Fucoidin as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of seaweed functional products, and particularly relates to fucoidin as well as a preparation method and application thereof, in particular to application of the fucoidin in food and preparation of medicines for adjuvant treatment of age-related maculopathy. According to the invention, alginate lyase is adopted to carry out enzymolysis on seaweed, and the fucoidin with the molecular weight of 20-60 kDa is obtained after treatment of multi-stage alcohol precipitation, hydrogen peroxide degradation and membrane interception. Experimental results show that the fucoidin has the effect of repairing retinal cells, can effectively improve maculopathy, can be used for preparing related products for treating age-related macular degeneration, and provides safer natural material support for treatment of age-related macular degeneration.

Description

Technical Field

[0001] This invention belongs to the technical field of seaweed functional products, specifically relating to a fucoidan, its preparation method and application, and particularly to the application of the fucoidan in food and in the preparation of drugs for adjuvant treatment of age-related macular degeneration. Background Technology

[0002] Age-related macular degeneration (AMD) is a degenerative disease that occurs in the macular region of the retina. It specifically refers to an eye disease in people over 50 years of age caused by damage to the macular structure, resulting in progressive loss of central vision, distorted vision, or central scotoma. It is one of the leading causes of blindness in the elderly worldwide. The macula, a key structure in the posterior pole of the retina, contains a large number of cone cells responsible for fine vision and color perception. Damage to its function directly affects patients' daily activities such as reading and driving, severely reducing their quality of life.

[0003] The etiology of AMD is not fully understood, but it is currently believed to be related to multiple factors, including genetics, aging, oxidative stress, chronic inflammation, vascular abnormalities, and systemic diseases. Based on clinicopathological features, AMD is mainly divided into two types: dry (atrophic) and wet (exudative). The core pathological mechanism of dry AMD is atrophy of the retinal pigment epithelial cells (RPE) in the macular region, thickening of Bruch's membrane, and formation of drusen, ultimately leading to loss of photoreceptor function and progressive vision decline. The core pathological mechanism of wet AMD is abnormal proliferation of choroidal neovascularization (CNV) that breaks through Bruch's membrane, invading the subretinal space, causing exudation, hemorrhage, and scar formation, resulting in rapid loss of central vision. Currently, treatment for wet AMD mainly focuses on inhibiting choroidal neovascularization and reducing exudation and hemorrhage, while there is a lack of targeted radical drugs for dry AMD.

[0004] Polysaccharides, as a class of natural macromolecular compounds, have advantages such as good biocompatibility, low toxicity, and multi-target action. They have shown good effects in oxidative stress, anti-inflammation, and anti-angiogenesis, and have become a research hotspot in recent years.

[0005] Fucoidan is a type of water-soluble sulfated polysaccharide mainly extracted from the cell walls and intercellular matrix of brown algae. Its core structure is based on L-fucose linked by α-(1→3) or α-(1→4) linkages, supplemented by neutral or acidic sugar residues such as galactose, xylose, and glucuronic acid. The molecule is also rich in sulfate ester groups. This unique structure endows it with many biological activities, which is the core basis for its application value.

[0006] As a natural marine bioactive substance, fucoidan has advantages such as low toxicity, good biocompatibility, and wide availability. Its biological functions have been confirmed by a large number of studies, including immune regulation, anti-tumor (CN118580387A), anticoagulation (CN110437288A), lipid-lowering (CN116023522A), anti-inflammatory (CN117343206A, CN115028750A), whitening and antioxidant (CN109055460A), and intestinal flora regulation (CN119144679A). It has broad application prospects in the fields of food, medicine, health products, and cosmetics.

[0007] Currently, there are several published research reports on the application of Lycium barbarum polysaccharides in the treatment of AMD, but there are no reports on the application of fucoidan in AMD. Summary of the Invention

[0008] To address the above-mentioned technical problems, this invention provides a fucoidan, its preparation method, and its application, particularly relating to the application of this fucoidan in food and in the preparation of drugs for adjuvant treatment of age-related macular degeneration.

[0009] The technical solution of the present invention is as follows: A method for preparing fucoidan specifically includes the following steps: S1 Seaweed Pretreatment: The seaweed is washed, impurities removed, dried, and crushed to obtain seaweed powder; S2 enzymatic hydrolysis: Add water to seaweed powder, then add alginate lyase, and perform enzymatic hydrolysis at 45-60℃ and pH 6.0-7.0 for 3-6 hours. After the reaction, centrifuge the hydrolysate and collect the supernatant. The weight of alginate lyase added is 0.5%-5% of the weight of seaweed powder. S3 gradient ethanol precipitation: Concentrate the supernatant obtained in S2, remove the precipitate, add 70% ethanol for ethanol precipitation, then centrifuge to remove the precipitate to obtain crude fucoidan; add 30% ethanol to the obtained crude fucoidan for ethanol precipitation, centrifuge to remove the precipitate; finally add 70% ethanol for ethanol precipitation, centrifuge to remove the precipitate, vacuum dry to obtain a dry sample. S4 Degradation: Add hydrogen peroxide solution and ascorbic acid solution to the dried sample obtained in S3, and react at 45~60℃ for 0.5~3 h. The mass-volume ratio of dried sample to hydrogen peroxide solution is 1 g: 8~15 mL, the concentration of hydrogen peroxide solution is 60~240 mM, the amount of ascorbic acid added is 0.106~0.423 g, and the concentration of ascorbic acid solution is 40~160 mM. S5 Retention and Purification: The reaction solution after S4 degradation is centrifuged to remove the precipitate, the supernatant is subjected to membrane separation, the membrane retention liquid is collected, and vacuum dried to obtain the fucoidan.

[0010] In the above-mentioned preparation method provided by the present invention, preferably, the seaweed mentioned in S1 is selected from any one of wakame seaweed, kelp, Sargassum seaweed, Sargassum fusiforme, and kelp.

[0011] Preferably, the mass-to-volume ratio of seaweed powder to water in S2 is 1 g: 10~20 mL.

[0012] Preferably, the mass-to-volume ratio of seaweed powder to water in S2 is 1 g: 12~18 mL.

[0013] Preferably, the concentration of the hydrogen peroxide solution in S4 is 60-150 mM, and the concentration of the ascorbic acid solution is 40-90 mM.

[0014] Preferably, the pore size of the membrane during membrane separation in S5 is 1000 Da.

[0015] Furthermore, the fucoidan products obtained by the above method also fall within the protection scope of this invention.

[0016] Preferably, the fucoidan comprises, by mass percentage, the following monosaccharides: Fucose 25.88%~45.78%, xylose 0.59%~3.61%, glucose 1.98%~9.64%, mannose 0.42%~6.65%, rhamnose 3.71%~10.80%, galactose 10.60%~30.61%, glucuronic acid 0.95%~3.48%.

[0017] Preferably, the number-average molecular weight of the fucoidan is in the range of 20 to 60 kDa, and the sulfate content in the fucoidan is 21.21% to 38.94%.

[0018] Furthermore, the present invention also provides the application of the fucoidan in the preparation of drugs for adjuvant treatment of age-related macular degeneration.

[0019] Preferably, in the application described, the form of the drug involved includes, but is not limited to, any of the following: oral liquid, capsule, solid granule powder, eye patch, eye drop, and eye spray.

[0020] Preferably, the present invention also provides the application of the fucoidan in food, wherein the food contains the fucoidan and conventionally used additives in food, including but not limited to sweeteners, thickeners and stabilizers.

[0021] Adding the aforementioned fucoidan to food can not only improve vision and alleviate age-related macular degeneration, but also provide some degree of health benefits.

[0022] The beneficial effects of this invention are as follows: (1) A process for preparing fucoidan is provided, wherein seaweed is enzymatically hydrolyzed by alginate lyase, purified by multi-stage alcohol precipitation, and finally degraded. The fucoidan obtained has a molecular weight of 20~60 kDa and contains active ingredients such as antioxidant, anti-inflammatory and anti-angiogenic activities. (2) A new application of fucoidan has been developed, namely, its application in the preparation of products with the effect of preventing and alleviating age-related macular degeneration. The experimental results of this invention show that fucoidan can significantly increase the thickness of the outer nuclear layer (ONL) cells of the retina, effectively alleviate retinal pigment epithelial cell damage, and improve macular degeneration. Attached Figure Description

[0023] Figure 1 HE staining images of the retinas of mice in each group in this invention; Figure 2 These are OCT scans of mice in each group in this invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0025] Example 1 A method for preparing fucoidan, the specific steps of which are as follows: S1 Seaweed Pretreatment: Wash the kelp, remove impurities, dry it at 60℃ until the moisture content is ≤12%, and crush it to obtain kelp powder; S2 enzymatic hydrolysis: Water was added to kelp powder at a mass-to-volume ratio of 1 g: 15 mL, followed by the addition of alginate lyase. Enzymatic hydrolysis was carried out at 45 ℃ and pH 7.0 for 4 h. After the reaction, the hydrolysate was centrifuged at 4000 rpm for 15 min, and the supernatant was collected. The alginate lyase added accounted for 0.75% of the weight of the kelp powder. S3 gradient ethanol precipitation: Concentrate the supernatant obtained in S2, remove the precipitate, add 70% ethanol for ethanol precipitation, then centrifuge to remove the precipitate to obtain crude fucoidan; add 30% ethanol to the obtained crude fucoidan for ethanol precipitation, centrifuge to remove the precipitate; finally add 70% ethanol for ethanol precipitation, centrifuge to remove the precipitate, vacuum dry to obtain a dry sample. S4 Degradation: 120 mM hydrogen peroxide solution and 80 mM ascorbic acid solution were added to the dried sample obtained in S3, and the reaction was carried out at 60℃ for 1 h. The mass-volume ratio of the dried sample to the hydrogen peroxide solution was 1 g: 10 mL, and the amount of ascorbic acid added was 0.1761 g. S5 Retention and Purification: The reaction solution after S4 degradation is centrifuged to remove the precipitate, and the supernatant is separated using a 1000 Da membrane. The membrane retentate is collected and vacuum dried to obtain the fucoidan.

[0026] Comparative Example 1 Unlike Example 1, in the S4 degradation process, only hydrogen peroxide solution (120 mM) was used for degradation, and the other steps were the same as in Example 1; Comparative Example 2 Unlike Example 1, in the S4 degradation process, the concentration of hydrogen peroxide solution was 60 mM and the concentration of ascorbic acid solution was 60 mM, while the other steps were the same as in Example 1. Experimental Example 1 The structures of the fucoidan obtained in Example 1 and the comparative examples were analyzed and characterized as follows: 1-1 Determination of total sugar content of fucoidan Accurately weigh a certain amount of fucoidan sample and fucose standard, react them sequentially with phenol and concentrated sulfuric acid, and then measure the absorbance using a spectrophotometer. Plot a standard curve using fucose as the standard, and calculate the total sugar content in the sample. The results are shown in Table 1.

[0027] Table 1 Total sugar content in fucoidan Total sugar content (%) Example 1 52.73 Comparative Example 1 40.30 Comparative Example 2 42.57

[0028] 1-2 Determination of sulfate content in fucoidan The sample was hydrolyzed with a strong acid to completely release bound sulfate ions into free SO4²⁻. The hydrolysate was mixed with barium chloride reagent, and the weight of the barium sulfate precipitate was measured after the reaction. The mass percentage of sulfate ions in the sample was calculated, and the results are shown in Table 2.

[0029] Table 2. Sulfate content in fucoidan Sulfate content (%) Example 1 31.28 Comparative Example 1 21.09 Comparative Example 2 26.67

[0030] 1-3 Determination of molecular weight of fucoidan A GPC system equipped with a differential refractive index detector (RID) or a multi-angle laser light scattering detector (MALLS) was used. Standard curves were plotted using a series of dextran standards with known molecular weights. Fucoidan samples were dissolved in the mobile phase (phosphate buffer) and injected. The molecular weight was calculated by the retention time, and the results are shown in Table 3.

[0031] Table 3. Molecular weight of fucoidan samples Molecular weight (kDa) Example 1 51.057 Comparative Example 1 240.647 Comparative Example 2 167.932

[0032] 1-4 Determination of Monosaccharide Composition Fucoidan samples were completely acid-hydrolyzed under specific conditions (110°C, 2–4 h). The hydrolysate was neutralized, dried, and then derivatized with PMP reagent. A reversed-phase C18 column was used with gradient elution using phosphate buffer-acetonitrile as the mobile phase, and detection was performed with a UV detector. Qualitative analysis was performed by comparing the retention times with standard monosaccharide derivatives, and quantification was performed using the external standard method. The mass ratios of each monosaccharide were calculated, and the results are shown in Table 4.

[0033] Table 4 Monosaccharide composition in fucoidan Fucose content (%) Mannose content (%) Glucuronic acid content (%) Rhamnose content (%) Glucose content (%) Glucose content (%) Galactose content (%) Example 1 38.713 0.435 1.095 8.334 8.404 2.910 29.114 Comparative Example 1 28.201 3.754 7.081 3.708 5.351 2.792 19.568 Comparative Example 2 33.534 5.495 3.245 2.021 9.084 4.648 30.389

[0034] Experiment Example 2 Application of fucoidan prepared in Example 1 and the comparative examples in the preparation of remedies for age-related macular degeneration.

[0035] Male C57BL / 6J mice (7 weeks old), all individuals were healthy and showed no ocular surface abnormalities. They were acclimatized for one week.

[0036] The selected mice were randomly divided into three groups according to their body weight: a control group, a model group, and a fucoidan group, with 10 mice in each group, for a total of 50 mice. Except for the control group, the other two groups were injected with 8 units of 40 mg / kg NaIO3 once via the tail vein to establish the dAMD model.

[0037] (1) Blank group: 12 hours after modeling, 5 μL of PBS buffer was dropped into the eyes, and the treatment frequency was once a day; (2) Model group: 12 hours after modeling, 5 μL of PBS buffer was dropped into the eyes, and the treatment frequency was once a day; (3) Fucoidan (Example 1) group: 12 h after modeling, 5 μL of fucoidan solution (20 mg / mL) was instilled into the eyes once a day; (4) Comparative Example 1: 12 h after modeling, 5 μL of fucoidan solution (20 mg / mL) was instilled into the eyes once a day; (5) Comparative Example 2: 12 h after modeling, 5 μL of fucoidan solution (20 mg / mL) was instilled into the eyes once a day.

[0038] All groups were kept in the same environment with 12 / 12 hours of light / dark, room temperature (20±2)℃, relative humidity 50%~65%, and good ventilation. They were fed standard mouse feed and allowed free access to food and water.

[0039] 2-1 HE staining (hematoxylin and eosin staining) to detect mouse retinal morphology Mouse eyeballs were harvested at 0, 7, and 14 days after treatment, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 5 µm. Paraffin sections were stained using a hematoxylin and eosin staining kit (Auragene, Hunan, China) according to the manufacturer's instructions. All stained images were taken using an optical microscope (Nikon, Melville, NY) to observe the retinal region. Figure 1 .

[0040] from Figure 1 The results show that, after treatment with fucoidan (Example 1), the retinal inner layer (INL) and outer nuclear layer (ONL) of the macular mice were more tightly packed than those in the model group, and the total retinal thickness was greater. The retinal pigment epithelium was also more tightly packed than that in the model group, with significant differences at 7 and 14 days of treatment, indicating that the fucoidan prepared by the method in Example 1 has a significant effect on improving macular degeneration. The fucoidan groups prepared in Comparative Examples 1 and 2 showed no significant changes compared to the model group.

[0041] 2-2 OCT Mouse Fundus Examination and Retinal Thickness Measurement After 14 days of drug administration, mice were anesthetized by isoflurane inhalation. Compound tropicamide eye drops were instilled into the mice's eyes for 10 minutes to dilate their pupils. The mice were then secured, and their corneas were protected with medical-grade sodium hyaluronate. After fitting a contact lens, OCT was performed to observe the fundus. Cross-sectional images of the mouse retina were obtained, with the optic disc as a landmark. The retinal thickness at a distance of 1000 μm from the optic nerve was measured. Figure 2 .

[0042] according to Figure 2 The results were measured and analyzed using IMAGE J software, and the results are shown in Table 5.

[0043] Table 5. Thickness of retinal cell layer in mice of each group Group / Thickness (µm) Blank group Model group Fucoidan group Comparative Example 1 Comparative Example 2 Internal limiting membrane-ganglionic cell layer 10.5 5.3 10.5 6.1 7.8 Ganglion cell layer - inner network layer 65.9 39 58 40.2 49.8 Inner network layer - kernel layer 27 11.9 25.5 12.6 19.4 Kernel layer - External network layer 16.5 0.5 15.7 1.1 9.7 Outer network layer - outer core layer 53.9 19.2 50.5 22.3 37.2 outer nuclear layer - outer membrane 0 0 3.3 2.1 3.2 External membrane - rod and cone layers 43.3 19.6 38.1 21.3 29.6 Rods and cones - pigment epithelium 26.3 10.5 26.3 11.5 19.6 retina 243.4 106 227.9 117.2 176.3

[0044] Table 5 and Figure 2Data showed that the average total retinal thickness in the control group was 243.4 µm, in the model group it was 106 µm, in the fucoidan group (Example 1) it was 227.9 µm, in Comparative Example 1 it was 117.2 µm, and in Comparative Example 2 it was 176.3 µm. It can be seen that after fucoidan treatment, the total retinal thickness of macular mice increased compared to the model group. The thickness of the outer nuclear layer did not differ significantly from the control group. While Comparative Examples 1 and 2 showed an improvement trend compared to the model group, the improvement effect was significantly weaker than that of Example 1. This indicates that the fucoidan prepared in this invention has the effect of repairing retinal cell damage and can effectively improve macular degeneration.

[0045] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for preparing fucoidan, characterized in that, The steps include the following: S1 Seaweed Pretreatment: The seaweed is washed, impurities removed, dried, and crushed to obtain seaweed powder; S2 enzymatic hydrolysis: Add water to seaweed powder, then add alginate lyase, and perform enzymatic hydrolysis at 45-60℃ and pH 6.0-7.0 for 3-6 hours. After the reaction, centrifuge the hydrolysate and collect the supernatant. The weight of alginate lyase added is 0.5%-5% of the weight of seaweed powder. S3 gradient ethanol precipitation: Concentrate the supernatant obtained in S2, remove the precipitate, add 70% ethanol for ethanol precipitation, then centrifuge to remove the precipitate to obtain crude fucoidan; add 30% ethanol to the obtained crude fucoidan for ethanol precipitation, centrifuge to remove the precipitate; finally add 70% ethanol for ethanol precipitation, centrifuge to remove the precipitate, vacuum dry to obtain a dry sample. S4 Degradation: Add hydrogen peroxide solution and ascorbic acid solution to the dried sample obtained in S3, and react at 45~60℃ for 0.5~3 h. The mass-volume ratio of dried sample to hydrogen peroxide solution is 1 g: 8~15 mL, the concentration of hydrogen peroxide solution is 60~240 mM, the amount of ascorbic acid added is 0.106~0.423 g, and the concentration of ascorbic acid solution is 40~160 mM. S5 Retention and Purification: The reaction solution after S4 degradation is centrifuged to remove the precipitate, the supernatant is subjected to membrane separation, the membrane retention liquid is collected, and vacuum dried to obtain the fucoidan.

2. The preparation method according to claim 1, characterized in that, The seaweed mentioned in S1 is selected from any one of wakame, kelp, Sargassum fusiforme, and kelp.

3. The preparation method according to claim 1, characterized in that, The concentration of the hydrogen peroxide solution in S4 is 60~150 mM, and the concentration of the ascorbic acid solution is 40~90 mM.

4. The preparation method according to claim 1, characterized in that, The membrane pore size described in S5 is 1000 Da during membrane separation.

5. Fucoidan prepared by any one of the preparation methods described in claims 1 to 4.

6. The fucoidan as described in claim 5, characterized in that, The fucoidan, by mass percentage, comprises the following monosaccharides: Fucose 25.88%~45.78%, xylose 0.59%~3.61%, glucose 1.98%~9.64%, mannose 0.42%~6.65%, rhamnose 3.71%~10.80%, galactose 10.60%~30.61%, glucuronic acid 0.95%~3.48%.

7. The fucoidan as described in claim 5, characterized in that, The number-average molecular weight of the fucoidan is in the range of 20 to 60 kDa, and the sulfate content in the fucoidan is 21.21% to 38.94%.

8. The use of the fucoidan as described in claim 5 in the preparation of drugs for adjuvant treatment of age-related macular degeneration.

9. The application as described in claim 8, characterized in that, The drug forms include, but are not limited to, any of the following: oral liquid, capsules, solid granules, eye patches, eye drops, and eye sprays.

10. The application of fucoidan in food as described in claim 5, characterized in that, The food contains the fucoidan, as well as commonly used food additives, including but not limited to sweeteners, thickeners, and stabilizers.

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