Application of mussel polysaccharide in preparation of product for treating and / or preventing inflammatory bowel disease

Abstract

The invention discloses application of mussel polysaccharide in preparation of a product for treating and / or preventing IBD (infectious bursal disease). The structure of the mussel polysaccharide is alpha-pyranoid glucan formed by glucosidic bonds of (1-> 4)-alpha-D-Glc and (1-> 2)-alpha-D-Glc, the molecular weight is 300-3500 kDa, and the mussel polysaccharide shows remarkable inhibition of DSS-induced body weight loss, diarrhea, hemafecia and other symptoms in an in-vivo model, and recovers the colon length. Moreover, the mussel polysaccharide can also increase the mRNA expression level of an antioxidant gene in the colon of a model animal, inhibit oxidative stress and reduce the loss of tight junction protein. The results indicate that the mussel polysaccharide is an active substance of the IBD prevention and treatment medicine and food, and has value in development and utilization of marine biological resources.

Description

Technical Field

[0001] This invention relates to the fields of food or pharmaceutical technology, and in particular to the use of a mussel polysaccharide in the preparation of products for the treatment and / or prevention of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of relapsing, chronic, nonspecific inflammatory bowel diseases characterized by immune dysregulation, altered gut microbiota, and disrupted intestinal permeability. It is characterized by recurrent flare-ups and remissions, and includes two distinct types: ulcerative colitis (UC) and Crohn's disease (CD). The etiology of IBD is not fully understood, and its pathogenesis is highly complex. Currently, clinical understanding suggests that the main pathogenic mechanisms involve multiple factors, including the external environment, gut microbiota dysregulation, and abnormal immune function, acting together to disrupt the intestinal epithelial barrier, increasing mucosal permeability and facilitating antigen passage. Prolonged antigen stimulation of intestinal tissue leads to an overreaction and misrecognition of antigens by the intestinal mucosal immune barrier to gut microbiota and their products. This triggers the activation of macrophages and lymphocytes, releasing a series of cytokines and inflammatory mediators that activate the body's immune response, ultimately resulting in tissue damage and clinical manifestations such as abdominal pain, diarrhea, bloody stools, weight loss, and extraintestinal complications like arthritis and joint pain.

[0003] The recurrent attacks and complex pathological evolution of IBD pose a continuous challenge to clinical prevention and treatment, making the research and development of related therapeutic drugs and functional foods a focus of attention. Natural polysaccharides, as an important class of bioactive substances, are widely distributed in plants, animals, microorganisms, and marine organisms, possessing broad prospects for medicinal development. Mussels, a marine shellfish used for both food and medicine, mainly inhabit shallow sea areas among rocks and are widely distributed in the Bohai and Yellow Seas of China. Due to their rich nutritional value and significant medicinal potential, mussels have been extensively farmed globally, resulting in considerable production. Therefore, in-depth development of the medicinal value of mussel polysaccharides is of great significance. Mussel polysaccharides obtained through different extraction processes exhibit significant differences in structural composition and bioactivity, specifically in the degree of polymerization, molecular weight, and the composition of monosaccharides and the mode of glycosidic bond linkage. According to existing literature, high-molecular-weight dextran MP-I (molecular weight 1.35 × 10⁻⁶) has shown significant differences in these properties. 6 Da, α-(1→4)-D-glucan (with an average of one α-D-glucan branched at the C-6 position every 8 residues along the main chain) has significant anti-tumor effects and can alleviate acute liver injury; oligodextrose MP (molecular weight 4250 Da, with a 1,4-glycosidic main chain containing 1,6-glycosidic side chains) has broad immunomodulatory and antioxidant stress effects and can protect against IBD; high-glucan (MP-A) has only been reported to have lipid-regulating activity, and there are no reports of its activity in alleviating IBD. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this invention provides an application of mussel polysaccharide in the preparation of products for the treatment and / or prevention of inflammatory bowel disease (IBD). Mussel polysaccharide is a high-glucan with a 1,4-glycosidic backbone and 1,2-glycosidic side chains, exhibiting significant protective activity against IBD. It can effectively alleviate symptoms such as weight loss, diarrhea, and bloody stools in model animals, and this effect is independent of the intestinal flora, possibly by inhibiting oxidative stress and inflammatory responses to improve colitis.

[0005] The technical solution of this invention is as follows: The purpose of this invention is to provide the application of mussel polysaccharide in the preparation of products for the treatment and / or prevention of inflammatory bowel disease.

[0006] In one embodiment of the present invention, mussel polysaccharide is a white powder, readily soluble in water and dimethyl sulfoxide, insoluble in organic solvents such as ethanol and acetone, and shows a positive result in the sulfuric acid-phenol reaction. It is an α-pyranose-type dextran with a structure consisting of (1→4)-α-D-Glc and (1→2)-α-D-Glc glycosidic bonds, and has a molecular weight of 300-3500 kDa.

[0007] In one embodiment of the present invention, the mussel is the purple mussel (Mussel simonii). M. edulis , M. galloprovincialis Thick-shelled mussels ( M. unguiculatus, M. coruscus ) or jade mussels ( Perna viridis ).

[0008] In one embodiment of the present invention, mussel polysaccharide exists in the form of organic acid salt or inorganic acid salt; The organic acids are acetic acid, malic acid, maleic acid, citric acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, or oxalic acid. The inorganic acids are hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, or nitric acid.

[0009] In one embodiment of the present invention, inflammatory bowel disease is a mouse model of acute and chronic ulcerative colitis (UC) induced by DSS.

[0010] In one embodiment of the present invention, the product is food, health food, special medical food, or medicine.

[0011] In one embodiment of the present invention, the pharmaceutical product further includes a pharmaceutically acceptable carrier, excipient, or excipient.

[0012] In one embodiment of the invention, treatment and / or prevention of inflammatory bowel disease includes combating symptoms such as weight loss, diarrhea, and / or rectal bleeding.

[0013] The beneficial technical effects of this invention are as follows: This invention utilizes DSS-induced acute and chronic ulcerative colitis mouse models and found that mussel polysaccharide administration significantly alleviated symptoms such as weight loss, diarrhea, and bloody stools in the model animals, and restored colon length. Furthermore, mussel polysaccharide can increase the mRNA expression level of antioxidant genes in the colon of the colitis animal model, inhibit oxidative stress, and reduce the loss of tight junction proteins. Attached Figure Description

[0014] Figure 1 The HPGPC elution curve of mussel polysaccharide prepared in Example 1; Figure 2 The ultraviolet absorption spectrum of mussel polysaccharide prepared in Example 1; Figure 3 The infrared spectrum of mussel polysaccharide prepared in Example 1; Figure 4 The mussel polysaccharide prepared in Example 1 1 H-NMR spectrum; Figure 5 The mussel polysaccharide prepared in Example 1 13 C-NMR spectrum; Figure 6 DEPT spectrum of mussel polysaccharide prepared in Example 1; Figure 7 The HMQC spectrum of mussel polysaccharide prepared in Example 1; Figure 8 The mussel polysaccharide prepared in Example 1 1 H- 1 H COSY spectrum; Figure 9 The TOCSY spectrum of mussel polysaccharide prepared in Example 1; Figure 10 The HMBC spectrum of mussel polysaccharide prepared in Example 1; Figure 11 The therapeutic effects of mussel polysaccharide on acute and chronic ulcerative colitis in mice; Figure 12 The therapeutic effect of mussel polysaccharide on acute ulcerative colitis in pseudo-sterile mice; Figure 13 The expression levels of tight junction-related proteins in mouse colon tissue; Figure 14 The expression level of mRNA of genes related to oxidative stress in mouse colon. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0016] In the examples, the mussel polysaccharide raw material was self-made.

[0017] Example 1 1000 g of dried mussels (dried mussels) from Shengsi, Zhoushan were crushed into coarse powder, mixed with 5 times their volume of water, and heated to 100 ℃ for 2 h. 0.2% alkaline protease powder (pH 8.0) was added to the filtrate, and the mixture was kept at 50 ℃ for 3 h and then inactivated at 80 ℃. 3 times their volume of 95% ethanol was added to the filtrate to precipitate polysaccharides. The polysaccharide precipitate was washed, dehydrated, filtered, and vacuum dried to obtain 130 g of crude mussel polysaccharide. The crude mussel polysaccharide was dissolved in 1.2 L of water, centrifuged at 12000 rpm to remove insoluble matter, and ultrafiltered using a membrane with a molecular weight cutoff of 300 kDa. The concentrate was freeze-dried under vacuum to obtain 110 g of mussel polysaccharide MCG.

[0018] Test example: (1) Structural identification of mussel polysaccharides Total sugar content determination: Accurately measure glucose solution (100 g / L) μ 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of glucose polysaccharide (g / mL) were placed in 10 mL stoppered test tubes, and water was added to each tube to bring the volume to 1.0 mL. 1 mL of 6% phenol solution was precisely added to each tube, followed immediately by 5 mL of sulfuric acid. Using water as a blank, the optical density at 490 nm was measured using a UV spectrophotometer. A standard curve was plotted with glucose concentration on the x-axis and optical density on the y-axis to obtain the linear regression equation. 1.0 mL (100 g / mL) of the mussel polysaccharide solution prepared in Example 1 was precisely measured. μ The total sugar content was 96% (g / mL) and determined in parallel according to the above standard curve plotting method.

[0019] Determination of Glucuronic Acid Content: Accurately measure 50 μL of glucuronic acid reference standard. μ 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of a sulfuric acid-borax solution (g / mL) were placed in stoppered test tubes, and water was added to each tube to bring the volume to 0.5 mL. After pre-cooling, 3 mL of sulfuric acid-borax solution was added to each tube, and the tubes were heated at 100°C for 10 min. After cooling in an ice bath, 100 g / mL of sulfuric acid-borax solution was added to each tube. μ L-carbazole reagent, after shaking well, is heated at 100℃ for 15 min, cooled to room temperature, and a blank is prepared by replacing the corresponding reagent in the sample with water. The optical density value is measured at 530 nm. A standard curve is plotted with glucose concentration on the x-axis and optical density value on the y-axis. 0.5 mL (50 mg / L) of the mussel polysaccharide solution prepared in Example 1 is accurately measured. μ The optical density of the mussel polysaccharide prepared in Example 1 was measured in parallel according to the above standard curve plotting method (g / mL), and the optical density value was found to be comparable to that of the blank control, indicating that there was no obvious uronic acid component in the mussel polysaccharide.

[0020] Determination of relative molecular mass: High performance liquid chromatography-size exclusion chromatography (HPGPC) was used, employing a TSK-gel GMPWXL column (300 mm × 7.8 mm, size exclusion limit 5 × 10⁻⁶). 6 The relative molecular mass distribution of mussel polysaccharides was determined using the following chromatographic conditions: mobile phase: 0.05 mol / L sodium nitrate solution; flow rate: 0.6 mL / min; column temperature: 35 ℃; sample concentration: 10 mg / mL; injection volume: 20 μL; detector: differential refractive index detector (35 ℃). The HPGPC elution curve of mussel polysaccharides prepared in Example 1 is shown below. Figure 1 As shown. A broadly distributed dextran standard with a known molecular weight (Agilent Technologies, catalog number: PSS-DXTB1.5M, Mw = 1.52 × 10⁻⁶) was used. 6 Da; PSS-MEDXTB2M, Mw = 2.05×10 6 Retention time correction was performed using a membrane (Da) to determine that the molecular weight of the main component of the mussel polysaccharide was between 1000 kDa and 2050 kDa. To further verify the molecular weight range of the polysaccharide using ultrafiltration experiments: when using an ultrafiltration membrane with a molecular weight cutoff of 1000 kDa, the main component of the polysaccharide was still retained, while using an ultrafiltration membrane with a pore size of 0.22... μ When filtered through a membrane with a molecular weight cutoff of m (corresponding to a molecular weight cutoff of approximately 2200 kDa), the polysaccharide can completely permeate, and the results of the ultrafiltration experiment are consistent with those of the HPGPC analysis. Considering the precision of the filter membrane and the wide range of molecules that can permeate in the ultrafiltration experiment, the relative molecular weight of this mussel polysaccharide is determined to be approximately 300 kDa to 3500 kDa.

[0021] Ultraviolet spectroscopy analysis: The mussel polysaccharide prepared in Example 1 showed no ultraviolet absorption spectrum in the range of 200 nm to 400 nm, as shown below. Figure 2 As shown, it conforms to the structural characteristics of polysaccharides.

[0022] Infrared spectroscopy analysis: The KBr pellet method was used for determination. The infrared spectrum of mussel polysaccharide prepared in Example 1 is as follows: Figure 3 As shown, 3393.7 cm -1 The peak of the stretching vibration of OH is shown at 2928.9 cm⁻¹. -1 The CH stretching vibrations in the methine and methylene groups are shown at 1155.4 cm⁻¹. -1 1080.4 cm -1 1020.7 cm -1 The characteristic skeletal vibrations of the sugar ring COH and COC are observed at 1419.4 cm⁻¹. -1 1368.7cm -1 This indicates in-plane deformation vibrational absorption of the sugar ring OH and at 576.7 cm⁻¹.-1 The out-of-plane deformation vibration of the OH group is absorbed. 847.2 cm -1 The absorption at 928.6 cm⁻¹ indicates that the sugar ring is in the α-configuration. -1 The absorption at 1651.9 cm⁻¹ indicates that the sugar ring is in the D-configuration. -1 The absorption at the site is caused by the association of trace amounts of water with hydroxyl groups, a common feature in polysaccharides. The ultraviolet and infrared spectra of mussel polysaccharides show typical... α -D-pyranose dextran structural characteristics.

[0023] Nuclear magnetic resonance spectroscopy (600 MHz, D2O): The mussel polysaccharide prepared in Example 1... 1 H-NMR spectrum as follows Figure 4 As shown, in δ End groups are present at 5.23 ppm. 1 The signal of H is located in a lower field ( δ H The concentration was >5.0 ppm, and the broad single peak indicates that the mussel polysaccharide has an α-configuration. δ The signals at 3.0~4.0 ppm are the H2~H6 signals of the sugar rings. 13 C-NMR spectrum as follows Figure 5 As shown, in δ A signal of end-group C1 is present at 99.5 ppm, and it is located at a higher field. δ C <102 ppm), in compliance with α -Characteristics of configurational dextran, and its 1 Consistent with the judgment in the H-NMR spectrum. Appearing in the high-field region. δ 60.1 ppm, in the DEPT spectrum ( Figure 6 The negative peak indicates that the corresponding C signal is a methylene (-CH2), which may be the signal of C6 of the sugar ring; δ Signals in the range of 69.0~77.2 ppm should be sugar ring C2~C5 signals.

[0024] The structure of mussel polysaccharides was further determined using 2D nuclear magnetic resonance (NMR) technology. The CH correspondence was determined by HMQC spectroscopy. Figure 7 );pass 1 H- 1 H-COSY spectra determine the proton linking order, starting from the end-group H signal ( δ 5.23 ppm), which allows for the continuous detection of signals from other adjacent H values ​​( Figure 8 The TOCSY spectrum was used to confirm the attribution of proton signals in the same spin system. Figure 9 ). Regarding mussel polysaccharides 1 H and 13C signal attribution discovery C2 ( δ 76.4 ppm) and C4 ( δ The chemical shift (77.2 ppm) clearly shifted to a lower field, indicating the presence of (1→2) and (1→4) glycosidic bonds in mussel polysaccharides. HMBC mapping was used to further investigate the chemical shift. 13 C signal and 1 H signal is assigned ( Figure 10 This was verified and the connection mode between sugar rings was further confirmed. In the HMBC spectrum, H1 (…) can be seen... δ 5.23 ppm) and C2 ( δ 77.2 ppm), C1 ( δ 99.8 ppm) and H2 ( δ Coupling-related signal of 3.43 ppm; H1 ( δ 5.23 ppm) and C4 ( δ 76.4 ppm), C1 ( δ 99.5 ppm) and H4 ( δ The coupling correlation signal of 3.52 ppm indicates the presence of (1→2) and (1→4) glycosidic bonds in mussel polysaccharides, which is consistent with the judgment that the chemical shifts of C2 and C4 are significantly shifted to the lower field.

[0025] The above spectral and wavelength analyses determined that the mussel polysaccharide is (1→4)- α -D-Glc and (1→2)- α Composed of -D-Glc glycosidic bonds α -Pyranotype dextran with a relative molecular weight of 300 kDa-3500 kDa.

[0026] (2) Evaluation of the efficacy of mussel polysaccharide in inflammatory bowel disease ① Evaluation of the therapeutic effect of mussel polysaccharide on ulcerative colitis This invention utilizes DSS-induced acute and chronic ulcerative colitis (UC) mouse models to evaluate the pharmacological activity of mussel polysaccharides. Experimental results show that mussel polysaccharides can significantly improve symptoms such as weight loss, diarrhea, hematochezia, and colonic tissue pathological damage induced by DSS modeling.

[0027] A. Experimental Principle: DSS-induced colonic injury in C57BL / 6J mice is a classic mouse model of ulcerative colitis (UC), mimicking the main clinical features, biochemical indicators, and pathological changes of UC. This model is also widely used in the study of inflammatory bowel disease (IBD), especially with long-term chronic circulating administration, and can also mimic some features of Crohn's disease. This invention uses this induction protocol as an animal model to evaluate the treatment or prevention of IBD, particularly ulcerative colitis, with natural polysaccharides.

[0028] B. Experimental Materials and Methods A) Animal source: C57BL / 6J mice (6-7 weeks old) were purchased from Vital River.

[0029] B) Animal rearing conditions: Animals were housed in SPF-grade animal facilities. Temperature: 22-24 ºC; Humidity: 45-80%; Light: 150-300 Lx, 12-hour day-night cycle. Their rearing, administration of medication, and euthanasia were strictly in accordance with animal welfare guidelines (referencing the AAALAC guidelines).

[0030] C) Animal Grouping and Administration: C57BL / 6J mice were acclimatized for one week, and modeling began at 8 weeks of age. Acute UC modeling was established as follows: Mice were divided into a blank control group, a model control group, a low-dose model drug group, and a high-dose model drug group, with 8 mice in each group. Mice in the model control group and the model drug group were given free access to 3% DSS sterilized water for 5 days, then switched to normal drinking water. Mice in the blank control group received normal drinking water for 7 days. Chronic UC modeling was established as follows: Mice were divided into a blank control group, a model control group, a low-dose model drug group, and a high-dose model drug group, with 8 mice in each group and similar average body weight. Mice in the model control group and the model drug group were given free access to 2% DSS sterilized water for 7 days, then switched to normal drinking water for 14 days. A cycle of 21 days was performed, for a total of 3 cycles. Mice in the blank control group received normal drinking water for 63 days. Mice in each group were administered a solvent (0.1% NaCl, blank control group and model control group) via intraperitoneal injection between 9:00 and 11:00 AM daily, along with 200 mg / kg mussel polysaccharide (low-dose group) and 500 mg / kg mussel polysaccharide (high-dose group). After the experiment, mice were dissected, and the length of the colon to rectum was measured. Rectal tissue was taken 1 cm from the anus, fixed with 4% paraformaldehyde, and prepared into paraffin sections. The remaining colonic tissue was flash-frozen in liquid nitrogen and stored at -80°C.

[0031] C. Observation indicators: A) The Disease Activity Index (DAI) score is a comprehensive score based on weight change, stool characteristics, and rectal bleeding, which can be used to assess the severity of enteritis.

[0032] Mice in each group were weighed daily, and their weight was recorded. The weight change was calculated based on the weight on day 0. A weight loss of 1% to 5% was scored as 1 point, a weight loss of 5% to 10% as 2 points, a weight loss of 10% to 20% as 3 points, and a weight loss of ≥20% as 4 points.

[0033] The characteristics of the feces of mice in each group were observed and scored daily. Normal fecal shape was scored as 0 points, soft but formed feces as 1 point, soft feces as 2 points, loose or wet feces as 3 points, and wet feces adhering to the anus as 4 points.

[0034] The rectal bleeding status of mice in each group was recorded daily. A fecal occult blood test kit was used to detect the degree of occult blood in the mice's feces, and scores were assigned according to the kit instructions: 0 points for negative, 1 point for weakly positive, 2 points for positive, 3 points for visible bloodstains in the feces, and 4 points for rectal bleeding.

[0035] B) Hematoxylin-eosin (HE) staining and pathological scoring were performed on the distal colorectal tissue. Using an automated dehydrator, the tissue was sequentially passed through eight gradient alcohol stages, three xylene stages, and three paraffin stages according to a preset program. After dehydration, the tissue was removed and embedded into paraffin blocks using an automated embedding machine. The paraffin blocks were then thinly sliced ​​using a semi-automatic microtome to prepare sections. Staining was performed using a fully automated staining machine, and the slides were mounted using an automated mounting machine. After scanning the slides, pathological scoring was performed. Scoring was conducted on five dimensions: crypt loss, neutrophil infiltration in the lamina propria, degree of inflammation, extent of inflammation, and surface epithelial integrity. Crypt loss was scored from 0 to 4 points, neutrophil infiltration in the lamina propria from 0 to 3 points, degree of inflammation from 0 to 3 points, extent of inflammation from 0 to 3 points, and surface epithelial integrity from 0 to 3 points.

[0036] D. Experimental Results: The results of the DAI scores (acute model) for each group are shown in Table 1 below; Table 1

[0037] like Figure 11 As shown in Table 1, in the acute model, compared with the non-model group, the model group showed a significant decrease in body weight on day 5 of modeling and a significant increase in DAI score on day 7 (Table 1). The colon length at dissection was also significantly reduced. The high- and low-dose mussel polysaccharide groups restored the body weight loss, reduced the DAI score, and increased colon length. Pathological scoring based on HE staining showed that the model group had more severe pathological damage compared with the non-model group, while the high-dose mussel polysaccharide group significantly reduced this phenomenon. In the chronic model, compared with the non-model group, the model group's body weight remained at a lower level, and mussel polysaccharide administration had no effect on this indicator; however, high-dose mussel polysaccharide administration significantly improved colon length and pathological score in the chronic model mice. These experimental results indicate that mussel polysaccharide has a therapeutic effect on DSS-induced acute and chronic colitis in mice.

[0038] ② Pseudo-sterile animal experiments to detect the dependence of the therapeutic effect of the natural product mussel polysaccharide on ulcerative colitis on intestinal flora. This invention established a pseudo-germ-free mouse model using continuous antibiotic gavage for two weeks and a DSS-induced acute ulcerative colitis mouse model to evaluate whether the pharmacological activity of mussel polysaccharide depends on the gut microbiota. Experimental results showed that mussel polysaccharide could still improve DAI scores and alleviate colonic shortening after gut microbiota clearance, indicating that mussel polysaccharide does not depend on the gut microbiota to exert its effects.

[0039] A. Establishment of a pseudo-germ-free mouse model combined with acute ulcerative colitis: An antibiotic mixture was prepared using ampicillin (200 mg / kg), metronidazole (200 mg / kg), neomycin (200 mg / kg), and vancomycin (100 mg / kg) in sterile saline. Mice were administered the antibiotics via gavage to establish a pseudo-germ-free mouse model. Based on this, a DSS (Diverterless Superposition of Suppressants) model of acute ulcerative colitis was further established.

[0040] B. Experimental Materials and Methods A) Animal source: C57BL / 6J mice (6-7 weeks old) were purchased from Vital River.

[0041] B) Animal rearing conditions: Animals are housed in SPF-grade animal facilities. Temperature: 22-24ºC; Humidity: 45-80%; Light: 150-300 Lx, 12-hour day-night cycle. Their rearing, administration of medication, and euthanasia are strictly in accordance with animal experimentation and welfare guidelines (referencing the AAALAC guidelines).

[0042] C) Animal Grouping and Administration: C57BL / 6J mice were acclimatized for one week, and modeling began at 8 weeks of age. One week prior to modeling, mice were administered antibiotics via gavage, followed by UC modeling using 2.5% DSS. Antibiotic administration continued throughout the modeling process. Animal groups included a blank control group, a pseudo-sterile blank control group, a 2.5% DSS group, a pseudo-sterile 2.5% DSS modeling group, a 2.5% DSS + mussel polysaccharide administration group (500 mg / kg), and a 2.5% DSS + pseudo-sterile mussel polysaccharide administration group (500 mg / kg).

[0043] C. Observation indicators: Same as test case — (2) Evaluation of the efficacy of mussel polysaccharide in inflammatory bowel disease — ① Evaluation of the therapeutic effect of mussel polysaccharide on ulcerative colitis — C. Observation indicators — A) Disease activity index (DAI) score.

[0044] D. Experimental Results: The DAI score results for each group (day 14) are shown in Table 2 below; Table 2

[0045] like Figure 12 As shown, in untreated mice, DSS modeling increased the DAI score (Table 2) and decreased colon length in the model group (Model), while mussel polysaccharide administration reduced the DAI score and restored colon length. In antibiotic-treated mice (pseudo-germ-free mice), DSS modeling also increased the DAI score (Table 2) and decreased colon length in the model group (Model+ABX), while mussel polysaccharide administration again reduced the DAI score and restored colon length. These results indicate that, compared with normal model mice, mussel polysaccharide also improved key pathological indicators of ulcerative colitis in antibiotic-treated pseudo-germ-free mice, namely changes in DAI score and colon length. This suggests that the therapeutic effect of mussel polysaccharide on DSS-induced ulcerative colitis in mice does not depend on the regulation of gut microbiota. Although gut microbiota dysbiosis is considered a key factor in the pathogenesis of ulcerative colitis (UC), and many drugs work by restoring microbiota balance, this experiment reveals that the therapeutic effect of mussel polysaccharide may depend on other mechanisms of action.

[0046] ③ Effects of mussel polysaccharides on tight junction proteins in the colon This invention uses Western blotting to detect the effect of the natural product mussel polysaccharide on tight junction proteins in colon tissue. The results show that high doses of mussel polysaccharide can significantly restore the expression of tight junction proteins in colon tissue.

[0047] A. Materials and Methods A) Colon tissue samples: Mouse colon tissue was removed from a -80°C ultra-low temperature freezer and placed in liquid nitrogen or dry ice (to prevent freeze-thaw cycles). A colon sample approximately 0.5 cm in length was picked up using ophthalmic forceps and placed in a 1.5 mL centrifuge tube. The colon weight was weighed and recorded, maintaining each sample weight between 20-30 mg. Ten times the sample weight of RIPA lysis buffer (containing 1% PMSF protease inhibitor and 0.1% totipotent nuclease) and 3 grinding beads were added to each tube. Homogenization was performed using a homogenizer at 70 Hz for 45 s three times to obtain a homogenate. For membrane protein detection, sonication was performed using a cell disruptor, followed by incubation on ice for 30 min, followed by shaking for 30 sec every 5 min to ensure complete cell lysis. The cells were then centrifuged at 4°C and 10,000 rpm for 5 min. The supernatant was the protein solution; it was transferred to new centrifuge tubes and aliquoted for cryopreservation. All operations were performed at low temperatures. B) Protein concentration quantification: Prepare standard curve samples of various concentrations and add them to 96-well plates. Separately, add the sample to be tested to each 96-well plate, 20 μL per well. μ L. Prepare the BCA colorimetric solution according to the ratio, adding 180 μL to each well. μ For samples with a large number of samples, a multi-channel pipette should be used to add the sample, shortening the addition time. Incubate at 37℃ for 10-20 min, and detect the absorbance value at 562 nm using a microplate reader. Plot a standard curve based on the protein concentration and OD value of the standard curve, and calculate the protein concentration of the sample to be tested and the concentration of the sample before dilution based on the standard curve. Dilute different concentrations of protein samples to the same concentration (1-2 mg / mL) with 5×SDS (sample loading buffer), boil in a 95℃ metal bath for 10 min, then cool on ice and store at -80℃ for later use. C) Western blotting steps a. Prepare SDS-PAGE electrophoresis gel: Determine the required gel pore size by checking the size of the target protein band. Wash the 1.5 mm glass plate, install the gel casting rack, and add double-distilled water to check for leaks. According to the SDS-PAGE separating gel and stacking gel preparation systems, add the appropriate volumes of double-distilled water, 30% acrylamide mixture, 4×Tris-HCl-SDS separating gel buffer (pH 8.8), 4×Tris-HCl-SDS stacking gel buffer (pH 6.8), and 10% APS solution, respectively, and mix thoroughly. Invert the gel casting rack onto a paper towel to dry. Add the appropriate volume of TEMED to the separating gel mixture and mix thoroughly. Use a 5 mL pipette to add the mixture between the two glass plates, then slowly add 1 mL of anhydrous ethanol. Incubate at room temperature for 30 min. A clear separation occurred between the separating gel and anhydrous ethanol. The anhydrous ethanol was discarded, and the mixture was rinsed twice with double-distilled water. It was then inverted and air-dried on paper towels. An appropriate volume of TEMED was added to the stacking gel mixture and mixed thoroughly. This mixture was then added between two glass plates, and a comb was inserted. The mixture was allowed to stand at room temperature for 30 minutes. The comb was then removed, and the mixture was placed in the electrophoresis tank. Electrophoresis buffer was added, and the mixture was ready for subsequent sample loading. b. Electrophoresis: Thaw the sample at 4℃, mix well, centrifuge at 10000 rpm for 3 min, and take 10 samples. μ Add L supernatant to the gel wells, add markers to the wells on both sides, and use 10 μL for the blank wells. μ Add L 1X SDS loading buffer and start electrophoresis. Set the voltage to 66V. Once the sample has moved below the stacking gel, adjust the voltage to 110V. Stop electrophoresis once the target bands separate. c. Transfer: Immerse the PVDF membrane completely in methanol until it changes color completely. Place the PVDF membrane, transfer clip, sponge pad, filter paper, and gel into the transfer buffer. Seal the transfer clip in the following order: transfer clip white plate, sponge pad, filter paper, PVDF membrane, gel, filter paper, sponge pad. Place the transfer clip in the electrophoresis tank, fill it with transfer buffer, set the current to 220 mA, and transfer for 2 hours.

[0048] d. Blocking: Prepare a 5% (w / v) skim milk blocking solution using PBST solution and mix thoroughly. After transfer, remove the PVDF membrane and place it in the skim milk blocking solution for 1 hour. e. Primary antibody incubation: Dilute the primary antibody with commercially available primary antibody diluent according to the recommended ratio in the instructions, and incubate overnight at 4°C; after incubation, wash three times with PBST solution for 10 min each time. f. Secondary antibody incubation: Select a suitable secondary antibody based on the species of the primary antibody. Dilute the secondary antibody with commercially available secondary antibody dilution solution at a ratio of 1:10000 and incubate at room temperature for 1 h. After incubation, wash three times with PBST solution for 10 min each time. g. Development: Use a pipette to draw 1 mL of developer A, change the pipette tip and draw 1 mL of developer B, mix well; remove most of the buffer solution from the PVDF membrane with absorbent paper, place it in the luminescent solution, and then perform development and exposure.

[0049] D) Data Processing: Image J was used for protein quantification, and Graphpad Prism 8.0.1 software was used for statistical analysis. Figures and tables are presented as mean ± standard error (SEM). Independent samples were compared using independent samples t-tests, and multiple groups were analyzed using one-way ANOVA.

[0050] B. Experimental Results like Figure 13As shown, in the acute model, the protein levels of tight junction proteins E-cadherin and Occludin were significantly reduced in the model group (compared to the unmodeled group), while high-dose mussel polysaccharide administration significantly restored this phenomenon. In the chronic model, compared to the unmodeled group, E-cadherin was significantly reduced, and Occludin showed a decreasing trend, while compared to the model group, the protein levels of E-cadherin and Occludin were significantly increased in the high-dose mussel polysaccharide group. These results indicate that mussel polysaccharide can alleviate the loss of E-cadherin and Occludin proteins in both acute and chronic models.

[0051] ④ Effects of mussel polysaccharides on oxidative stress genes in the colon This invention uses real-time quantitative fluorescence PCR technology to detect the effect of the natural product mussel polysaccharide on the mRNA expression level of key genes for oxidative stress in colon tissue.

[0052] A. Experimental Materials and Methods A) RNA extraction from colon tissue: Accurately weigh 10mg~20mg of colon tissue; using the MolPure® FlashCell / Tissue Total RNA Kit, add two steel balls to a centrifuge tube containing the tissue, and add 500 mL of lysis buffer LB. Homogenize using a homogenizer. Transfer the homogenate to a DNA removal / RNA adsorption universal column, centrifuge at 13,000 rpm for 1 min, and collect the filtrate containing RNA (gDNA on the column, RNA in the filtrate). Add 0.5 times the volume of the filtrate (approximately 250 μL) of anhydrous ethanol to the filtrate and mix by pipetting. Add the entire mixture to a new DNA removal / RNA adsorption universal column, centrifuge at 13,000 rpm for 30 s, and discard the filtrate. Add 700 μL of protein removal buffer, centrifuge at 13,000 rpm for 30 seconds at room temperature, discard the filtrate, and return the DNA removal / RNA adsorption universal column to the 2 mL collection tube. Add 500 μL of protein removal buffer. µ L rinsing solution Centrifuge at 13,000 rpm for 30 seconds and discard the filtrate. Repeat the steps, placing the DNA removal / RNA adsorption universal column back into the 2 mL collection tube. Centrifuge the empty column at 13,000 rpm for 2 minutes to remove residual wash buffer. Place the DNA-clearing RNA-adsorption universal column into a new 1.5 mL RNase-free centrifuge tube, and add 30-50 μL of [unspecified substance] to the center of the membrane. µIncubate with LRNase-free H2O at room temperature for 1 min, then centrifuge at 13,000 rpm for 1 min. Collect the filtrate, which is the RNA solution. The sample can be stored at -80℃ for a long time.

[0053] B) Real-time quantitative PCR: a. Detect the concentration of extracted RNA using a microplate reader; b. Reverse transcribe the RNA into cDNA, using a reverse transcription system (20... μ L), as shown in Table 3 below. Reverse transcription process: i. 37°C, 15 min; ii. 85°C, 5 s; iii. Maintain 10°C. c. Quantitative PCR: The reaction system (per well) is shown in Table 4 below. Quantitative PCR process: i. 50°C, 2 min; ii. 95°C, 20 s; iii. 95°C, 5 s; iv. 60°C, 30 s; iii-iv cycles 40 times. Primer sequence listing is shown in Table 5 below.

[0054] Table 3

[0055] Table 4

[0056] Table 5

[0057] C) Data Processing: The 2^-△△Ct method was used. i. △Ct = Ct (target gene) - Ct (internal reference gene); ii. -△△Ct = -(△Ct (drug-treated group) - △Ct (model group)); iii. Powering -△△Ct by 2, i.e., 2^-△△Ct, yields the fold relationship between the drug-treated group and the solvent group. Data are expressed as mean ± standard deviation (mean ± sem), and t-tests were used for statistical analysis.

[0058] B. Experimental Results like Figure 14 As shown, in the acute model, compared with the non-model group, the colon tissue of the model group... Spink4 The mRNA level was significantly reduced. Sod1 , Cox17 , Prdx6 and Cox7a1 The mRNA levels tended to decrease, while the high and low doses of mussel polysaccharide significantly increased them. Sod1 and Cox7a1 mRNA levels increased in the high-dose mussel polysaccharide group. Cox17 mRNA. Mussel polysaccharide administration in an acute model showed... Prdx6 and Spink4 The mRNA was unaffected. In the chronic model, compared with the non-model group, the colon tissue of the model group was... Sod1 , Cox17 , Prdx6 and Cox7a1 The mRNA level was significantly reduced. Spink4 The mRNA levels tended to decrease, while the high-dose mussel polysaccharide group showed a significant increase. Sod1 , Cox17 , Prdx6 , Cox7a1 and Spink4 The study results indicate that mussel polysaccharide can restore antioxidant stress genes in colon tissue in both acute and chronic models. Sod1 , Cox17 , Prdx6 , Cox7a1 and Spink4 The expression.

[0059] In summary, this invention reveals that mussel polysaccharides can inhibit the production of inflammatory factors in the colon of ulcerative colitis (UC) animal models, increase the mRNA expression level of antioxidant genes, inhibit oxidative stress, and ultimately reduce the loss of tight junction proteins, thereby maintaining the intestinal barrier, improving pathological damage, and alleviating the occurrence and development of colitis. Moreover, this process may be independent of the gut microbiota. Therefore, mussel polysaccharides can be used to prepare drugs for the treatment of IBD, especially ulcerative colitis.

[0060] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. Use of mussel polysaccharide in the preparation of a product for treating and / or preventing inflammatory bowel disease.

2. Use according to claim 1, characterized in that, The structure of mussel polysaccharide is α-pyran type glucan composed of (1→4)-α-D-Glc and (1→2)-α-D-Glc glycosidic bond, and the molecular weight is 300-3500 kDa.

3. Use according to claim 1, characterized in that, The inflammatory bowel disease is a DSS-induced acute and chronic ulcerative colitis (UC) mouse model.

4. Use according to claim 1, characterized in that, The product is a food, a health food, a special medical food or a drug.

5. Use according to claim 4, characterized in that, The drug further comprises a pharmaceutically acceptable carrier, excipient or adjuvant.

6. Use according to claim 4, characterized in that, The drug is an oral preparation or an injection preparation.

7. The use according to claim 1, characterized in that, The mussel polysaccharide exists in the form of an organic acid salt or an inorganic acid salt.

8. Use according to claim 7, characterized in that, The organic acid is acetic acid, malic acid, maleic acid, citric acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid or oxalic acid; The inorganic acid is hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid.

9. The use according to claim 1, characterized in that, The treatment and / or prevention of inflammatory bowel disease includes counteracting symptoms such as weight loss, diarrhea and / or hematochezia.

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