Low molecular weight fucoidan and its preparation method and application

Abstract

The application discloses low-molecular-weight fucoidan and a preparation method and application thereof, and the low-molecular-weight fucoidan contains 52% to 65% polysaccharide, 32% to 47% sulfate, 0 to 2% glucuronic acid and 0 to 1% protein, and has an average molecular weight of 2.0 to 4.5 kDa. The low-molecular-weight fucoidan prepared by a specific extraction and separation degradation method can resist high-fat-induced obesity, including reducing the contents of fasting blood glucose, total cholesterol, triglyceride and low-density lipoprotein cholesterol, and promoting the relative abundance of obesity negatively correlated flora and inhibiting the relative abundance of obesity positively correlated flora, thereby preventing the development of obesity, and having great application prospect as a candidate drug for preventing and / or treating obesity.

Description

(I) Technical Field

[0001] This invention belongs to the field of polysaccharide technology, specifically relating to a low molecular weight fucoidan, its preparation method, and its application in the preparation of anti-obesity drugs. (II) Background Technology

[0002] Obesity is a chronic disease that can easily lead to diabetes, hypertension, cardiovascular disease, and many other illnesses. Various factors induce obesity, such as high-fat, high-sugar, and high-calorie diets, genetic mutations, metabolic disorders, and gut microbiota imbalance. In 2022, the global obese population exceeded 1 billion, with 43% of adults being overweight. China's obese population has exceeded 90 million, and in 2020, the economic losses due to overweight amounted to US$283.3 billion. This figure is projected to reach US$458.913 billion by 2025. Obesity has become the fifth leading cause of death worldwide, with overweight individuals causing 5 million deaths from obesity-related diseases. Therefore, finding a drug that can effectively prevent and alleviate obesity is of significant clinical importance.

[0003] Brown algae are an important type of economically important seaweed, including kelp, wakame, Sargassum fusiforme, and staghorn kelp. Kelp (Saccharina japonica) is distributed in coastal provinces such as Fujian, Shandong, Liaoning, Jiangsu, Zhejiang, and Guangdong. Kelp has functions such as anti-radiation, prevention and treatment of goiter, and lowering blood pressure, blood lipids, and blood sugar. Among them, kelp polysaccharides, especially highly sulfated fucoidan, have been reported to have the most biological activities, including antioxidant, anti-inflammatory, anti-tumor, prebiotic, and immunomodulatory effects. However, modern research shows that fucoidan, due to its large molecular weight and complex composition, affects its absorption and metabolism in vivo, resulting in low bioavailability. (III) Summary of the Invention

[0004] The purpose of this invention is to provide a low molecular weight fucoidan, its preparation method, and its application. A low molecular weight fucoidan with a high degree of sulfation is efficiently extracted from kelp. This low molecular weight fucoidan can alleviate weight gain in mice with high fat and high sugar induced obesity and regulate related intestinal flora. It is expected to be developed into an anti-obesity functional food or sugar drug, and significantly improve the bioavailability of fucoidan.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a low molecular weight fucoidan, wherein the low molecular weight fucoidan comprises 52% to 65% polysaccharide by mass, 32% to 47% sulfate by mass, 0% to 2% glucuronic acid by mass, and 0% to 1% protein by mass; and the average molecular weight ranges from 2.0 to 4.5 kDa, preferably from 2.5 to 3.5 kDa.

[0007] Furthermore, the polysaccharide includes fucose, mannose, galactose and glucose, with a molar ratio of 90-95:2-5:3-5:0-1, preferably 92.92:3.00:3.76:0.36.

[0008] Furthermore, the low molecular weight fucoidan preferably contains 41.48% polysaccharide and 37.7% sulfate.

[0009] The low molecular weight fucoidan of this invention has an α-fucose main chain linked by 1→3, and includes (1→3,4)-α-fucose, (1→2,3)-α-fucose, (1→4)-β-mannose, (1→6)-β-galactose, and other branches. Sulfate groups are substituted at the C-2 and C-4 positions of the fucose.

[0010] Secondly, the present invention provides a method for preparing the aforementioned low molecular weight fucoidan, the method comprising the following steps:

[0011] a. Extraction of fucoidan: Dried kelp powder was extracted with calcium chloride aqueous solution at 80-120℃. The extract was concentrated, dialyzed, precipitated with alcohol, and centrifuged. The precipitate was redissolved and dried to obtain calcium-extracted fucoidan.

[0012] b. Purification of fucoidan: Dissolve the calcium-extracted fucoidan prepared in step a in water, add disodium ethylenediaminetetraacetate (EDTA-2Na), adjust the pH to 6.0, add sodium chloride, precipitate with alcohol, and centrifuge; repeat the process of dissolving the precipitate in water, adding sodium chloride, precipitating with alcohol 1-2 times, centrifuging, redissolving the precipitate in water, adjusting the pH to 6.0, filtering through a 0.2 μm filter membrane, and drying to obtain purified fucoidan;

[0013] c. Preparation of low molecular weight fucoidan: Dissolve the purified fucoidan prepared in step b in deionized water a, add anhydrous copper sulfate, adjust the pH to 7-8 (preferably 7.5), then add hydrogen peroxide, and degrade in a water bath at 50-70℃ for 4-6 hours (preferably 60℃ for 5 hours). After the reaction is complete, cool to room temperature, add NaOH to remove copper ions, centrifuge, adjust the pH of the supernatant to neutral, dialyze, dry the retentate, and redissolve it in deionized water b. Purify by anion exchange chromatography, elute by gravity with deionized water c, 0.2M, 0.5M, and 1.0M sodium chloride aqueous solutions for 2-5 column volumes (preferably 3), collect the eluent of 0.5M or 1.0M sodium chloride aqueous solution, concentrate to 50% of the original volume, dialyze, dry the retentate, and obtain the low molecular weight fucoidan.

[0014] Furthermore, in step a, the mass concentration of the calcium chloride aqueous solution is 0.1-1.5%, preferably 1%; the volume of the calcium chloride aqueous solution used is 5-15 mL / g based on the mass of the kelp powder, preferably 10 mL / g.

[0015] Furthermore, in step a, the extraction temperature is 100℃, and the extraction is repeated 2-3 times, each time for 3 hours. Dialysis is performed using a 3kDa molecular weight cutoff membrane dialysis bag.

[0016] Furthermore, the kelp powder in step a is obtained by drying and pulverizing fresh kelp and then passing it through a 100-mesh sieve.

[0017] Further, step a is carried out as follows: Add a 1% calcium chloride aqueous solution to the dried kelp powder, extract at 100℃ for 3 hours, repeat the extraction twice, filter, combine the filtrates, cool to room temperature and concentrate to 20-30% of the original volume, dialyze in pure water with a 3kDa dialysis bag for 72 hours, change the water every 12 hours, add anhydrous ethanol to the effluent to a final ethanol volume concentration of 75%, let stand at room temperature for alcohol precipitation overnight, centrifuge, redissolve the precipitate in water and freeze dry at -40℃ for 48 hours to obtain calcium-extracted fucoidan.

[0018] Further, in step b, the amount of water used for dissolution each time is 10-30 mL / g based on the mass of the calcium-extracted fucoidan, preferably 20 mL / g; the mass ratio of disodium ethylenediaminetetraacetate to calcium-extracted fucoidan is 0.01-0.1:1, preferably 0.05:1; the mass ratio of sodium chloride to calcium-extracted fucoidan each time is 0.1-1:1, preferably 0.4-0.48:1; and the volume of anhydrous ethanol used for each alcohol precipitation is 10-30 mL / g based on the mass of the calcium-extracted fucoidan, preferably 16-20 mL / g.

[0019] Further, step b is performed as follows: Dissolve the calcium-extracted fucoidan prepared in step a in water, stir and dissolve for 60 min in a water bath at 40–45 °C, add EDTA-2Na, adjust the pH to 6.0, add sodium chloride a, and precipitate with anhydrous ethanol a at room temperature, followed by a first centrifugation; add water again to the first precipitate, stir vigorously for 60 min to dissolve completely, add sodium chloride b, and precipitate with anhydrous ethanol b at room temperature, followed by a second centrifugation; redissolve the second precipitate in water, add sodium chloride c, and precipitate with anhydrous ethanol c at room temperature, followed by a third centrifugation; redissolve the third precipitate in water, adjust the pH to 6.0, and filter through a 0.2 μm filter. After membrane drying, purified fucoidan was obtained. The amount of water used for each dissolution was 20 mL / g based on the mass of calcium-extracted fucoidan. The mass ratio of EDTA-2Na to calcium-extracted fucoidan was 0.05:1. Sodium chloride a, b, and c are all sodium chloride, named to indicate the amount used in different steps, and the letters themselves have no meaning. The mass ratio of sodium chloride a to calcium-extracted fucoidan was 0.48:1. The mass ratio of sodium chloride b and c to calcium-extracted fucoidan was 0.4:1. The volume of anhydrous ethanol a was 20 mL / g based on the mass of calcium-extracted fucoidan. The volume of anhydrous ethanol b and c was 16 mL / g based on the mass of calcium-extracted fucoidan.

[0020] Furthermore, in step c, deionized water a, b, and c are all deionized water. They are named for convenience in describing the different amounts used in different steps, and the letters themselves have no meaning. The volume of deionized water a and b is 10-30 mL / g based on the mass of purified fucoidan, preferably 10-15 mL / g (more preferably, the volume of deionized water a is 15 mL / g based on the mass of purified fucoidan, and the volume of deionized water b is 10 mL / g based on the mass of purified fucoidan); the mass ratio of anhydrous copper sulfate to purified fucoidan is 0.01-0.1:1, preferably 0.08:1; the volume concentration of hydrogen peroxide is 30%, and the final concentration added is 8-10%, preferably 9%; the final concentration of NaOH used to remove copper ions is 0.3M.

[0021] Furthermore, in step c, each dialysis session uses a 100Da dialysis bag in deionized water. All drying processes involve freeze-drying at -40°C for 48 hours.

[0022] Furthermore, in step c, the anion exchange chromatography uses a DEAE agarose gel Fast-Flow anion exchange column.

[0023] Thirdly, the present invention provides the use of the aforementioned low molecular weight fucoidan in the preparation of products for the prevention and / or treatment of obesity, the products including food or pharmaceuticals.

[0024] Fourthly, the present invention provides a product for preventing and / or treating obesity, wherein the product is the low molecular weight fucoidan or a food or medicine containing the low molecular weight fucoidan.

[0025] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0026] This invention prepares low molecular weight fucoidan through specific extraction and separation degradation methods. The low molecular weight fucoidan contains 52%–65% polysaccharide and 32%–47% sulfate. The polysaccharide mainly comprises fucose, mannose, galactose, and glucose; its molecular weight is 2.5–3.5 kDa, with sulfate groups substituted at the C-2 and C-4 positions of the fucose group. Furthermore, the low molecular weight fucoidan prepared by this invention can combat hyperlipidemia-induced obesity, including reducing fasting blood glucose, total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels. It also promotes the relative abundance of obesity-negative bacteria and inhibits the relative abundance of obesity-positive bacteria, thereby preventing the development of obesity. It shows great promise as a candidate drug for the prevention and / or treatment of obesity. (iv) Description of the attached drawings

[0027] Figure 1 This is a characteristic high-efficiency gel permeation chromatogram of low molecular weight fucoidan.

[0028] Figure 2 This is the mass spectrometry (MRM) image of low molecular weight fucoidan.

[0029] Figure 3 This is the characteristic infrared spectrum of low molecular weight fucoidan.

[0030] Figure 4 This is the TIC plot of low molecular weight fucoidan by GC-MS.

[0031] Figure 5 Characteristics of low molecular weight fucoidan 13 C NMR spectrum.

[0032] Figure 6 Characteristics of low molecular weight fucoidan 1 H NNMR spectrum.

[0033] Figure 7 The effects of the low molecular weight fucoidan prepared in Example 1 on the body weight change curve (a), cumulative body weight (b), liver weight (c), and epididymal fat weight (d) of mice in a high-fat induced obesity model (* represents P<0.05; ** represents P<0.01 vs. model group; #### represents P<0.0001 vs. control group).

[0034] Figure 8 The graph shows the changes in blood glucose and blood lipids in a high-fat induced obesity mouse model using low molecular weight fucoidan prepared in Example 1, with fasting blood glucose (a), total cholesterol (b), and triglycerides (c).

[0035] Figure 9 The effects of low molecular weight fucoidan prepared in Example 1 on the α diversity of gut microbiota in a high-fat induced obesity mouse model: ace index (a), Chao 1 index (b), faith_pd index (c).

[0036] Figure 10 The effects of low molecular weight fucoidan prepared in Example 1 on the phylum level of the intestinal flora in a high-fat induced obesity mouse model: Bacteroidetes (a), Verrucous Microbes (b), Campylobacteria (c), and Proteobacteria (d).

[0037] Figure 11The effects of the low molecular weight fucoidan prepared in Example 1 on the gut microbiota levels in a high-fat induced obesity mouse model are as follows: g__Akkemansia;s__uncultured_bacterium(a), g__Muribaculaceae;s__uncultured_bacterium(b), f__Oscillospiraceae;__;__(c), g__Rikenellacear_RC9_gut_group(d), g__Blautia;s__Lachnospiraceae_bacterium(e), g__Eubacterium_fissicatena_group;s__uncultured_bacterium(f). (V) Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0039] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the examples are commercially available.

[0041] Material:

[0042] The kelp was purchased from Rongcheng City, Shandong Province.

[0043] equipment:

[0044] High-performance gel permeation chromatography was performed using TSK G5000 pwxl (7.8×300mm, Tosoh Corporation, Japan) and TSK G3000 pwxl (7.8×300mm, Tosoh Corporation, Japan) columns in series, and standard curves were prepared using a series of standard dextrans with different molecular weights.

[0045] High performance liquid chromatography (HPLC) was performed using a Waters e2695 HPLC system (Waters Corporation, USA).

[0046] High performance liquid chromatography-mass spectrometry (LC-MS) analysis was performed using an AB Exion LC-Triple Quad 3500 LC-MS system (AB SCIEX, USA).

[0047] Infrared analysis was performed using a Nicolet 6700 Fourier transform infrared spectrometer (Thermo Scientific, USA).

[0048] The gas chromatograph-mass spectrometer used was a 7890A-7000B GC-MS system (Agilent Technologies, USA).

[0049] Nuclear magnetic resonance (NMR) analysis was performed using an AVANCE III 500MHz NMR spectrometer (Bruker, Switzerland).

[0050] Example 1: Preparation of low molecular weight fucoidan

[0051] a. Fucoidan extraction:

[0052] After drying, the kelp was mechanically pulverized and passed through a 100-mesh sieve. 200g of the powder was added to 2L of 1% calcium chloride aqueous solution and extracted at 100℃ for 3h. The mixture was filtered, and the filter cake was extracted twice more. The filtrates were combined and cooled to room temperature (25-30℃). The mixture was concentrated to 1.6L (27% of the filtrate volume) by rotary evaporation at 60℃. The mixture was dialyzed in pure water for 72h using a 3kDa molecular weight cutoff membrane dialysis bag, with the water changed every 12h. The cutoff liquid was then added to anhydrous ethanol until the final ethanol concentration was 75%. The mixture was allowed to stand at room temperature for alcohol precipitation overnight. The supernatant was removed by centrifugation, and the precipitate was reconstituted with water and freeze-dried at -40℃ for 48h to obtain 5.2g of calcium-extracted fucoidan (yield 2.6%).

[0053] b. Fucoidan purification:

[0054] Take 5g of the calcium-extracted fucoidan prepared above, dissolve it in 100mL of deionized water, stir and dissolve in a water bath at 40-45℃ for 60min, add 0.25g of disodium ethylenediaminetetraacetate (EDTA-2Na), adjust the pH to 6.0 with 0.1M NaOH solution, add 2.4g of NaCl, then add 100mL of anhydrous ethanol, let stand at room temperature for alcohol precipitation overnight; centrifuge to remove the supernatant, redissolve the precipitate in 100mL of deionized water, stir vigorously for 60min to fully dissolve it, add 2g of... After fully dissolving NaCl, precipitate the precipitate overnight with 80 mL of anhydrous ethanol. Centrifuge to remove the supernatant, collect the precipitate, redissolve it in 100 mL of deionized water, stir to fully dissolve, add 2 g of NaCl, and then add 80 mL of anhydrous ethanol for precipitation overnight. Centrifuge to remove the supernatant, redissolve the precipitate in 100 mL of deionized water, adjust the pH to 6.0 with 1 M HCl, filter through a 0.2 μm filter membrane, and freeze-dry the filtrate at -40℃ for 48 h to obtain 2.56 g of fucoidan (yield 51.2%).

[0055] c. Preparation of low molecular weight fucoidan:

[0056] Dissolve 1 g of the prepared fucoidan in 15 mL of deionized water, add 80 mg of anhydrous copper acetate, adjust the pH to 7.5 with 2 M NaOH, add 30% H2O2 aqueous solution to a final volume concentration of 9%, incubate at 60 °C for 5 h, cool to room temperature after the reaction is complete, add 0.3 M NaOH to remove copper ions, centrifuge to remove the precipitate, take the supernatant, adjust the pH to neutral, dialyze in pure water using a 100 Da dialysis bag, freeze-dry the retentate at -40 °C for 48 h, and obtain 0.62 g of degraded low molecular weight fucoidan (yield 62%). 500 mg of degraded low molecular weight fucoidan was dissolved in 10 mL of deionized water and purified by DEAE agarose gel Fast-Flow anion exchange chromatography column. The column was washed with pure water, 0.2 M, 0.5 M, and 1.0 M NaCl aqueous solutions for 3 column volumes each. The 1.0 M eluent was collected, concentrated to 50% of its original volume by rotary evaporation, and dialyzed against pure water for 48 h using a 100 Da molecular weight cutoff membrane. The cutoff solution was lyophilized at -48 °C to obtain 240 mg of low molecular weight fucoidan (yield 48%).

[0057] d. Identification and analysis of the structure of low molecular weight fucoidan:

[0058] (1) Polysaccharide content determination

[0059] Using glucose as a standard, the total sugar content was determined by the phenol-sulfuric acid method, indicating that the total sugar content of low molecular weight fucoidan was 41.48%. Using potassium sulfate solution as a standard, the sulfate content was determined by the gelatin-barium chloride method, indicating that the sulfate content was 37.70%. Using glucuronic acid as a standard, the glucuronic acid content was determined by the m-hydroxybiphenyl method, indicating that the glucuronic acid content was 0. In addition, the protein content was determined by the Coomassie brilliant blue method to be 0.

[0060] (2) Determination of relative molecular weight

[0061] The relative molecular weight of the low molecular weight fucoidan was determined to be 2.6 kDa by high performance gel permeation chromatography (HPGPC), and its purity was over 95% (e.g., Figure 1 ).

[0062] (3) Determination of monosaccharide composition and content

[0063] The monosaccharide composition and content were determined by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) after complete hydrolysis. 2 mg of low molecular weight fucoidan was dissolved in 1 mL of 4 M TFA (trifluoroacetic acid) aqueous solution, sealed, and hydrolyzed at 110 °C for 6 h. After cooling to room temperature and drying, 0.2 mL of methanol was added, and the mixture was dried again. This process was repeated three times. 100 μL of 0.1 M NaOH aqueous solution was added, and the volume was adjusted to 1 mL with water to obtain the hydrolyzed sample. 400 μL of the hydrolyzed sample was added to 450 μL of 0.3 M NaOH aqueous solution and 450 μL of 0.5 M MPa methanol solution. The mixture was reacted at 70 °C for 30 min, cooled to room temperature, and then neutralized with 450 μL of HCl. The mixture was extracted three times with chloroform, the lower layer was discarded, and the upper layer was filtered through a 0.2 μm membrane and analyzed by mass spectrometry (MRM). Chromatographic conditions: ACQUITY Premier HSS T3 Column (2.1 × 100 mm, 1.8 μm); mobile phase: A acetonitrile: B water (5 mM ammonium acetate) = 75:25; flow rate: 0.3 mL / min; column temperature: 40 °C; mass spectrometry conditions: ESI negative source; spray voltage: -4500 V; temperature: 600 °C. Results are shown below. Figure 2 As shown, the results indicate that low molecular weight fucoidan mainly contains fucose, mannose, galactose and glucose, with a molar ratio of 92.92:3.00:3.76:0.36.

[0064] (4) Infrared spectroscopy detection

[0065] Infrared spectra were detected using a Nicolet 6700 Fourier transform infrared spectrometer (Thermo Scientific, USA). Figure 3 ), 3502.11cm -1 The absorption peak is for the OH stretching vibration, at 2988.84 cm⁻¹. -1 The absorption peak for the CH stretching vibration is 1027.25 cm⁻¹. -1 The peak at 1216.31 cm⁻¹ represents the vibrational absorption peak of the glycosidic bonds COH and COC. -1 The peak value is the stretching vibration peak of S=O, at 960.82 cm⁻¹. -1 The absorption peak is due to the asymmetric vibration of COS, at 827.80 cm⁻¹. -1 This is an absorption peak associated with the COS symmetric vibration.

[0066] (5) Detection of glycosidic bond linkage

[0067] Low molecular weight fucoidan was desulfurized using cation exchange resin, and then methylated before GC-MS detection.

[0068] 732# cation exchange resin was packed into a chromatography column and soaked in a 10% sodium chloride aqueous solution at room temperature for 24 hours. It was then rinsed with water until colorless. 1 mol / L hydrochloric acid was slowly passed through the resin for 2-3 column volumes. After the acid was completely introduced, the resin was soaked for 1 hour and then rinsed with water until the pH of the effluent was 5. 50 mg of low molecular weight fucoidan was loaded into 5 mL of deionized water. After loading, the solution was allowed to stand and acidify for 30 min. Three column volumes of deionized water were used for elution. The pH of the eluent was adjusted to 9 with pyridine. The solution was concentrated under reduced pressure to 10% of its original volume and freeze-dried at -40°C for 48 h. The resulting sample was dissolved in 3 mL of a mixed solvent of 89% DMSO, 10% methanol, and 1% pyridine. The solution was incubated at 110°C for 4 h, and the reaction was terminated by adding water. The solution was dialyzed in pure water using a 3 kDa dialysis bag for 48 h. The retentate was concentrated under reduced pressure to 10% of its original volume and then freeze-dried at -40°C for 48 h to obtain 12 mg of desulfurized polysaccharide. 10 mg of the desulfurized polysaccharide was added to 5 mL of DMSO and stirred until fully dissolved. 0.1 g of the solution was then added... NaOH was added, and the mixture was stirred for 2 hours under an anhydrous and oxygen-free environment with argon gas. 1 mL of iodomethane was added in an ice-water bath, and the reaction continued for another 2 hours. 2 mL of water was then added to terminate the reaction. An equal volume of chloroform was added, and the mixture was shaken thoroughly to extract the polysaccharide. This process was repeated three times. The upper aqueous layer was removed, and the mixture was concentrated to 20% of its original volume by rotary evaporation and then freeze-dried at -40°C for 48 hours to obtain 2 mg of methylated polysaccharide. 2 mg of methylated polysaccharide was transferred to an ampoule, and 4 mL of 2 mol / L TFA aqueous solution was added. The mixture was reacted at 100°C for 6 hours. 4 mL of methanol was added to remove the TFA, and the polysaccharide was dissolved in 3 mL of water. 20 mg of methylated polysaccharide was then added. NaBH4 was reacted at room temperature for 3 hours. 1 mL of glacial acetic acid was added to neutralize the reaction, followed by 1 mL of methanol. The mixture was then evaporated to dryness, and another 1 mL of methanol was added. Finally, 1 mL of methanol was added, and the mixture was evaporated to dryness again. The mixture was then dried at 60°C for 2 hours. 2 mL of glacial acetic anhydride and 2 mL of pyridine were added, and the reaction was continued at 100°C for 1 hour. The reaction was terminated by adding 1 mL of deionized water. The mixture was extracted three times with chloroform, and the chloroform layer was collected and analyzed by GC-MS. The TIC chromatogram is shown below. Figure 4 Database comparison results show that this low molecular weight fucoidan is mainly composed of a backbone linked by α-(1→3)-L-fucoside bonds, and contains branches with (1→3,4)α-fucose, (1→2,3)α-fucose, (1→4)β-mannose, and (1→6)β-galactose.

[0069] (6) Nuclear magnetic resonance analysis

[0070] 100 mg of low molecular weight fucoidan was dissolved in 0.5 mL of D2O, freeze-dried at -40 °C for 24 h, and the process was repeated three times. The resulting solution was analyzed using a Bruker Avance 500 MHz nuclear magnetic resonance spectrometer. 13 In C NMR ( Figure 5The δ97.5 signal belongs to 1,3-α-Fuc4S, the δ96.9 signal belongs to 1,3-α-Fuc2,4S, the δ89.4 signal belongs to 1,3-α-Fuc2S, the δ78~δ60 signals belong to the C2~C5 signal peaks of α-Fuc, and the δ16.0 signal belongs to the C6 signal peak of α-Fuc. 1 In the H NMR spectrum ( Figure 6 The signal at δ5.47 is assigned to 1,3-α-Fuc2S, the signal at δ5.36 is assigned to 1,3-α-Fuc4S, the signal at δ5.09 is assigned to 1,3-α-Fuc2,4S, and the signals at δ4.72 and δ4.41 are characteristic signals of β-glycosidic bonds, indicating that this polysaccharide contains 1,4-β-Man and 1,6-β-Gal.

[0071] Example 2: Anti-obesity activity of low molecular weight fucoidan

[0072] (1) Construction of obesity model

[0073] This animal experiment followed the guidelines for the management and use of laboratory animals and was approved by the Animal Ethics Committee of Zhejiang University of Technology. Fifteen 6-week-old male C57BL / 6J mice were purchased from Spifort (Beijing) Biotechnology Co., Ltd. The animal center provided a temperature of 22-26℃, a relative humidity of 50-60%, a 12-hour light / dark cycle, and free access to food and water.

[0074] After one week of acclimatization, the animals were randomly divided into three groups: a control group, a model group, and an experimental group (LMWF), with five animals in each group. The control group was fed a low-fat, sugar-free diet (3.6 kcal / g, TP23303, Nantong Trofi), while the model and experimental groups were fed sterile water and a high-fat, sugar-free diet (60% high-fat, sugar-free diet, 5.1 kcal / g, TP23301, Nantong Trofi). Each group was fed for four weeks to establish an obesity model.

[0075] (2) Low molecular weight fucoidan anti-obesity treatment

[0076] From weeks 5 to 8, the experimental group was administered low molecular weight fucoidan prepared according to the method in Example 1 by gavage at a dose of 50 mg / kg daily. The control group and the model group were administered the same amount of water by gavage. During the experiment, body weight was measured weekly. After fasting for 12 hours at the end of the fourth and eighth weeks, fasting blood glucose was measured via the tail vein using a glucometer. After the eighth week, the mice were fasted for 12 hours, then sacrificed, and blood, liver, epididymis, and contents of the cecum were collected for physiological indicators and intestinal flora detection.

[0077] (3) Measurement of physiological indicators

[0078] Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were determined according to the standard operating procedures provided by Nanjing Jiancheng Commercial Biochemical Reagent Kit. Results are as follows: Figure 7 As shown in Figures a and b, with increasing high-fat diet feeding time, the body weight of mice in the model group increased rapidly after eight weeks of feeding, with a cumulative body weight significantly higher than that of mice on a low-fat diet (P<0.0001). After gavage administration of polysaccharide at a dose of 50 mg / kg in the fourth week, the rate of body weight gain in mice administered low molecular weight fucoidan was significantly lower than that in the model group (P<0.01). Figures c and d show that with the body weight gain induced by the high-fat diet, the liver weight and epididymal fat weight of mice in the model group also increased significantly (P<0.0001, P<0.0001). Gavage administration of low molecular weight fucoidan can effectively improve obesity induced by a high-fat diet in mice.

[0079] Effects on blood sugar and blood lipids, such as Figure 8 As shown, after four weeks of high-fat diet feeding, the fasting blood glucose in the model group was much higher than that in the low-fat diet control group (P<0.001). a shows that gavage administration of low molecular weight fucoidan for four weeks can reduce blood glucose concentration (P<0.0001); b and c show that compared with the low-fat diet, the serum TC and TG levels of mice fed the high-fat diet in the model group were increased, while the serum TC and TG levels in the experimental group were lower (P<0.01, P<0.0001). The experimental group had no significant effect on HDL-C levels, but it could reduce LDL-C levels (P<0.01).

[0080] (4) 16S rRNA analysis of gut microbiota

[0081] Microbial DNA was extracted from mouse cecal contents using the KAPA HiFi HotStart ReadyMix kit. The V3-V4 region of the 16S rRNA gene was amplified using primers 338F (5'-CCTACGGGNGGCWGCAG-3') and 806R (5'-GACTACHVGGGTATCTAATCC-3'). PCR conditions were set as follows: 95°C pre-denaturation for 3 min, 95°C denaturation hold for 0.5 min, 55°C annealing for 0.5 min, 72°C hold for 0.5 min, for 25 cycles, followed by a final extension at 72°C for 5 min. An amplicon library was prepared using the TruSeq DNA Sample Preparation Kit. The amplicon library was then analyzed using a Qubit Fluorometer. Before sequencing, amplicon purification was performed using Ampure XP Beads, quantification was performed using the KAPA Illumina Library Quantification Kit, and sequencing was finally performed by Hangzhou Kaitai Biotechnology Co., Ltd. on their Illumina Hiseq PE250 platform. Bioinformatics analysis of the sequencing data was performed using the microbiome analysis platform QIIME2 2022.2 (QuantitativeInsights Into Microbial Ecology, USA). The q2-dada2 plugin was used to remove sequencing noise and perform quality filtering on the sequencing data, removing low-quality sequences and selecting unique sequences. The sequences were classified into species using the classifier silval38-ssu-nr99-338f-806r-classifier to generate a feature table for subsequent analysis.

[0082] (5) Analysis of gut α-diversity results

[0083] Assessing the impact of gut α-diversity, such as Figure 9 As shown in Figures a and b, compared with the control group, the ace and Chao1 indices of mice in the high-fat diet model group were decreased (P<0.0001, p<0.0001). After four weeks of gavage administration of polysaccharides, the ace and Chao1 indices of the experimental group significantly increased, almost returning to the control group level. Figure c shows that compared with the control group, the faith_pd index of mice in the model group was decreased (P=0.003), while the faith_pd index of the experimental group was slightly increased compared with the model group. These results indicate that a high-fat diet significantly reduces the diversity and number of species in the gut microbiota of mice, while low molecular weight fucoidan can significantly restore the gut microbiota dysbiosis caused by a high-fat diet.

[0084] (6) Analysis of gut microbiota at the phylum level

[0085] The effects of low molecular weight fucoidan on the gut microbiota at the phylum level mainly include Firmicutes, Bacteroidetes, Desulfurized Bacteria, Verrucous Microbes, Deferobacteria, and Campylobacteria. For example... Figure 10 As shown in Figure a, the relative abundance of Bacteroidetes in the experimental group was significantly higher than that in the control group (P<0.0001), and low molecular weight fucoidan significantly increased the relative abundance of Bacteroidetes (P<0.0001); Figure b shows that the model group and the experimental group had no significant effect on Verrucous Microbes; Figure c shows that the experimental group could improve or reverse the increase in Campylobacteria abundance caused by a high-fat diet; Figure d shows that the experimental group could improve the increase in Proteobacteria abundance caused by a high-fat diet. These results indicate that a high-fat diet leads to an increase in the abundance of Firmicutes, Deferrobacteria, Campylobacteria, and Proteobacteria, while decreasing the abundance of Bacteroidetes, Desulfobacteria, and Verrucous Microbes. Gavage administration of low molecular weight fucoidan can effectively improve the intestinal flora at the phylum level.

[0086] (7) Analysis of gut microbiota at the species level

[0087] The effects of low molecular weight fucoidan on gut microbiota at the species level, such as Figure 11 As shown in Figures a, b, and c, a high-fat diet induces a decrease in the relative abundance of *g__Akkermansia*, *g__Muribaculaceae*, and *f__Oscillospiraceae*, which are significantly alleviated by gavage administration of low molecular weight fucoidan. Simultaneously, Figure d shows that a high-fat diet increases the abundance of *g__Rikenellaceae*, which is effectively reduced by gavage administration of low molecular weight fucoidan. Figures e and f show that gavage administration of low molecular weight fucoidan can improve the abundance reduction of *g__Blautia* and *g__Eubacterium* induced by a high-fat diet. These results indicate that low molecular weight fucoidan has a regulatory effect on the gut microbiota of mice fed a high-fat diet at the genus level.

[0088] In summary, the test results show that low molecular weight fucoidan can reduce obesity induced by a high-fat diet, including reducing fasting blood glucose, total cholesterol, triglycerides and low-density lipoprotein cholesterol levels. At the same time, it promotes the relative abundance of obesity-negative bacteria and inhibits obesity-positive bacteria, thereby preventing the development of obesity. This low molecular weight polysaccharide may be a potential carbohydrate drug for the prevention and / or treatment of obesity.

[0089] Example 3: Preparation and performance testing of low molecular weight fucoidan

[0090] 1. Polysaccharide preparation

[0091] The fucoidan prepared in step b of Example 1 is designated as polysaccharide A.

[0092] In step c of Example 1, the final concentration of H2O2 added was changed to 4.5%, and the collection of 1.0M elution fraction was changed to collection of 0.5M elution fraction. All other operations were the same, and low-sulfated low molecular weight fucoidan (yield 32%) was obtained, which was denoted as polysaccharide B.

[0093] In step c of Example 1, the final concentration of H2O2 added was changed to 4.5%, and other operations were the same. The high-sulfated low molecular weight fucoidan (52%) was denoted as polysaccharide C.

[0094] In Example 1, step c, the collection of 1.0M elution fraction was changed to the collection of 0.5M elution fraction, while other operations remained the same. The resulting low molecular weight fucoidan was denoted as polysaccharide D.

[0095] 2. Structural analysis and identification

[0096] The polysaccharides A, B, C, and D from step 1 were detected using the method described in Example 1, and the results are as follows:

[0097] (1) Polysaccharide content determination

[0098] The total sugar contents of polysaccharides A, B, C, and D were 60.97%, 45.33%, 41.74%, and 43.53%, respectively; the sulfate contents were 20.15%, 22.56%, 29.81%, and 27.21%, respectively; the glucuronic acid contents were 2.5%, 2.1%, 0.2%, and 0%, respectively; and the protein contents determined by the Coomassie brilliant blue method were 0.07%, 0.11%, 0.01%, and 0%, respectively.

[0099] (2) Determination of relative molecular weight

[0100] The relative molecular masses of polysaccharides A, B, C, and D are 345.6 kDa, 16.6 kDa, 15.8 kDa, and 2.9 kDa, respectively.

[0101] (3) Determination of monosaccharide composition and content

[0102] The molar ratios of fucose, mannose, galactose, and glucose are as follows:

[0103] A: 66.43: 25.63: 5.52: 1.41

[0104] B: 61.87: 23.66: 12.62: 1.2

[0105] C:74.20:19.85:5.48:8.71

[0106] D:91.88:2.85:3.61:0.42

[0107] (4) Infrared spectroscopy detection

[0108] All sugars are at 3493-3502cm. -1 The absorption peaks for the stretching vibration of OH are observed at 2988-2989 cm⁻¹. -1 The weak absorption peak at 1216-1219 cm⁻¹ is due to the CH stretching vibration. -1 The absorption peak is due to the S=O stretching vibration, 960-962 cm⁻¹. -1 The absorption peak indicates the presence of a COS asymmetric stretching motion peak.

[0109] (5) Detection of glycosidic bond linkage

[0110] Polysaccharides A, B, and C are mainly composed of a backbone linked by α-(1→3)-L-fucoside bonds and β-(1→4)-D-mannoside bonds. The backbone also contains α-(1→4)-L-fucoside bonds, α-(1→3,4)-L-fucoside bonds, α-(1→2,3)-L-fucoside bonds, β-(1→6)-D-galactoside bonds, and β-(1→3)-D-glucoside bonds. Polysaccharide D is mainly composed of a backbone linked by α-(1→3)-L-fucoside bonds.

[0111] (6) Nuclear magnetic resonance analysis

[0112] exist 13 In C NMR, the δ97.5 signal is assigned to 1,3-α-Fuc4S, the δ96.9 signal to 1,3-α-Fuc2,4S, the δ89.4 signal to 1,3-α-Fuc2S, the δ78~δ60 signals to the C2~C5 signal peaks of α-Fuc, and the δ16.0 signal to the C6 signal peak of α-Fuc. 1 In the 1H NMR spectrum, the signal at δ5.47 is assigned to 1,3-α-Fuc2S, the signal at δ5.36 is assigned to 1,3-α-Fuc4S, the signal at δ5.09 is assigned to 1,3-α-Fuc2,4S, and the signals at δ4.72 and δ4.41 are characteristic signals of β-glycosidic bonds, indicating that the polysaccharides A, B, C, and D all contain 1,4-β-Man and 1,6-β-Gal.

[0113] The above results indicate that as the concentration of hydrogen peroxide increases, the molecular weight of the obtained fucoidan gradually decreases, and the proportion of mannose gradually decreases; the sulfate content of the fraction eluted with low concentration NaCl in the DEAE agarose gel Fast-Flow anion exchange chromatography column purification is lower than that of the fraction eluted with high concentration NaCl.

[0114] 3. Anti-obesity activity of fucoidan

[0115] The anti-obesity properties of polysaccharides A, B, C, and D from step 1 were tested using the method described in Example 2. The results are as follows:

[0116] (1) Measurement of physiological indicators

[0117] Polysaccharides A, B, C, and D all effectively inhibited the rate of weight gain in mice, but their effects were not as good as those of the low molecular weight fucoidan prepared in Example 1. Polysaccharides B and C reduced blood glucose concentration (P<0.01, P<0.01), while polysaccharides A and D showed no significant difference in their reducing effects, but their effects were not as good as those of the low molecular weight fucoidan prepared in Example 1. Polysaccharides A, B, C, and D all reduced serum total cholesterol (TC) levels, and polysaccharides A, C, and D all reduced serum triglyceride (TG) levels, but their effects were not as good as those of the low molecular weight fucoidan prepared in Example 1.

[0118] (2) Analysis of gut α-diversity results

[0119] Polysaccharides A, B, C, and D all had relatively small effects on gut microbiota dysbiosis.

[0120] (3) Analysis of gut microbiota at the phylum level

[0121] Polysaccharides A, B, and C all increased the relative abundance of Bacteroidetes at the phylum level, comparable to the effects of polysaccharide D and Example 1; their enrichment ability for Verrucomicrobia was relatively weak, lower than that of polysaccharide D and Example 1; their effects on Campylobacteria were similar, with no significant reversal; in Proteobacteria, they reduced the abundance increase caused by a high-fat diet, which was similar to the results of the low molecular weight polysaccharide prepared in Example 1, while polysaccharide D could not reduce the abundance of Proteobacteria.

[0122] (4) Analysis of gut microbiota at the species level

[0123] Polysaccharides A, B, and C could not increase the abundance reduction of *g* Akkermansia s_uncultured_bacterium caused by a high-fat diet. Polysaccharide D was more effective than polysaccharides A, B, and C, but less effective than in Example 1. Regarding *g* Muribaculaceae s_uncultured_bacterium, polysaccharides A and B could not reverse the abundance reduction caused by a high-fat diet. Polysaccharides C and D could increase its abundance, but less effectively than in Example 1. Polysaccharides A, B, and D could increase the abundance of *f* Oscillospiraceae, with effects similar to Example 1. Polysaccharide C was less effective than the other groups. Polysaccharides A, B, C, and D could all reduce the abundance increase of *g* Rikenellaceae RC9_gut_group caused by a high-fat diet. Polysaccharide D was more effective than A, B, and C, but less effective than in Example 1. Polysaccharides A, B, C, and D could all increase the abundance of *g* Odoribacter, with polysaccharides B and D showing the best effects, superior to Example 1.

[0124] The low molecular weight fucoidan prepared in Example 1 showed the best effect, while the undegraded fucoidan showed the worst effect, followed by the degradation effect of 4.5% hydrogen peroxide. Under the same molecular weight conditions, the higher degree of sulfation was better than the lower degree of sulfation.

[0125] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope defined by the claims of the present invention; and such modifications or substitutions are still within the scope defined by the claims of the present invention.

Claims

1. A method for preparing low molecular weight fucoidan for the prevention and / or treatment of obesity, characterized in that, The method includes the following steps: a. Extraction of fucoidan: Dried kelp powder was extracted with calcium chloride aqueous solution at 80-120℃. The extract was concentrated, dialyzed, precipitated with alcohol, and centrifuged. The precipitate was redissolved and dried to obtain calcium-extracted fucoidan. b. Purification of fucoidan: Dissolve the calcium-extracted fucoidan prepared in step a in water, add disodium ethylenediaminetetraacetate, adjust the pH to 6.0, add sodium chloride, precipitate with alcohol, and centrifuge; repeat the process of dissolving the precipitate in water, adding sodium chloride, precipitating with alcohol 1-2 times, centrifuging, redissolving the precipitate in water, adjusting the pH to 6.0, filtering through a 0.2 μm filter membrane, and drying to obtain purified fucoidan; c. Preparation of low molecular weight fucoidan: Dissolve the purified fucoidan prepared in step b in deionized water (a), add anhydrous copper sulfate, adjust the pH to 7-8, then add hydrogen peroxide, and degrade in a water bath at 50-70℃ for 4-6 hours. After the reaction is complete, cool to room temperature, add NaOH to remove copper ions, centrifuge, adjust the pH of the supernatant to neutral, dialyze, dry the retentate, and redissolve in deionized water (b). Purify by anion exchange chromatography, eluting with deionized water (c), 0.2 M, 0.5 M, and 1.0 M sodium chloride aqueous solutions for 2-5 column volumes each by gravity. Collect the eluent of 0.5 M or 1.0 M sodium chloride aqueous solution, concentrate to 50% of the original volume, dialyze, dry the retentate, and obtain the low molecular weight fucoidan with a molecular weight of 2.6-2.

9. kDa; the mass ratio of anhydrous copper sulfate to purified fucoidan is 0.01-0.1:1; the hydrogen peroxide volume concentration is 30%, and the final concentration is 8-10%; the NaOH used to remove copper ions is added to a final concentration of 0.3 M.

2. The method as described in claim 1, characterized in that, In step a, the mass concentration of the calcium chloride aqueous solution is 0.1-1.5%; the volume of the calcium chloride aqueous solution used is 5-15 mL / g based on the mass of the kelp powder.

3. The method as described in claim 1, characterized in that, Step a is carried out as follows: Add 1% calcium chloride aqueous solution to dried kelp powder, extract at 100℃ for 3 h, repeat extraction twice, filter, combine the filtrates, cool to room temperature and concentrate to 20-30% of the original volume, dialyze in pure water with a 3 kDa dialysis bag for 72 h, change the water every 12 h, add anhydrous ethanol to the effluent to a final ethanol volume concentration of 75%, let stand at room temperature for alcohol precipitation overnight, centrifuge, redissolve the precipitate in water and freeze dry at -40℃ for 48 h to obtain calcium-extracted fucoidan.

4. The method as described in claim 1, characterized in that, In step b, the amount of water used for each dissolution is 10-30 mL / g based on the mass of the calcium-extracted fucoidan; the mass ratio of disodium ethylenediaminetetraacetate to calcium-extracted fucoidan is 0.01-0.1:1; the mass ratio of sodium chloride to calcium-extracted fucoidan is 0.1-1:1; and the volume of anhydrous ethanol used for each alcohol precipitation is 10-30 mL / g based on the mass of the calcium-extracted fucoidan.

5. The method as described in claim 1, characterized in that, Step b is carried out as follows: Dissolve the calcium-extracted fucoidan prepared in step a in water, stir and dissolve for 60 min under a water bath at 40~45℃, add EDTA-2Na, adjust the pH to 6.0, add sodium chloride a, precipitate with anhydrous ethanol a at room temperature, and centrifuge for the first time. The first precipitate was added to water again and stirred vigorously for 60 min to dissolve completely. Sodium chloride b was added, and the mixture was precipitated with anhydrous ethanol b at room temperature and centrifuged a second time. The second precipitate was redissolved in water, sodium chloride c was added, and the mixture was precipitated with anhydrous ethanol c at room temperature and centrifuged a third time. The third precipitate was redissolved in water, the pH was adjusted to 6.0, filtered through a 0.2 μm filter membrane, and dried to obtain purified fucoidan.

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