A mussel polysaccharide, and a preparation method and application thereof

Abstract

The application discloses a mussel polysaccharide and a preparation method and application thereof. The mussel polysaccharide MP-I with a novel structure is prepared from Mytilus coruscus through the process steps of water extraction and concentration, enzymatic deproteinization, alcohol precipitation and anion exchange, and the like. Animal experiments prove that the mussel polysaccharide MP-I prepared by the application can effectively improve intestinal flora and relieve intestinal inflammation by inhibiting the expression of inflammatory signal pathways such as JAK2 / STAT3 and NF-kappa B. Therefore, the mussel polysaccharide MP-I is a natural and safe drug or food for improving inflammatory bowel disease, can effectively improve the inflammatory bowel disease, is safe, non-toxic and free of side effects, the raw material is abundant, the steps are simple, the cost is low, and the effect is remarkable.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a mussel polysaccharide, its preparation method, and its application. Background Technology

[0002] Mussels (Mytilus spp.) are a group of marine bivalve mollusks widely distributed in the intertidal zone to shallow sea areas, belonging to the phylum Mollusca, class Bivalvia, and family Mytilidae. They are an important aquaculture species. Their adaptation to the high osmotic pressure, high salinity, and complex microecological environment of the ocean endows their metabolic products with unique structures and significant biological functions. Recent studies have shown that mussels are rich in proteins, polysaccharides, bioactive peptides, and various trace elements. Among these, natural polysaccharides derived from mussels, due to their good water solubility and complex spatial conformation, exhibit a wide range of functional activities in the biomedical field, such as anti-inflammatory, antioxidant, immunomodulatory, and anti-tumor effects. These biological effects provide enormous application potential for mussel polysaccharides in chronic disease intervention, immune disease regulation, and functional food development. Traditional research has focused on the basic physicochemical properties of mussel polysaccharides and the activity evaluation of crude extracts. However, with the development of polysaccharide chemistry and structural biology, researchers have gradually realized that the biological activity of polysaccharides is closely related to their monosaccharide composition, glycosidic bond linkage, branched structure, and spatial conformation. Recent studies have revealed that novel mussel polysaccharides with specific linkages (such as 2,4-Glc and 4,6-Glc) and unique spatial conformations not only retain the biological functions of their sources but also exhibit more significant anti-inflammatory or immunomodulatory effects. The correlation between structural novelty and functional activity has become a current research hotspot. Therefore, developing and identifying novel mussel polysaccharides with well-defined structural characteristics and clear functional targets not only enriches the structural types and activity profiles of marine natural products but also provides new ideas for the development of marine functional biomaterials. These polysaccharides, as marine functional foods, drug lead compounds, or intervention factors for inflammation-related diseases, have significant scientific value and practical application prospects in the high-value utilization of marine resources and green pharmaceutical research and development.

[0003] Inflammatory bowel disease (IBD) is an immune-mediated, chronic, relapsing inflammatory bowel disease. Based on its pathogenesis and location, it can be divided into Crohn's disease and ulcerative colitis, both characterized by disruption of the structure and function of the intestinal epithelial barrier. Reducing the high incidence of IBD has become a major challenge. Therefore, finding an effective drug or functional food to improve inflammatory bowel disease is of great importance. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a novel mussel polysaccharide MP-I that can improve inflammatory bowel disease.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the mussel polysaccharide MP-I.

[0006] The final technical problem to be solved by the present invention is to provide the application of the mussel polysaccharide MP-I in the preparation of drugs to improve inflammatory bowel disease.

[0007] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a mussel polysaccharide with α-1,4-glycosidic bonds as the main chain and α-1,6-glycosidic bonds as the branches. On average, there is one α-1,6-glucose branch for every 10 glucose main chains. Its structural formula is shown below: .

[0008] The mussel polysaccharide has an α-pyranotropic dextran configuration and a relative molecular weight of 312~533 kDa.

[0009] The present invention also includes a method for preparing mussel polysaccharide, comprising the following steps: extracting and filtration mussel meat with water to obtain a concentrated solution, then adding papain for enzymatic hydrolysis, followed by high-speed centrifugation to remove protein and enzyme, and taking the supernatant; precipitating the supernatant with alcohol, washing, and centrifuging to obtain the precipitate, which is the crude mussel polysaccharide; reconstituted the crude mussel polysaccharide, separating it first through a DEAE exchange column and then further separating and purifying it through a Sepharose CL-6B molecular sieve chromatography column to obtain the final product.

[0010] The mussel meat to water mass-volume ratio is 20:1 to 100:1 g / L, and preferably, it is 50:1 g / L.

[0011] The mass-to-volume ratio of papain to concentrate is 0.5 g / 100 mL to 5 g / 100 mL, and preferably, the mass-to-volume ratio of papain to concentrate is 1 g / 100 mL.

[0012] The eluent obtained by separating the crude mussel polysaccharide using a DEAE exchange column is a NaCl solution with gradient concentrations of 0M NaCl, 0.1M NaCl, 0.2M NaCl, and 0.5M NaCl.

[0013] Among them, the mussel polysaccharide obtained by Sepharose CL-6B molecular sieve chromatography was eluted isocratically with 0.05M NaCl solution.

[0014] The present invention also includes the application of the mussel polysaccharide in the preparation of a drug for the prevention or treatment of inflammatory bowel disease, wherein, preferably, the inflammatory bowel disease includes colitis.

[0015] The colitis mentioned includes, but is not limited to, ulcerative colitis.

[0016] The mussel polysaccharide prepared by this invention can significantly improve ulcerative colitis induced by sodium dextran sulfate (DSS) in mice.

[0017] The mussel polysaccharide of this invention can be administered rectally, achieving therapeutic effects at a dosage of 10 mg / kg, and exhibits significant efficacy advantages compared to mussel polysaccharides prepared by other methods. The high dose of this invention (20 mg / kg) is comparable to that of a positive control drug. This mussel polysaccharide alleviates intestinal inflammation by inhibiting the expression of inflammatory signaling pathways such as JAK2 / STAT3 and NF-κB.

[0018] The mussel polysaccharide of this invention regulates the composition of the intestinal microbiota in mice with DSS-induced colitis.

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention uses thick-shelled mussels as raw material and prepares a novel mussel polysaccharide through water extraction and concentration, enzymatic protein decomposition, alcohol precipitation, and anion exchange. Animal experiments have shown that the mussel polysaccharide prepared by this invention can effectively improve intestinal flora, act as a prebiotic, and alleviate intestinal inflammation by inhibiting the expression of inflammatory signaling pathways such as JAK2 / STAT3 and NF-κB. Therefore, this mussel polysaccharide is a natural and safe drug or food for improving inflammatory bowel disease. It can effectively improve inflammatory bowel disease, is safe, non-toxic, and has no side effects. The raw materials are abundant, the process is simple, the cost is low, and the effect is significant. Attached Figure Description

[0020] Figure 1 The above is the HPGPC chromatogram of mussel polysaccharide MP-I of the present invention;

[0021] Figure 2 The infrared spectrum analysis of mussel polysaccharide MP-I of the present invention is shown below;

[0022] Figure 3 The figure shows the effect of the mussel polysaccharide MP-I of the present invention on the disease activity index of DSS-induced ulcerative colitis mice;

[0023] Figure 4 The figure shows the effect of the mussel polysaccharide MP-I of the present invention on the body weight of mice with DSS-induced ulcerative colitis.

[0024] Figure 5 The figure shows the effect of mussel polysaccharide MP-I intervention of the present invention on colon length in mice with DSS-induced ulcerative colitis;

[0025] Figure 6 This is representative of the effect of mussel polysaccharide MP-I intervention of the present invention on colon length in mice with DSS-induced ulcerative colitis;

[0026] Figure 7 The figure shows the effect of mussel polysaccharide MP-I intervention of the present invention on the histopathological results of colon tissue in mice with DSS-induced ulcerative colitis;

[0027] Figure 8 This invention compares the mussel polysaccharide MP-I (MP-1) of this invention with the mussel polysaccharide MP-II (MP-2) reported in the literature in improving DSS-induced ulcerative colitis in mice.

[0028] Figure 9 The effect of the invented mussel polysaccharide MP-I on the expression of inflammatory signaling pathways in Caco-2 cells stimulated by TNF-α.

[0029] Figure 10 The figure shows the effect of mussel polysaccharide MP-I on the intestinal flora of mice with DSS-induced ulcerative colitis. Detailed Implementation

[0030] Example 1: Preparation of mussel polysaccharide MP-I

[0031] Take 250g of thawed mussel meat (purchased from various aquaculture farms in Rizhao, Shandong), homogenize it, add 20 times the amount of water (about 5L), heat to a gentle boil for 2 hours, filter through 4 layers of gauze, and concentrate to 500mL by rotary evaporation. Add 1% papain (5g / 500mL) (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: G2227454) to the concentrate, incubate in a 60℃ water bath for 3 hours, and gently boil for 15 minutes to inactivate the enzyme and obtain the enzymatic hydrolysate. Add 500mL of Sevage reagent (chloroform: n-butanol = 4:1) to the enzymatic hydrolysate and shake vigorously for 10 minutes. After standing and separating the layers, discard the lower layer. Add 1 / 4 volume (250mL) of Sevage reagent to the supernatant, centrifuge at 4000rpm for 10 minutes, and repeat until no protein layer remains. Concentrate the solution to 200-300mL, add 750ml of pure ethanol, let stand for 2 hours, centrifuge at 4000rpm for 10 minutes, and discard the supernatant. The precipitate was washed twice with 750 ml of 75% ethanol and once with acetone. After centrifugation, crude polysaccharide was obtained, dried, and weighed to obtain 5 g of mussel crude polysaccharide.

[0032] Dissolve 1.5g of crude mussel polysaccharide in 5mL of pure water to a concentration of 0.3g / mL. Centrifuge at 4000rpm for 5min and collect the supernatant mussel polysaccharide solution. Prepare elution buffers of varying concentrations: 0.1M NaCl, 0.2M NaCl, and 0.5M NaCl, pH=7. Prepare an equilibration buffer containing 2M NaCl and 0.2M NaOH, pH=13. First, wash the DEAE Sepharose FF anion exchange column (packing company: Cytiva; packing model: Cytiva SwedenAB SE-75184 Uppsala Sweden; column height 27cm, inner diameter 2.6cm, packing volume 90ml) with the equilibration buffer (2M NaCl, 0.2M NaOH) for 2-3 column volumes, then wash with pure water until the mobile phase pH=7. Next, 5 mL of the crude mussel polysaccharide solution was loaded onto the plate at a flow rate of 1 mL / min. Elution was performed sequentially with four gradients of elution buffers: 0 M NaCl, 0.1 M NaCl, 0.2 M NaCl, and 0.5 M NaCl. Each gradient elution took approximately 6 hours. The eluent was collected, with a total elution time of 24 hours. 200 μL of the eluent from each tube was added to 200 μL of phenol solution (5% by mass). Then, 1 mL of concentrated sulfuric acid was added to each sugar tube on an ice pack. After mixing, the tube was boiled for 20 minutes and then immediately cooled for 20 minutes. 200 μL of the solution from each sugar tube was transferred to a 96-well plate (n=3, three parallel samples) and detected using a microplate reader at a wavelength of 490 nm. Obvious sugar peaks appeared in elution solutions with concentrations of 0M NaCl and 0.1M NaCl. After averaging the values ​​of each tube, elution curves were plotted. The peak sugar tubes of the eluents eluted with 0M NaCl and 0.1M NaCl were collected and freeze-dried to obtain two types of mussel polysaccharides. The mussel polysaccharide eluted with 0M NaCl weighed 600 mg with a yield of 40%, while the polysaccharide eluted with 0.1M NaCl weighed 1 g with a yield of 70%.

[0033] To further improve purity, the two polysaccharides needed to be purified using a Sepharose CL-6B molecular sieve chromatography column. After washing the Sepharose CL-6B molecular sieve chromatography column with pure water, 100 mg of each of the two freeze-dried mussel polysaccharides were dissolved in 5 mL of pure water to a concentration of 20 mg / mL. Samples were then loaded, with each loading being 2.5 mL, for a total loading of 5 mL. Elution was then performed using 0.05 M NaCl solution at a constant flow rate of 0.3 mL / min, with one tube added every 10 minutes for a total of 40 tubes. The sugar distribution in these 40 tubes was measured using the sulfuric acid-phenol method. Samples containing sugar were then freeze-dried to obtain the final two mussel polysaccharides.

[0034] The mussel polysaccharide eluted with 0M NaCl weighed 75 mg with a purity of 89%. It is a dextran with a molecular weight of 194–246 kDa, predominantly composed of α-1,4-glycosidic bonds with a small amount of α-1,6-glycosidic bonds as branches, and a repeating unit of 3. Its content was relatively low, with a total yield of 30%. The polysaccharide eluted with 0.1M NaCl weighed 85 mg with a purity of 99%. It is a dextran with a molecular weight of 312–533 kDa, predominantly composed of α-1,4-glycosidic bonds with a small amount of α-1,6-glycosidic bonds as branches, and a repeating unit of 9. Its content was relatively high, with a total yield of 56%. Considering the purity and yield issues, the mussel polysaccharide MP-I eluted with 0.1M NaCl was used for subsequent studies.

[0035] Example 2: Determination of total sugar content of mussel polysaccharide MP-I

[0036] The total sugar content of mussel polysaccharides was determined using the sulfuric acid-phenol method. A 5% phenol solution and a 0.1 mg / mL standard glucose solution were prepared. 10 mg of the mussel polysaccharide MP-I sample prepared in Example 1 was accurately weighed, transferred to a 100 mL volumetric flask, and diluted with water to a concentration of 0.1 mg / mL. This solution was then used as the sample determination solution.

[0037] Pipette 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, and 0.6 ml of 0.1 mg / ml standard glucose solution into colorimetric tubes, respectively, and add distilled water to each tube to a final volume of 2.0 ml. Add 1.0 ml of 5% phenol to the solution, then quickly add 5.0 ml of concentrated sulfuric acid. Vortex to mix thoroughly, let stand for 10 minutes, and then place the colorimetric tubes in a shaking, constant-temperature metal bath at 80°C for 15 minutes. Measure the absorbance at 490 nm using a UV-Vis spectrophotometer. Use 1.0 ml of water as a blank, following the same colorimetric procedure. Plot the glucose concentration on the x-axis and the absorbance value on the y-axis to create a standard curve.

[0038] Pipette 1.0 ml of the sample solution into a colorimetric tube and follow the steps above to measure the absorbance value.

[0039] Based on the steps described above, a standard curve is constructed, yielding the equation: y = 0.065x - 0.006, R0. 2 = 0.999, the total sugar content of mussel polysaccharides is 99.08%.

[0040] Table 1 Total sugar content of mussel polysaccharide MP-I

[0041]

[0042] Example 3: Determination of the molecular weight of mussel polysaccharide MP-I

[0043] The molecular weight and distribution of mussel polysaccharide MP-I prepared in Example 1 were determined by HPGPC. High-performance gel permeation chromatography (HPLC) was used for detection using a tandem column. The chromatographic conditions were as follows: Instrument: Waters HPLC system; Detector: Waters differential detector; Column: Three 8×300 mm polymer-based water-soluble SEC (GFC) columns in series; Mobile phase: 0.05 M NaCl solution; Flow rate: 0.65 ml / min; Column temperature: 40 ºC; Injection volume: 30 μl. The molecular weight results of mussel polysaccharide MP-I are shown in Table 2.

[0044] Table 2. Molecular weight results of mussel polysaccharide MP-I

[0045]

[0046] like Figure 1 The mussel polysaccharide MP-I purified in Example 1 is a homogeneous polysaccharide, and its molecular weight is 312~533kDa, as shown in Table 2.

[0047] Example 4: Determination of the monosaccharide components of mussel polysaccharide MP-I

[0048] The monosaccharide fraction of mussel polysaccharide MP-I prepared in Example 1 was determined by high performance liquid chromatography. 5 mg of the sample was added to 1 mL of 2M trifluoroacetic acid (TFA) solution and heated at 121 °C for 2 hours. Nitrogen gas was then introduced, and the mixture was dried. Methanol was added for washing, and the mixture was dried again. This methanol washing process was repeated 2-3 times to obtain a 5 mg / mL polysaccharide hydrolysate.

[0049] Take 0.2 mL of 5 mg / mL polysaccharide hydrolysate into a stoppered conical centrifuge tube, add 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution for PMP derivatization, vortex mix, and react in a 70 ℃ water bath for 1 h. After the reaction is complete, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the added sodium hydroxide, add 1 mL of chloroform and vortex extract 3 times to remove excess PMP, discard the chloroform layer, take 0.3 mL and add water to make up to 1 mL.

[0050] The chromatographic conditions were as follows: Instrument: Thermo U3000 HPLC system; Detector: DAD detector; Column: ZORBAX EclipseXDB-C18; Mobile phase: acetonitrile: phosphate buffer (potassium dihydrogen phosphate 12 g / L, pH adjusted to 6.8 with 2 M NaOH) isocratic elution, with a volume ratio of acetonitrile to phosphate buffer of 17:83; Flow rate: 0.8 mL / min; Column temperature: 30 ºC; Detection wavelength: 250 nm; Injection volume: 10 μL. The monosaccharide components of mussel polysaccharide MP-I are shown in Table 3.

[0051] Table 3. Results of monosaccharide composition of mussel polysaccharide MP-I

[0052]

[0053] The monosaccharide component of this mussel polysaccharide MP-I is 100% glucose.

[0054] Example 5: Methylation determination of mussel polysaccharide MP-I

[0055] The methylation determination of mussel polysaccharide MP-I prepared in Example 1 was performed on an Agilent 7890A-5977B gas chromatography-mass spectrometry (GC-MS) system. The sample was pretreated according to the following steps: 1 mg of mussel polysaccharide MP-I was dissolved in 1 ml of DMSO; 30 mg of NaOH was added and incubated for 30 min; 250 μl of iodomethane solution was added, nitrogen was introduced, and the reaction was carried out in the dark for 1 h; another 250 μl of iodomethane solution was added and the reaction was carried out for 1 h; 1 ml of water and 2 ml of dichloromethane were added, vortexed, centrifuged, and the aqueous phase was discarded; the mixture was washed three times with water; the lower dichloromethane phase was aspirated and dried under nitrogen; 1 ml of 2M TFA was added, and the reaction was carried out at 121 °C for 120 min; the mixture was dried under nitrogen at 30 °C; 1 ml of freshly prepared 1 M NaBD4 (Anhui Zesheng Technology Co., Ltd.; Product No.: E0900270250; 1 M NaBD4 was prepared by dissolving in ammonia water) was added. The sample was incubated with magnetic stirring at room temperature for 2.5 hours; the reaction was terminated by adding 300 μl of acetic acid, and dried under nitrogen. It was then dried twice under nitrogen at 40°C with 2 ml of 5% (vol / vol) acetic acid-methanol solution (5% (vol / vol) acetic acid dissolved in methanol is used to remove excess reducing agent NaBD4 and its products), followed by washing the sample with 2 ml of methanol and drying twice under nitrogen at 40°C. 1.5 mL of acetic anhydride was added, vortexed, and the reaction was carried out at 100°C for 2.5 h. 2 ml of water was added and the mixture was allowed to stand for 10 min. 1 mL of dichloromethane was added, vortexed, centrifuged, and the aqueous phase was discarded. The washing was repeated three times with water. The lower dichloromethane phase was collected and analyzed.

[0056] The chromatographic parameters were as follows: Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas was high-purity helium (purity not less than 99.999%), flow rate was 1.0 mL / min, and the injection port temperature was 260℃. The injection volume was 1 μL, split injection was used, the split ratio was 10:1, and the solvent delay was 2.2 min. The temperature program was: 50℃ held for 1.0 min, increased to 130℃ at 50℃ / min, increased to 230℃ at 3℃ / min, and held for 2 min.

[0057] The mass spectrometry parameters are as follows: Agilent Technologies (China) Co., Ltd. quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source has an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode is full scan (SCAN), and the mass scan range (m / z) is 30-600.

[0058] The glycosidic bond linkage mode of mussel polysaccharide MP-I was analyzed based on the methylation results, and the results are shown in Table 4:

[0059] Table 4. Analysis of glycosidic bond linkage modes of mussel polysaccharide MP-I

[0060]

[0061] The mussel polysaccharide MP-I has an α-1,4-glycosidic bond as the main chain and an α-1,6-glycosidic bond as the side chain in a ratio of 9:2, with an average of 9 glucose molecules between two side chains.

[0062] Example 6 Infrared Spectroscopic Analysis of Mussel Polysaccharide MP-I

[0063] Example 1 prepared mussel polysaccharide MP-I and analyzed it using Fourier transform infrared spectroscopy. The steps are as follows: 2 mg of dried mussel polysaccharide MP-I sample was weighed into a mortar, 200 mg of KBr powder was added and ground evenly, then compressed into tablets. The sample was scanned using a Fourier transform infrared microscope with a wavelength range of 4000~400 cm⁻¹. -1 Record the infrared spectrum.

[0064] like Figure 2The infrared spectral analysis results of mussel polysaccharide MP-I are as follows: The polysaccharide sample MP-I exhibits a characteristic absorption peak at 3397.21 cm⁻¹. -1 A strong and broad absorption peak exists at 2929.46 cm⁻¹, which is the strong absorption peak of the OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The nearby moderate-intensity peak is the absorption peak of the CH stretching vibration of the methine (-CH2); 1647.17 cm⁻¹ -1 The absorption peak at 1450-1200 cm⁻¹ is the hydration vibration peak of polysaccharides. -1 The absorption peak is at 1416.77 cm⁻¹. -1 1367.48 cm -1 1239.01 cm -1 The peak at 1154.961 cm⁻¹ represents the angular vibration absorption of CH₄, which, along with the stretching vibration of CH₄, constitutes the characteristic absorption of the sugar ring; 1154.961 cm⁻¹ represents the stretching vibration of pyranose COC; 1150-1010 cm⁻¹ -1 The two strong absorption peaks between, at 1081.21 cm⁻¹ -1 1023.02 cm -1 The presence of pyranoside was confirmed again at point 1, and the absorptions at these three sites were due to the bending vibrations of the CO bond in the COH or COC structure; 929.98 cm⁻¹ -1 The peak at 848.48 cm⁻¹ shows the asymmetric ring stretching vibration of the pyran ring. -1 The peak represents the CH-angle vibration of the diastereomer of the α-terminal group of pyranose; 762.32 cm⁻¹ -1 The peaks of symmetrical ring stretching vibrations of the pyran ring are shown.

[0065] The above results further demonstrate that mussel polysaccharide MP-I has a typical pyranose ring structure and exhibits the characteristics of α-glycosidic bonds, 848 cm⁻¹ -1 The characteristic peaks at this location are consistent with the α-1,4-glycosidic and α-1,6-glycosidic bond linkages observed in methylation analysis. (1150-1010 cm⁻¹) -1 The COC vibration peak supports the presence of the glucose pyran ring.

[0066] Example 7: Effect of mussel polysaccharide MP-I on DSS-induced ulcerative colitis in mice

[0067] (1) Experimental animals: 40 male C57BL / 6J mice, aged 6-8 weeks and weighing 20±2 g, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.

[0068] (2) Animal grouping: After one week of acclimatization, animals were randomly grouped into 5 groups (8 animals / group): Control group, DSS group, 5-ASA treatment group, low-dose mussel polysaccharide treatment group (10 mg / kg) and high-dose mussel polysaccharide treatment group (20 mg / kg).

[0069] (3) Animal modeling and drug administration: The control group mice received standard drinking water, while the drinking water of the other groups was supplemented with 3% DSS to induce acute ulcerative colitis for 7 days. One day after modeling, the low-dose mussel polysaccharide group was slowly injected 100 μL of mussel polysaccharide MP-I (10 mg / kg) into the rectum every day, the high-dose mussel polysaccharide group was slowly injected 100 μL of mussel polysaccharide MP-I (20 mg / kg) into the rectum every day, and the 5-ASA positive control group was slowly injected 100 μL of 5-ASA (20 mg / kg) into the rectum every day.

[0070] (4) DAI score: During the experiment, the three clinical symptoms of UC were assessed daily according to DAI, including the percentage of weight loss, stool viscosity, and rectal bleeding. The DAI score is the sum of the daily weight loss score, stool viscosity score, and rectal bleeding score. DAI score indicators: the percentage of weight loss (0 points: no change in weight, 1 point: 1%-5% decrease, 2 points: 5%-10% decrease, 3 points: 10%-20% decrease, 4 points: >20%), stool consistency (0 points: normal, 2 points: slightly moist but not attached to the perianal area, 4 points: diarrhea), and rectal bleeding (0 points: normal, 2 points: slight bleeding, 4 points: hematochezia) are the sum of the three scores.

[0071] (5) Animal sampling: Eight hours after the last administration, mice were euthanized by cervical dislocation. The peritoneal cavity of the mice was exposed, and the colon and rectum were separated. The length of the colon and rectum was measured and photographed. Subsequently, pre-cooled physiological saline was repeatedly injected into the intestinal lumen to remove the contents. Approximately 1 cm of the distal colon was excised and fixed in 4% paraformaldehyde solution for histopathological examination.

[0072] (6) Experimental results:

[0073] ① Disease Activity Index (DAI) score for mouse colitis: See details below. Figure 3 and Figure 4 Compared to the control group, mice given 3% DSS in their drinking water exhibited acute clinical symptoms, including weight loss, altered fecal characteristics, and severe bloody stools, which were reflected in increased DAI scores. Compared to the model group mice, mussel polysaccharide significantly improved the clinical symptoms and reduced DAI scores in a dose-dependent manner.

[0074] ② Measurement of mouse colon length: See details below. Figure 5 and Figure 6 Compared with the control group, mice treated with DSS showed significant congestion, extensive mucosal ulceration, and shortening of the colon and rectum. Compared with the model group, rectal injection of high-dose mussel polysaccharide significantly reversed intestinal congestion and shortened colon and rectum, suggesting that mussel polysaccharide can improve the severity of UC, and that high-dose mussel polysaccharide MP-I has a protective effect comparable to that of the positive control drug.

[0075] ③ Histopathological evaluation of mouse colon tissue: See details below. Figure 7 Compared with the control group mice, DSS intervention led to damage to the colonic epithelial mucosa, necrosis of crypt structures, and extensive infiltration of inflammatory cells. However, compared with the model group, rectal injection of mussel polysaccharide significantly alleviated the damage to colonic tissue structure and reduced inflammatory cell infiltration, suggesting that mussel polysaccharide MP-I has a protective effect against DSS-induced colonic tissue pathological damage, and that high doses of mussel polysaccharide MP-I have a protective effect comparable to that of the positive control drug.

[0076] Example 8: Comparison of the effects of the mussel polysaccharide MP-I (MP-1) of the present invention and the mussel polysaccharide MP-II (MP-2) reported in the literature on improving DSS-induced ulcerative colitis in mice.

[0077] (1) According to the literature (Mar Drugs. 2021 Aug 20;19(8):468.), mussel polysaccharide lipopolysaccharide stimulated RAW264.7 cells and dextran sulfate sodium (DSS) induced mouse ulcerative colitis model, its structure is {DGlcpa1-4-[DGlcpa1-6DGlcpa4-6DGlcpa]}n. We prepared this mussel polysaccharide according to the literature report. The specific method is as follows: 700g of fresh mussel meat was extracted with hot water (100℃) for 5h. Insoluble substances were removed by filtration, and the filtrate was deproteinized four times using the Sevag method. After being placed at 4℃ overnight, three times the volume of cold ethanol was added to precipitate the free oligosaccharide material. The precipitate was then centrifuged (3000g*10min), washed with enol, acetone and ether in sequence, and vacuum dried to obtain crude product (31g). The crude product was placed in a DEAE Sepharose anion exchange column (2.0 x 40 cm) and eluted sequentially with distilled water at a flow rate of 32 mL / h. The fraction (21 g) collected from the main peak was further separated on a Sephrose CL-6B column (1.0 x 100 cm) eluted with distilled water at a flow rate of 16 mL / h, and lyophilized to obtain 14.2 g of white powder MP-II.

[0078] (2) The efficacy of MP-I and MP-II in improving DSS-induced ulcerative colitis in mice was compared using the experimental protocol of Example 7. The specific experimental protocol is as follows:

[0079] a. Experimental animals: 40 male C57BL / 6J mice, aged 6-8 weeks and weighing 20±2 g, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.

[0080] b. Animal grouping: After one week of acclimatization, animals were randomly divided into 5 groups (8 animals / group): Control group, DSS group, 5-ASA treatment group, MP-I (20 mg / kg) and MP-II (20 mg / kg).

[0081] c. Animal modeling and drug administration: Control group mice received standard drinking water, while the drinking water of other groups was supplemented with 3% DSS to induce acute ulcerative colitis for 7 days. One day after modeling, the MP-I group received a slow rectal injection of 100 μL of mussel polysaccharide (20 mg / kg) daily, the MP-II group received a slow rectal injection of 100 μL of mussel polysaccharide (20 mg / kg) daily, and the 5-ASA positive control group received a slow rectal injection of 100 μL of 5-ASA (20 mg / kg) daily.

[0082] d. DAI Score: During the experiment, daily assessments were conducted based on three clinical symptoms of UC (ulcerative colitis) according to the DAI, including the percentage of weight loss, stool consistency, and rectal bleeding. The DAI score is the sum of the daily weight loss score, stool consistency score, and rectal bleeding score. DAI scoring indicators: The sum of the percentage of weight loss (0 points: no change in weight, 1 point: 1%-5% decrease, 2 points: 5%-10% decrease, 3 points: 10%-20% decrease, 4 points: >20%), stool consistency (0 points: normal, 2 points: slightly moist but not adhering to the perianal area, 4 points: diarrhea), and rectal bleeding (0 points: normal, 2 points: slight bleeding, 4 points: rectal bleeding) is the DAI score.

[0083] e. Animal sampling: Eight hours after the last administration, mice were euthanized by cervical dislocation. The peritoneal cavity of the mice was exposed, and the colon and rectum were dissected. The length of the colon and rectum was measured and photographed. Subsequently, pre-cooled physiological saline was repeatedly injected into the intestinal lumen to remove the contents. Approximately 1 cm of the distal colon was excised and fixed in 4% paraformaldehyde solution for histopathological examination.

[0084] (3) Experimental results are as follows Figure 8 .

[0085] a. Disease Activity Index (DAI) score in mice with colitis: Compared with the control group, mice given 3% DSS in their drinking water exhibited acute clinical symptoms, including weight loss, altered fecal characteristics, and severe bloody stools, which were reflected in an increase in the DAI score. Compared with the model group mice, mussel polysaccharide significantly improved the clinical symptoms of the mice and reduced the DAI score, showing a certain dose-dependent effect.

[0086] b. Measurement of colon length in mice: Compared with the control group, mice treated with DSS showed significant congestion, extensive mucosal ulceration, and shortened colon length. Compared with the model group, rectal injection of MP-I significantly reversed intestinal congestion and shortened colon length, suggesting that mussel polysaccharide can improve the severity of UC, and MP-I was significantly more effective than MP-II.

[0087] c. Histopathological evaluation of mouse colon tissue: Compared with the control group, DSS intervention caused damage to the colonic epithelial mucosa, necrosis of crypt structures, and extensive infiltration of inflammatory cells. Compared with the model group, rectal injection of MP-I significantly alleviated the damage to colonic tissue structure and reduced the infiltration of inflammatory cells. Furthermore, the effect of MP-I was significantly higher than that of MP-II, suggesting that MP-I has a protective effect against DSS-induced histopathological damage to the colon.

[0088] In summary, the mussel polysaccharide MP-I prepared in this invention has a better effect on improving DSS-induced ulcerative colitis in mice than the mussel polysaccharide MP-II.

[0089] Example 9: Effects of mussel polysaccharide MP-I on the expression of inflammatory signaling pathways in Caco-2 cells stimulated by TNF-α.

[0090] Caco-2 cells were divided into groups of 5 × 10 5 Cells were seeded at a density of [number] cells / mL into 6-well plates. After complete adherence, the medium was replaced with DMEM incomplete medium. Cells were divided into four groups: control group, model group, low-dose MP-I group (50 µg / mL), and high-dose MP-I group (100 µg / mL), with three replicates per group. When the cell density reached 80%, 2 mL of DMEM incomplete medium was added to the control group and model group. The low-dose and high-dose MP-I groups prepared in Example 1 were added to 2 mL of DMEM incomplete medium containing 50 µg / mL and 100 µg / mL of MP-I, respectively. After culturing for 24 h, modeling was performed. During modeling, 2 mL of DMEM incomplete medium was added to the control group, and 2 mL of DMEM incomplete medium containing 50 ng / mL TNF-α was added to the model group and drug-treated group. The cells were incubated at 37°C in a constant temperature incubator with 5% CO2 and saturated humidity for 24 h.

[0091] See protein immunoblotting results Figure 9 The results showed that TNF-α treatment significantly activated the JAK2 / STAT3 and NF-κB signaling pathways in Caco-2 cells. The expression levels of p-JAK2 / JAK2, p-STAT3 / STAT3, p-IKKα / IKKα, and p-NF-κB / NF-κB were significantly upregulated compared to the control group, while the expression levels of mussel polysaccharide MP-I were significantly downregulated. This suggests that mussel polysaccharide MP-I may alleviate intestinal inflammation by inhibiting the expression of inflammatory signaling pathways such as JAK2 / STAT3 and NF-κB.

[0092] Example 10: Effects of mussel polysaccharide MP-I on DSS-induced intestinal flora in mice

[0093] Fecal samples from mice in the control group, model group, and mussel polysaccharide MP-I (20 mg / kg) administration group (Example 7) were sent to Beijing Biomarker Biotechnology Co., Ltd. Small fragment libraries were constructed and sequenced using the Illumina Novaseq sequencing platform and the paired-end sequencing method. Reads were spliced, filtered, clustered, or denoised, and species annotation and abundance analysis were performed to reveal the species composition of the samples. Further alpha diversity analysis, beta diversity analysis, significant species difference analysis, and correlation analysis were conducted to explore the differences between samples.

[0094] Based on the obtained feature abundance table, the number of OTUs in each group was calculated. Venn diagrams were used to present the number of common and unique OTUs in each group to study the species composition of each sample. The control group had 1017 unique OTUs, the model group had 835 unique OTUs, and the mussel polysaccharide group had 1845 unique OTUs, used to study the species composition of each sample. The results showed that the species composition of the mussel polysaccharide MP-I group was significantly different from that of the model group. Figure 10Alpha diversity of gut microbiota was sequenced. ACE, Chao1, and Shannon alpha diversity indices were used to characterize the normality of gut microbiota. Results showed that mussel polysaccharide significantly improved the alpha diversity of the model group. The ACE, Chao1, and Shannon indices of the gut microbiota in the model group mice were significantly lower than those in the normal group, indicating that the alpha diversity of the gut microbiota in colitis mice was disrupted. After mussel polysaccharide MP-I intervention, the ACE, Chao1, and Shannon indices of the gut microbiota in mice significantly increased, indicating that mussel polysaccharide MP-I has a significant regulatory effect on the alpha diversity of the gut microbiota in DSS-induced acute ulcerative colitis mice. Principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) analysis showed that the samples in the model group were closely spaced and significantly clustered, and significantly separated from the control group samples. The MP-I samples in the mussel polysaccharide group clustered more closely with those in the control group, suggesting that mussel polysaccharide intervention can restore the β diversity of the mouse gut microbiota to some extent. The microbial community composition changes identified 10 bacterial phyla. Firmicutes and Bacteroidetes were the dominant phyla, accounting for approximately 70% of the total. Compared to the control group, the relative abundance of Firmicutes in the model group was reduced, indicating that the intervention of mussel polysaccharide MP-I reversed the decrease in the relative abundance of Firmicutes and the increase in the relative abundance of Bacteroidetes in the model group, and significantly reduced their ratio (F / B). This suggests that mussel polysaccharide MP-I can regulate the gut microbiota composition in mice with DSS-induced colitis. Visualization results of phylogenetic branching / taxonomic tree and LEfSe analysis showed that the most significantly enriched species in the feces of control group mice were... lachnospiraceae (Trichophyton) genus. Paraprevotella The genus *Paraplevocybe* is the core taxonomic group in the model group; the core taxonomic group of the mussel polysaccharide group MP-I is... Turicibacter (Zurich bacillus) genus.

Claims

1. The application of mussel polysaccharide in the preparation of drugs for the prevention or treatment of ulcerative colitis, characterized in that, The mussel polysaccharide has an α-1,4-glycosidic bond as the main chain and α-1,6-glycosidic bonds as the branches. On average, there is one α-1,6-glucose branch for every 10 glucose main chains. Its structural formula is shown below: ， The mussel polysaccharide has an α-pyranotropic dextran configuration and a relative molecular weight of 312-533 kDa. The total sugar content of the mussel polysaccharide is 99.08%, and the monosaccharide component is 100% glucose. The preparation method of the mussel polysaccharide includes the following steps: mussel meat is extracted with water, filtered, and concentrated to obtain a concentrated solution. Papain is then added for enzymatic hydrolysis. Following high-speed centrifugation to remove protein and enzyme, the supernatant is collected. The supernatant is precipitated with alcohol, washed, and centrifuged to collect the precipitate, which is the crude mussel polysaccharide. The crude mussel polysaccharide is reconstituted, separated using a DEAE exchange column, and then purified using a Sepharose CL-6B molecular sieve chromatography column. The eluent from the DEAE exchange column separation of the crude mussel polysaccharide is 0.1 M. The mussel meat was eluted with NaCl solution, and the mass-to-volume ratio of mussel meat to water was 20:1 to 100:1 g / L. The mass-to-volume ratio of papain to concentrate was 0.5 g / 100 mL to 5 g / 100 mL. The mussel polysaccharide obtained by Sepharose CL-6B molecular sieve chromatography was eluted isocratically with 0.05 M NaCl solution.

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