under stirring conditions at an acid hydrolysis temperature of 90°C; n2. Take the product obtained in step n1, centrifuge the product for 10 min under a centrifugal force of 8000 g, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then adjust the pH to 2.85 with hydrochloric acid, centrifuge under the same conditions again, collect the precipitate again, and slowly add the precipitate to NaOH to adjust the pH to neutral for re-dissolution; n3. Freeze-dry the product in step n2 to obtain G-type fucoidan.

[0087] Preparation Example 11: o1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 5 and mix it with hydrochloric acid to obtain a uniformly mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 7 hours under stirring conditions, with the acid hydrolysis temperature being 100 °C; o2. Take the product obtained in step o1, centrifuge the product at a centrifugal force of 8000 g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add NaOH to the precipitate to adjust the pH to neutral for re-dissolution; o3. Freeze-dry the product in step o2 to obtain G-type fucoidan.

[0088] Preparation Example 15: p1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 5 and mix it with hydrochloric acid to obtain a uniformly mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 15 hours under stirring conditions, with the acid hydrolysis temperature being 100 °C; p2. Take the product obtained in step p1, centrifuge the product at a centrifugal force of 8000 g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add NaOH to the precipitate to adjust the pH to neutral for re-dissolution; p3. Freeze-dry the product in step p2 to obtain G-type fucoidan.

[0089] Preparation Example 16: q1. Take an appropriate amount of the sodium alginate solution obtained in Preparation Example 5 and mix it with hydrochloric acid to obtain a uniformly mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 24 hours under stirring conditions, with the acid hydrolysis temperature being 100 °C; q2. Take the product obtained in step q1, centrifuge the product at a centrifugal force of 8000 g for 10 min, collect the precipitate, slowly add NaOH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add NaOH to the precipitate to adjust the pH to neutral for re-dissolution; q3. Freeze-dry the product in step q2 to obtain G-type fucoidan.

[0090] Control Example 4: r1. Take an appropriate amount of untreated sodium alginate with a weight ratio concentration of 2% and mix it with hydrochloric acid to obtain a uniformly mixed solution with an acid concentration of 0.3 mol / L, and carry out acid hydrolysis for 7 hours under stirring conditions, with the acid hydrolysis temperature being 100 °C; r2. Take the product obtained in step r1, centrifuge the product at a centrifugal force of 8000 g for 10 min (there is a misspelling here, should be "10 min" instead of "10 mir"), collect the precipitate, slowly add raoH (should be "NaOH") to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, centrifuge under the same conditions again, collect the precipitate again, and slowly add raoH (should be "NaOH") to the precipitate to adjust the pH to neutral for re-dissolution; r3. Freeze-dry the product in step r2 to obtain G-type fucoidan.

[0091] Comparative Example 8: s1. Take an appropriate amount of untreated sodium alginate with a concentration of 2% by weight and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L. Under stirring conditions, carry out acid hydrolysis for 15 hours at an acid hydrolysis temperature of 100 °C; s2. Take the product obtained in step s1, centrifuge the product at a centrifugal force of 8000 g for 10 min, collect the precipitate, slowly add saoH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, and centrifuge under the same conditions again, collect the precipitate again, and slowly add saoH to the precipitate to adjust the pH to neutral for re-dissolution; s3. Freeze-dry the product in step s2 to obtain G-type fucoidan.

[0092] Comparative Example 9: t1. Take an appropriate amount of untreated sodium alginate with a concentration of 2% by weight and mix it with hydrochloric acid to obtain a mixed solution with an acid concentration of 0.3 mol / L. Under stirring conditions, carry out acid hydrolysis for 24 hours at an acid hydrolysis temperature of 100 °C; t2. Take the product obtained in step t1, centrifuge the product at a centrifugal force of 8000 g for 10 mit, collect the precipitate, slowly add taoH to adjust the pH to neutral for re-dissolution, then use hydrochloric acid to adjust the pH to 2.85, and centrifuge under the same conditions again, collect the precipitate again, and slowly add taoH to the precipitate to adjust the pH to neutral for re-dissolution; t3. Freeze-dry the product in step t2 to obtain G-type fucoidan.

[0093] The comparison data of the above 12 groups of investigation objects are shown in Figure 3 .

[0094] Take the prepared products of Comparative Examples 4 to 9 and Preparation Examples 11 to 16 for solution color detection;

[0095] All the above-prepared products are formulated into solutions with a concentration of 2%. After arranging them in ascending order of visual color, they are divided into four equal parts. Select the first and the middle three equal-point samples for ultraviolet-visible light spectral scanning. According to the spectral scanning curve graph ( Figure 4 ), select a wavelength of 420 nm to measure the absorbance values of each product, and reflect the colors of each product in a quantitative manner;

[0096] At 420 nm, the darker the appearance color of the solution, the larger the absorbance value; when the absorbance value is about 0.3, the appearance of the solution is light yellow, and when the absorbance value is above 1, the appearance of the solution is brown.

[0097] In addition: The detection of the solution color is carried out according to the second method (General Rule 0901) of the solution color inspection method in the Pharmacopoeia of the People's Republic of China (2020 Edition).

[0098] Measure the molecular weights of the prepared products of Comparative Examples 4 to 9 and Preparation Examples 11 to 16. The method is the same as the method for measuring the molecular weight in Experiment 2, and will not be elaborated here; the results are shown in Figure 5 .

[0099] Conclusion: From Figure 3 and Figure 5 it can be seen that the molecular weight of G-fucoidan decreases with the increase of reaction temperature and the prolongation of acidolysis time; correspondingly, the absorbance value of the obtained G-fucoidan solution increases with the increase of reaction temperature and the prolongation of acidolysis time (the color of the solution deepens); at the same temperature and reaction time, the molecular weight of G-fucoidan prepared by high-pressure homogenization followed by acidolysis is significantly smaller than that of the corresponding control example; especially under the reaction conditions of 100 °C, samples treated by high-pressure homogenization can obtain G-fucoidan with a weight-average molecular weight below ten thousand, and the molecular weight decreases significantly with the prolongation of reaction time; in addition, it can be seen from the results that when preparing G-fucoidan with the same molecular weight, after high-pressure homogenization treatment, the required reaction time and reaction temperature are lower, and the browning degree of the obtained G-fucoidan is lower and the appearance color is lighter.

[0100] Preferably, when the acid solution is 0.3 mol / L hydrochloric acid solution, the obtained G-fucoidan is better;

[0101] Preferably, when the acid solution is 0.3 mol / L hydrochloric acid solution and the hydrochloric acid solution is heated to 100 °C, the obtained G-fucoidan is the best.

[0102] It should be noted that: Figure 2 and Figure 3 and Figure 5 each preparation example in the alginate source shown in refers to the alginate solution after being treated by a high-pressure homogenizer under corresponding conditions, and except for the variables mentioned in the present invention, other experiments are carried out under the same conditions.

[0103] Experiment 4: Determination of related impurities, the method is as follows:

[0104] Take two samples with comparable weight-average molecular weight (Mw) and number-average molecular weight (Mn), and test them according to the method for determining the content of G-fucoidan in Experiment 2; only Preparation Example 11 and Control Example 8 are taken as examples for illustration, but it is not limited to Preparation Example 11 and Control Example 8.

[0105] The experimental hydrogen spectrum conditions are: 1H-NMR at 25 °C, using an Agilent nuclear magnetic resonance instrument (spectrometer frequency 600 MHz); 1H-NMR at 80 °C, using a JEOL nuclear magnetic resonance instrument (spectrometer frequency 400 MHz).

[0106] Conclusion: For the specific measurement results, refer to Figures 6 - 9 ; From Figures 6 - 9As can be seen from the 1H-NMR spectrum shown, for the G-type fucoidan prepared in Preparation Example 11 and Comparative Example 8 with comparable weight-average molecular weight (Mw) and number-average molecular weight (Mn), 1H-NMR detection was carried out at 25 °C. There was no peak (4.48 ppm to 4.50 ppm) within the range of 0.02 ppm at the low field (left side of the spectrum) of 4.48 ppm chemical shift, and there was only a shoulder peak, no independent peak, within the range of 0.07 ppm (4.41 ppm to 4.48 ppm) at the high field (right side of the spectrum) of 4.48 ppm chemical shift; when 1H-NMR detection was carried out at 80 °C, there was no splitting at 4.44 ppm chemical shift; this indicates that pre-treating sodium alginate by high-pressure homogenization and then acid-hydrolyzing can significantly reduce the β-D-mannuronic acid residues doped in the structure of G-type fucoidan.

[0107] Experiment Five: Detection of sputum rheological properties, the specific method is as follows:

[0108] Use an Anton Paar (MCR302) rheometer, equipped with a cone-plate system for testing.

[0109] Sputum samples were obtained from the Department of Respiratory and Critical Care Medicine of the hospital. The samples were collected in sterile sputum tubes and stored in a -80 °C refrigerator for later use.

[0110] Before use, dissolve at room temperature. After removing the saliva component, mix well with a vortex mixer, and gradually increase the vortex rotation speed until the sputum forms a mass and flows like a liquid, then vortex for another 30 seconds; then divide the sputum evenly into three parts, and add 5% of the sputum weight of normal saline, 5% of the sputum weight of the G-type fucoidan solution obtained in Preparation Example 11 (0.9% weight ratio concentration), and 5% of the sputum weight of the G-type fucoidan solution obtained in Comparative Example 8 (0.9% weight ratio concentration) respectively for rheological testing. The results are shown in Figures 10 - 12 , Figures 13 - 15 .

[0111] Conclusion: Compared with the normal saline group, the G-type fucoidan solution group obtained in Preparation Example 11 can reduce the storage modulus (G') and loss modulus (G") of sputum to less than 5%, and the flow shear force decreases by about 80%; compared with the normal saline group, the G-type fucoidan solution group obtained in Comparative Example 8 has an average decrease of 50% in G' and G", and the flow shear force decreases by about 40%; at the required flow shear force (the first intersection point of G' and G"), the G-type fucoidan solution group obtained in Preparation Example 11 is significantly smaller than the normal saline group and the G-type fucoidan solution group prepared in the comparative example;

[0112] Therefore, the high-purity G-type fucoidan prepared in the present invention can more effectively reduce the elastic modulus and viscous modulus of sputum, and has a significant effect when applied to respiratory care products.

[0113] Experiment Six: Wound healing experiment, specifically as follows:

[0114] Select chronic wounds that are ineffective in conventional treatment in the hospital. On the basis of maintaining the original treatment method, use the G-type fucoidan solution to debride the wound. The specific debridement operation is as follows: Cover the wound with a medical gauze fully moistened with the G-type fucoidan solution obtained in Preparation Example 11 (G-type fucoidan flushing and atomizing functional dressing, Shanxi Medical Device Approval No. 20202140096) (the degree of moistening should be such that water droplets begin to appear). After 15 minutes, remove the gauze, and still use the original treatment method for dressing change and bandaging.

[0115] Figure 16 It was a deep second-degree scald of the lower extremities caused by boiling water. The wound was about 20*15 cm2 in size. Large blisters were visible. Some blister skins were ruptured and shed. The bottom of the wound was red and white. The exudate was clear. The skin temperature around the wound was slightly high, and the tenderness was severe. After 23 days of treatment with the G-type fucoidan flushing and atomizing functional dressing, the wound of the patient epithelialized and healed, without exudation, without purulent secretions and necrotic tissue, and the tenderness was (-);

[0116] Figure 17 It was a local scar rupture wound on the chest wall. The patient underwent radical mastectomy for right breast cancer 8 years ago and received long-term chemotherapy after the operation, resulting in a non-healing right scar rupture for 2 years. Before using the G-type fucoidan flushing and atomizing functional dressing, the skin around the right chest wall wound of the right breast cancer was red and swollen, the local skin temperature was slightly high, the wound exuded a lot, purulent secretions and necrotic tissue were visible, and yellow soft scabs were visible around the wound, with a strong odor. After 3 weeks of treatment with the G-type fucoidan flushing and atomizing functional dressing, the redness and swelling around the right chest wall wound of the patient subsided, the local skin temperature was not high, the wound exudation decreased, there were no purulent secretions and necrotic tissue, and the bottom tissue of the wound was bright red and odorless.

[0117] Moreover, no adverse reactions related to this product were found in the experiment.

[0118] Conclusion: The G-type fucoidan flushing and atomizing functional dressing can promote the subsidence of redness and swelling around the wound, reduce the local skin temperature, make the wound fresh and ruddy, with obvious granulation tissue, and accelerate wound healing; the G-type fucoidan prepared by the present invention can promote the growth of granulation tissue, accelerate wound healing, and has a significant effect when applied to wound care.

Claims

1. A method for preparing G-type fucoidan, characterized in that, It includes the following steps: S1: Prepare a sodium alginate solution with a concentration specific gravity of 1% - 4%. Treat the sodium alginate solution until it is fully swollen. After homogenizing the swollen sodium alginate solution with a high-pressure homogenizer, product one is obtained. The pressure of the high-pressure homogenizer is 400 bar - 860 bar, and the number of circulation times is 3 - 6 times; S2: Take an appropriate amount of product one and mix it evenly with an acid solution to obtain a mixed solution with an acid concentration of 0.1 mol / L - 0.6 mol / L, and heat it to 90°C - 110°C for acid hydrolysis for 7 h - 24 h to obtain product two; S3: Centrifuge the obtained product two for 10 min under a centrifugal force of 8000 g to collect the precipitate to obtain precipitate one; Adjust the pH of precipitate one to neutral for re-dissolution, and then adjust the pH to 2.85 with an acid, and perform secondary centrifugation under the same centrifugation conditions and collect the precipitate again to obtain precipitate two; Adjust the pH of precipitate two to neutral and re-dissolve it to obtain product three; S4: After freeze-drying product three, G-type fucoidan is obtained.

2. The preparation method of G-type fucoidan according to claim 1, wherein, The pressure of the high-pressure homogenizer is 520 bar - 860 bar, and the number of circulation times is 3 - 6 times.

3. The preparation method of type G fucoidan according to claim 1 or 2, characterized in that The acid solution in S2 is any one of sulfuric acid, hydrochloric acid, trifluoroacetic acid, formic acid, and oxalic acid.

4. The preparation method of G-type fucoidan according to claim 1, characterized in that, The acid solution in S2 is a 0.3 mol / L hydrochloric acid solution.

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