A low molecular weight chondroitin sulfate composition and its preparation method and application

Abstract

The present invention relates to a low-molecular-weight chondroitin sulfate composition, its preparation method, and application, aiming to address the problem that chondroitin sulfate products obtained by a single degradation method have limited functions and are difficult to synergistically regulate the intestinal barrier. The preparation method first prepares LMCSO by oxidative degradation; secondly, prepares LMCSH by hydrothermal degradation; and finally, prepares LMCSD by deamination degradation. Finally, the obtained LMCSO, LMCSH, and LMCSD are compounded in a weight ratio of 5:3:2 to obtain a low-molecular-weight chondroitin sulfate composition LMCSs-C. The composition LMCSs-C prepared by the present invention can synergistically improve intestinal barrier function through multiple mechanisms of action. It not only demonstrates significant intestinal barrier repair effects in vitro and in animal models, but also has good biocompatibility and safety, showing broad application prospects in maintaining intestinal health and intervening in related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biopolysaccharide preparation, and more specifically, to a low molecular weight chondroitin sulfate composition, a preparation method and an application thereof. Background Art

[0002] The intestinal barrier refers to the comprehensive defense mechanism of the intestinal mucosal system that prevents harmful factors from entering other tissues, organs or blood circulation of the body. Damage to the intestinal barrier can directly cause inflammatory substances to cross the epithelial barrier and activate the inflammatory response; and inflammation, in turn, further weakens the barrier function by destroying tight junctions and other means, causing intestinal damage. The maintenance of the intestinal barrier depends on three main barrier systems: the physical barrier composed of the mucus layer and epithelial cells, which is responsible for blocking the invasion of harmful substances; the immune barrier composed of intestinal immune cells and their secretory factors, which participates in pathogen recognition and immune regulation; the biological barrier mainly composed of intestinal flora and its metabolites, which plays an important role in maintaining microecological balance and barrier function.

[0003] Chondroitin sulfate (CS) is a sulfated glycosaminoglycan composed of repeating disaccharide units linked by β-(1→4) glycosidic bonds. Each unit consists of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) linked by β-(1→3) bonds. Its molecular weight typically ranges from 20 to 70 kDa. Studies have shown that CS exhibits multiple biological activities, including antioxidant, anti-inflammatory, immunomodulatory, and microbiome-modulating activities, suggesting its potential to enhance the intestinal barrier, but this area has received insufficient attention. Currently, low-molecular-weight chondroitin sulfate (LMCS), obtained by degradation of CS, exhibits superior bioavailability and bioactivity. However, due to different degradation mechanisms, LMCS prepared by different methods exhibit structural differences, which may lead to varying effects on the intestinal barrier (physical, immune, and biological). Therefore, studying the effects of different LMCS structures on intestinal barrier function and formulating them to optimize their enhancement is crucial for maintaining intestinal health. Summary of the Invention

[0004] The purpose of the present invention is to provide a low molecular weight chondroitin sulfate composition and a preparation method thereof, so that the prepared low molecular weight chondroitin sulfate composition can synergistically strengthen the physical barrier, immune barrier and biological barrier functions, and improve the application effect of low molecular weight chondroitin sulfate in enhancing intestinal barrier function.

[0005] In order to achieve the purpose and other advantages of the present invention, a method for preparing a low molecular weight chondroitin sulfate composition is provided, comprising: adding H2O2 and ascorbic acid to a final concentration of 20-30 mM to a 10 mg / mL chondroitin sulfate solution, adjusting the pH to 5.5±0.2, reacting at 30°C-40°C for 20-40 minutes, and purifying the resulting product through a 0.5 kDa ultrafiltration membrane and then freeze-drying to obtain an oxidative degradation product of chondroitin sulfate, LMCSO; adjusting the pH of the 10 mg / mL chondroitin sulfate solution to 4.0±0.2 with acetic acid, reacting at 100°C-120°C for 2-3 hours, and purifying the resulting product through a 0.5 kDa ultrafiltration membrane and then freeze-drying to obtain an oxidative degradation product of chondroitin sulfate, LMCSO; kDa ultrafiltration membrane purification and freeze-drying were performed to obtain the hydrothermal degradation product of chondroitin sulfate, LMCSH; 80 mg / mL chondroitin sulfate solution was mixed with hydrazine hydrate containing 2.5%-3.5% hydrazine sulfate in a volume ratio of 1:1, and deacetylation reaction was carried out at 85°C-95°C under nitrogen protection. After the deacetylation reaction was carried out for 8 hours, a sample was taken and immediately cooled to below -20°C in dry ice to terminate the reaction. The sample was then re-dissolved in deionized water and centrifuged at 4°C and 10,000 rpm for 10 minutes through a 3 kDa ultrafiltration centrifuge tube. The N-acetylgalactosamine content in the sample was determined by high performance anion exchange chromatography-pulsed amperometric detection and the degree of deacetylation DDA% was calculated. If DDA% If the content of N-acetylgalactosamine is ≥95%, the reaction is terminated immediately. Otherwise, the reaction is continued and samples are taken every hour to detect the content of N-acetylgalactosamine and calculate the degree of deacetylation DDA%, until DDA% ≥95%. The sampling, detection and calculation of the degree of deacetylation DDA% are completed within 60 minutes. The mixed solution after the deacetylation reaction is then dialyzed with alcohol to obtain a deacetylated product. The deacetylated product is reacted with a nitrite solution at 4-6°C for 30 minutes, the pH is adjusted to 8.0 to terminate the reaction, and then a NaBH4 solution is added to reduce the product at 45°C-55°C for 1.5-2.5 hours. The molar ratio of the deacetylated product to the nitrite solution and the NaBH4 solution is 1:1.1-1.3:1.8-2.2. The molar number of the deacetylated product is based on the molar number of free amino groups in the deacetylated product. The obtained product is then subjected to 0.5 The chondroitin sulfate deamination product LMCSD was obtained by purification with a kDa ultrafiltration membrane and freeze-drying; LMCSO, LMCSH and LMCSD were compounded in a weight ratio of 5:3:2 to obtain a low molecular weight chondroitin sulfate composition LMCSs-C.

[0006] Preferably, in the step of preparing LMCSO, ascorbic acid is first added to the chondroitin sulfate solution and mixed, and then the H2O2 solution is added dropwise under continuous stirring; after the H2O2 solution is added dropwise, the mixture is reacted at 35±1°C for 10-15 minutes, then heated to 38±1°C and continued to react for 10-15 minutes; the final concentrations of the H2O2 and ascorbic acid are both 25±0.5 mM.

[0007] Preferably, in the step of preparing LMCSH, the reaction process adopts programmed temperature control, first heating to 118±1°C at a rate of 120±5°C / min; then reacting at 118±1°C at a constant temperature for 60±5 minutes; then cooling to 100±2°C at a rate of 15±3°C / min; then reacting at a constant temperature for 60±5 minutes at 100±2°C; after the reaction is completed, the reaction vessel is immediately placed in an ice water bath, and the system temperature is reduced to below 25°C within 2 minutes.

[0008] Preferably, in the step of preparing LMCSD, when the content of N-acetylgalactosamine in the sample is determined using high performance anion exchange chromatography-pulsed amperometric detection, the detection conditions are: chromatographic column CarboPac PA20, flow rate 0.5 mL / min, NaOH gradient 2-100 mM.

[0009] Preferably, in the step of preparing LMCSD, the specific method of alcohol precipitation dialysis is: cooling the mixed solution after the deacetylation reaction to room temperature, slowly adding 4 times the volume of anhydrous ethanol pre-cooled to 4°C under stirring, and letting it stand at 4°C for 2 hours; collecting the precipitate, dissolving the precipitate in deionized water, placing it in a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyzing it in deionized water at 4°C for 24 hours, during which the water is changed at least 6 times.

[0010] The present invention also provides a low molecular weight chondroitin sulfate composition, which is prepared by the above preparation method, wherein the sulfate content in the low molecular weight chondroitin sulfate composition is 15.0-16.5%, and the uronic acid content is 28.0-29.0%.

[0011] The present invention also provides a pharmaceutical composition comprising the low molecular weight chondroitin sulfate composition and a pharmaceutically acceptable carrier.

[0012] Preferably, the pharmaceutical composition is used to treat diseases related to intestinal barrier dysfunction, including inflammatory bowel disease, irritable bowel syndrome or intestinal flora imbalance.

[0013] The present invention has at least the following beneficial effects:

[0014] First, the present invention ensures that each prepared LMCS product has the expected and differentiated molecular structural characteristics (such as specific glycosidic bond cleavage, terminal structure, and degree of deacetylation) by precisely controlling the condition parameters (concentration, pH, temperature, time, and molar ratio) of the three degradation processes: oxidation, hydrothermal, and deamination cleavage, and purification methods (0.5 kDa ultrafiltration). By real-time monitoring of the degree of deacetylation (DDA%) during the deamination cleavage process and precisely controlling the reaction endpoint, the accuracy and batch-to-batch consistency of the LMCSD structure are guaranteed. The three LMCS products are then compounded in an optimized 5:3:2 ratio, enabling the composition to effectively integrate the unique bioactive advantages of the three single-component LMCSs: LMCSO strengthens the mucus barrier, LMCSD promotes penetration and anti-inflammatory effects, and LMCSH stimulates GLP-1 secretion, resulting in a significant synergistic effect. This comprehensively and synergistically enhances the intestinal physical, immune, and biological barrier functions, providing a well-defined and controllable composition for effectively maintaining intestinal health.

[0015] Second, by first mixing ascorbic acid and chondroitin sulfate solution and then adding H2O2 solution dropwise under continuous stirring, the present invention effectively avoids violent or uncontrollable oxidation reactions caused by excessive local H2O2 concentration, thereby improving reaction uniformity and safety. The use of staged precise temperature control (first starting the reaction at a lower temperature and then slightly increasing the temperature to continue the reaction) helps to achieve the oxidative breakage of chondroitin sulfate molecules in a more gentle and controllable manner, reducing excessive degradation or the formation of by-products. The control of the final concentrations of H2O2 and ascorbic acid (25±0.5 mM) ensures the efficiency and specificity of the redox reaction. The synergistic effect of these factors can improve the structural uniformity of the LMCSO product, making it more stable and efficient in enhancing the physical barrier function of intestinal mucus.

[0016] Third, the present invention achieves precise control over the hydrothermal depolymerization process by setting a specific heating rate, reaction temperature (including a high-temperature section and a slightly lower constant-temperature section), and cooling rate, ultimately requiring rapid cooling in an ice-water bath. Dynamic temperature management can more effectively regulate the rate and extent of hydrolysis of the β-(1→4) glycosidic bond, avoiding excessive temperature fluctuations that lead to an excessively broad molecular weight distribution or uncontrollable structural damage. Rapid cooling can terminate the reaction in a timely manner and lock in the target molecular structure. The prepared LMCSH product has a more concentrated molecular weight and a more expected structure, and can exhibit superior and more stable activity in stimulating enteroendocrine cells to secrete GLP-1, exerting anti-inflammatory and metabolic regulatory functions.

[0017] Fourth, by limiting the use of high-performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and specifying the chromatographic column (CarboPac PA20) and detection conditions (flow rate, NaOH gradient), the present invention can rapidly, accurately, and specifically quantitatively detect the content of N-acetylgalactosamine in the reaction system, thereby accurately calculating the degree of deacetylation (DDA%). This enables real-time monitoring of the deacetylation reaction process, ensuring that DDA% ≥95%, and ultimately ensuring the high structural consistency and biological activity of the LMCSD product.

[0018] Fifth, the present invention ensures that the composition has appropriate electronegativity and molecular polarity by controlling the sulfate content (15.0-16.5%) and the uronic acid content (28.0-29.0%) within specific ranges, making the quality of the composition controllable and the function stable.

[0019] Sixth, the low molecular weight chondroitin sulfate composition of the present invention avoids the therapeutic limitations of a single component and achieves synergistic intervention on intestinal barrier dysfunction through the multi-target action mechanism of LMCSs-C: upregulating ZO-1 / Occludin / Claudin-1 expression to repair the physical barrier; promoting the proliferation of SCFAs-producing bacteria such as Bifidobacterium to improve the chemical barrier; inhibiting the colonization of Escherichia-Shigella and consolidating the immune barrier. It is particularly suitable for treating diseases related to intestinal barrier dysfunction and enhancing intestinal barrier function. Due to its uniform molecular weight and non-toxic residue, it has good oral bioavailability and safety.

[0020] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a three-dimensional CLSM image of FITC-labeled low molecular weight chondroitin sulfate in the mucus layer;

[0022] Figure 2 is the content of low molecular weight chondroitin sulfate in the lower chamber at a fixed permeation time point;

[0023] Figure 3 It is the result of the penetration of FD4 and phenol red into the mucus gel layer of the small intestine in vitro;

[0024] Figure 4 This is the result of the effect of LMCS treatment on the transepithelial electrical resistance of Caco-2 cell monolayers treated with DSS;

[0025] Figure 5 This is the result of the effect of LMCS treatment on the IL-8 secretion level of Caco-2 cells;

[0026] Figure 6 The results show the effect of LMCS treatment on the relative expression levels of tight junction proteins ZO-1, Claudin-1, and Occludin mRNA in Caco-2 cells;

[0027] Figure 7 is the apparent permeability coefficient of CS and LMCS on Caco-2 cell monolayer;

[0028] Figure 8 This is the result of the effect of LMCS treatment on the secretion levels of NO, IL-1β, and IL-6 in RAW264.7 cells;

[0029] Figure 9 This is the result of the effect of LMCS treatment on the GLP-1 secretion level of STC-1 cells;

[0030] Figure 10 The difference in intestinal flora species was analyzed based on the LDA score histogram;

[0031] Figure 11 is the concentration of short-chain fatty acids in the CS, LMCSO, LMCSD, and LMCSH groups after 24 h of in vitro fermentation;

[0032] Figure 12 The results show the effects of LMCS and LMCSs-C treatment on the length and pathological score of mouse colon;

[0033] Figure 13 This is the staining result of mouse colon tissue section;

[0034] Figure 14 The results show the effects of different ratios of LMCSs-C on the length and pathological scores of mouse colon;

[0035] Figure 15 The results show the effects of LMCSs-C prepared by different methods on the length and pathological scores of mouse colon. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below with reference to the embodiments and drawings so that those skilled in the art can implement the invention with reference to the description.

[0037] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0038] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0039] According to one embodiment of the present invention, when preparing the oxidative degradation product LMCSO, a chondroitin sulfate solution concentration of 10 mg / mL is used. The final concentrations of H2O2 and ascorbic acid can be 20 mM, 25 mM, or 30 mM. The target pH is adjusted to 5.5±0.2, and can be 5.3, 5.5, or 5.7. The reaction temperature can be 30°C, 35°C, or 40°C, and the reaction time can be 20 minutes, 30 minutes, or 40 minutes. The ultrafiltration equipment can use a spiral wound ultrafiltration membrane with a molecular weight cutoff of 0.5 kDa, and the freeze dryer can use a standard laboratory freeze dryer. Specifically, chondroitin sulfate can be dissolved in deionized water to prepare a 10 mg / mL solution. After adding ascorbic acid powder and stirring to dissolve, a 30% H2O2 solution is slowly added dropwise over 5 minutes while maintaining a constant temperature magnetic stirrer at 35°C. After the addition is complete, the pH is adjusted to 5.5±0.2 with 0.1 M HCl, and the reaction is continued for 30 minutes. The reaction solution was purified by a 0.5 kDa ultrafiltration membrane under a pressure of 0.2 MPa, and the permeate was collected and freeze-dried to obtain the LMCSO product.

[0040] To prepare the hydrothermal degradation product, LMCSH, a chondroitin sulfate solution concentration of 10 mg / mL was used. The target pH was adjusted to 4.0±0.2, and the actual range was 3.8-4.2. The reaction temperature could be 100°C, 110°C, or 120°C, and the reaction time could be 2 hours, 2.5 hours, or 3 hours. A polytetrafluoroethylene-lined autoclave could be used as the reaction vessel, and a programmed temperature oven could be used for the temperature control system. Specifically, a 10 mg / mL chondroitin sulfate aqueous solution was prepared, and the pH was adjusted to 4.0±0.2 with glacial acetic acid. The solution was transferred to an autoclave, sealed, and placed in a programmable temperature oven: the temperature was increased at a rate of 120°C / min to 118°C and held for 60 minutes; then the temperature was decreased at a rate of 15°C / min to 100°C and held for 60 minutes. Upon completion of the reaction, the autoclave was immediately immersed in an ice-water bath and cooled to below 25°C within 2 minutes. The reaction solution was purified by 0.5 kDa ultrafiltration and lyophilized to obtain LMCSH powder.

[0041] When preparing the deamination degradation product, LMCSD, a chondroitin sulfate concentration of 80 mg / mL was used. The hydrazine sulfate content in hydrazine hydrate could be 2.5%, 3.0%, or 3.5%. The deacetylation temperature could be 85°C, 90°C, or 95°C, and the reaction time was terminated when the DDA% was ≥95%. Four volumes of anhydrous ethanol were used for alcohol precipitation, and 10 kDa molecular weight cutoff dialysis tubing was used for dialysis. Specifically, an 80 mg / mL chondroitin sulfate solution was mixed with hydrazine hydrate containing 3.0% hydrazine sulfate in a 1:1 ratio by volume. The mixture was then filled with nitrogen and sealed in a pressure-resistant glass reactor. The reaction was allowed to proceed in an oil bath at 90°C. 2 mL samples were taken every 60 minutes and quenched with dry ice at -80°C. After reconstitution, the mixture was filtered through a 0.22 μm filter membrane and the residual N-acetylgalactosamine was determined using an ion chromatography system. When the DDA% was ≥95%, the reaction solution was cooled and four volumes of -20°C pre-cooled anhydrous ethanol were added. The reaction solution was then allowed to settle at 4°C for 2 hours. The precipitate was collected by centrifugation, dissolved in water, and placed in a 10 kDa dialysis bag for 24 hours at 4°C. The dialysate was mixed with a 0.5 M NaNO₂ solution at a molar ratio of 1:1.2 for free amino groups:NaNO₂. The reaction was allowed to proceed at 5°C for 30 minutes, and the pH was adjusted to 8.0. The product was then reduced with a 0.1 M NaBH₄ solution at 50°C for 2 hours. The product was then lyophilized through a 0.5 kDa ultrafiltration to obtain LMCSD.

[0042] The weight ratio of LMCSO, LMCSH, and LMCSD is 5:3:2. A V-type dry powder mixer can be used for mixing. Specifically, 50 g of freeze-dried LMCSO, 30 g of LMCSH, and 20 g of LMCSD were weighed and placed in the mixer hopper. Mix at 20 rpm for 30 minutes. Sieve through a 60-mesh sieve and dispense to obtain the LMCSs-C composition.

[0043] According to another embodiment of the present invention, ascorbic acid is preferentially added to the chondroitin sulfate solution. The H2O2 solution can be added at a rate of 0.5 mL / min, 1 mL / min, or 1.5 mL / min. A four-blade paddle stirrer can be used as the stirring device, mounted on the central axis of the reaction vessel. For pH monitoring, an online pH meter probe can be installed on the side wall of the reactor at a depth of 1 / 3 of the liquid surface. Specifically, a 10 mg / mL chondroitin sulfate solution can be added to a three-necked flask and maintained in a constant temperature water bath at 35°C. The stirrer is turned on (300 rpm), and ascorbic acid powder is added to a final concentration of 25 mM. Stir for 5 minutes until dissolved. A 30% H2O2 solution is added dropwise below the liquid surface using a constant flow pump at a rate of 1 mL / min.

[0044] The first stage temperature can be selected at 34°C, 35°C, or 36°C, and the reaction time can be selected from 10 minutes, 12 minutes, or 15 minutes. The second stage temperature can be selected from 37°C, 38°C, or 39°C, and the reaction time can be selected from 10 minutes, 12 minutes, or 15 minutes. The temperature control equipment can be a programmable constant-temperature water bath, with the temperature sensor placed at the geometric center of the reaction solution. Specifically, after the addition of H2O2, the reaction is maintained at 35°C for 12 minutes. The temperature is then raised to 38°C at a rate of 1°C / min and the reaction is continued for 12 minutes. The pH value is controlled to fluctuate between 5.4 and 5.6 during the reaction, and the reaction is immediately cooled in an ice bath to below 10°C. FTIR analysis shows that the product of the two temperature-controlled stages has a higher peak intensity at 1070 cm⁻¹.

[0045] The final concentrations of H2O2 and ascorbic acid can be 24.5 mM, 25.0 mM, or 25.5 mM. A precision pipette or electronic balance (0.1 mg accuracy) can be used as the measuring device. Specifically, weigh 0.176 g of ascorbic acid powder and use a microbalance to accurately measure to ±0.001 g. Measure 0.113 mL of a 30% H2O2 solution using a calibrated microsyringe to maintain an accuracy of ±1%. After addition, verify that the actual concentration is within the range of 25 ± 0.5 mM by iodine titration.

[0046] According to another embodiment of the present invention, the heating rate can be selected from 115°C / min, 120°C / min or 125°C / min. The target temperature can be selected from 116°C, 118°C or 120°C. The heating equipment can be a program oven, and the reaction vessel can be a stainless steel high-pressure reactor, which is assembled in the center of the oven cavity. The temperature sensor can be a K-type thermocouple, which is installed in the middle of the side wall of the reactor. Specifically, a chondroitin sulfate solution with a pH of 4.0 is placed in the high-pressure reactor and sealed. The oven is set to heat from 25°C to 118°C at a rate of 120°C / min, and the temperature of the inner wall of the reactor is monitored in real time. When it reaches 118°C, the constant temperature mode is automatically switched. The temperature record shows that the actual heating time is 58±2 seconds.

[0047] The constant temperature can be selected from 116°C, 118°C, or 120°C, and the duration can be selected from 55 minutes, 60 minutes, or 65 minutes. A spring-loaded safety valve can be used as the pressure control valve, installed at the vent port of the reactor lid. The thermocouple probe can be inserted halfway into the reaction solution. Specifically, maintain a constant temperature of 118°C for 60 minutes, recording the reaction solution temperature every 10 minutes. A pressure valve is used to maintain the internal pressure at a stable level of 0.25±0.02 MPa.

[0048] The cooling rate can be selected from 12°C / min, 15°C / min, or 18°C / min. The target temperature can be selected from 98°C, 100°C, or 102°C. The cooling system can utilize an air cooling module, installed in the rear duct of the oven. Specifically, air cooling is initiated at the end of the high-temperature period, decreasing the temperature to 100°C at a rate of 15°C / min. The air volume is adjusted during the cooling process to maintain a linear temperature drop. Ultraviolet spectroscopy testing indicates that the absorbance of melanoidin is ≤0.05.

[0049] The cryostat temperature can be selected at 98°C, 100°C, or 102°C, and the time can be selected at 55 minutes, 60 minutes, or 65 minutes. The quenching device can be a stainless steel ice-water bath, mounted on the workbench. The magnetic stirrer blade is placed at the bottom of the bath. Specifically, after reaching 100°C, the temperature is maintained for 60 minutes, and the reactor is immediately transferred to a 0-4°C ice-water bath. Stirring is started at 500 rpm, and the system temperature is reduced to below 25°C within 2 minutes. Product testing shows a particle size polydispersity index (PDI) of <0.2.

[0050] According to another embodiment of the present invention, the chromatographic column can be a CarboPac PA10, PA20, or PA30. The column temperature control range can be 30-40°C, and can be set to 35°C. The chromatographic column can be installed in the constant temperature chamber of an ion chromatograph. The sample injector can be an automatic sample injector, installed at the flow path interface between the pump and the chromatographic column. Specifically, the deacetylated sample is filtered through a 0.22 μm filter membrane, and 10 μL is injected. A CarboPac PA20 column (3×150 mm) is used, and the column temperature is maintained at 35°C. A pulsed amperometric detector with a gold working electrode can be used as the detector.

[0051] The flow rate can be selected from 0.4 mL / min, 0.5 mL / min, or 0.6 mL / min. A dual-plunger inline pump can be used as the infusion pump. A pulse dampener can be installed at the pump outlet, 15-20 cm from the column inlet. Specifically, the flow rate is set to 0.5 mL / min using the chromatographic workstation. The eluent is a NaOH solution prepared in ultrapure water, delivered to the system via an online degasser. Before operation, the actual flow rate error is calibrated using a flow meter. Three consecutive injections indicate a retention time deviation of ≤0.1 minute.

[0052] The starting concentration of NaOH can be 1 mM, 2 mM, or 3 mM, and the final concentration can be 80 mM, 100 mM, or 120 mM. A dual-channel proportional valve can be used as the gradient mixer, installed between the infusion pump and the injection valve. Specifically, set the gradient program: maintain 2 mM NaOH from 0 to 5 minutes; linearly increase to 100 mM from 5 to 20 minutes; and maintain 100 mM for 20 to 30 minutes. The gradient linear correlation coefficient is set to the threshold value R. 2 ≥0.995. Use a mixed sample of galactosamine and N-acetylgalactosamine to verify that the resolution is ≥1.5.

[0053] According to another embodiment of the present invention, the ethanol addition multiple can be 3 times, 4 times, or 5 times the volume. The ethanol pre-cooling temperature can be 0°C, 2°C, or 4°C. The standing time can be 1 hour, 2 hours, or 3 hours. The precipitation container can be a conical centrifuge bottle, placed on the inner shelf of a 4°C refrigerator. The stirring device can be a glass rod manual stirring or a magnetic stirrer. Specifically, the reaction solution after the deacetylation is cooled to below 25°C. Measure 4 times the volume of anhydrous ethanol and pre-cool it to 4°C. Slowly inject ethanol while stirring at 300 rpm for at least 5 minutes. After stopping stirring, move to a 4°C refrigerator and let it stand for 2 hours. The supernatant is collected and tested for hydrazine residue, which shows that the residual amount meets the safety threshold.

[0054] The molecular weight cutoff of the dialysis bag can be 8 kDa, 10 kDa, or 12 kDa. The dialysis time can be 20 hours, 24 hours, or 28 hours. The threshold for water changes should be set to no less than 6 times. The dialysis system can use a magnetic stirring water bath, which should be installed in a 4°C refrigerator; the dialysis bag holder should be installed in the center of the bath. Specifically, collect the alcohol precipitate and dissolve it in deionized water. Inject a dialysis bag with a molecular weight cutoff of 10 kDa and clamp both ends. Place it in 4°C deionized water and start magnetic stirring at 200 rpm. Change the dialysate every 4 hours, for a total of 6 water changes, for a total of 24 hours. The dialysate conductivity test shows that the ion residue meets the standard.

[0055] Alcohol precipitation must be performed before dialysis. The volume of deionized water used to dissolve the precipitate is controlled to be 1-2 times that of the precipitate. The amount of liquid in the dialysis bag does not exceed 80% of the volume. After the alcohol precipitation is completed, collect the precipitate by centrifugation at 5000 rpm for 10 minutes at 4°C. Add 1.5 times the volume of deionized water to the precipitate and vortex to dissolve it. When injecting into the dialysis bag, control the liquid level to be more than 3 cm away from the bag opening, remove bubbles and clamp it. The final product test shows that the recovery rate of the target component is in line with expectations. The order of alcohol precipitation followed by dialysis ensures the recovery of large molecular active components. The three work together to reduce the oxidation loss of free amino groups, control the residual hydrazine content below the safety limit, and meet the requirements of oral preparations.

[0056] Example 1:

[0057] The preparation of a low molecular weight chondroitin sulfate composition comprises the following steps:

[0058] (1) Preparation of low molecular weight chondroitin sulfate (LMCS): oxidative degradation method (LMCSO), hydrothermal degradation method (LMCSH) and deamination cleavage method (LMCSD).

[0059] LMCSO: To a 10 mg / mL chondroitin sulfate solution, add H2O2 and ascorbic acid, both at a final concentration of 25 mM, in sequence, adjust the pH to 5.5, and react at 35°C for 30 minutes. The resulting product is purified by a 0.5 kDa ultrafiltration membrane and lyophilized to obtain LMCSO, the oxidative degradation product of chondroitin sulfate.

[0060] LMCSH: A 10 mg / mL chondroitin sulfate solution was adjusted to pH 4.0 with glacial acetic acid, reacted at 110°C for 2.5 hours, and the resulting product was purified by a 0.5 kDa ultrafiltration membrane and freeze-dried to obtain the hydrothermal degradation product of chondroitin sulfate, LMCSH.

[0061] LMCSD: 80 mg / mL chondroitin sulfate solution was mixed with hydrazine hydrate containing 3.0% hydrazine sulfate in a volume ratio of 1:1. Deacetylation reaction was carried out at 90°C under nitrogen protection. After the deacetylation reaction was carried out for 8 hours, a sample was taken and immediately cooled to below -20°C in dry ice to terminate the reaction. The sample was then reconstituted with deionized water and centrifuged at 4°C and 10,000 rpm for 10 minutes through a 3 kDa ultrafiltration centrifuge tube. The N-acetylgalactosamine content in the sample was determined by high performance anion exchange chromatography-pulsed amperometric detection and the degree of deacetylation (DDA%) was calculated. If DDA% If the content of N-acetylgalactosamine is ≥95%, the reaction is terminated immediately. Otherwise, the reaction is continued and samples are taken every hour to detect the content of N-acetylgalactosamine and calculate the degree of deacetylation DDA%, until DDA% is ≥95%. The sampling, detection and calculation of the degree of deacetylation DDA% are completed within 60 minutes. Then, the mixture after the deacetylation reaction is cooled to room temperature, 4 times the volume of anhydrous ethanol precooled to 4°C is slowly added under stirring, and the mixture is allowed to stand at 4°C for 2 hours. After the standing period, the precipitate is collected by centrifugation at 5000 rpm for 10 minutes at 4°C, and the precipitate is dissolved in 1.5 times the volume of deionized water and loaded into a 10% molecular weight cut-off precipitate. kDa dialysis bag, placed in 4 ° C deionized water for 24 hours, during which the dialysate was replaced every 4 hours to obtain the deacetylated product, and then the deacetylated product was reacted with sodium nitrite solution at 4 ° C for 30 minutes, the pH was adjusted to 8.0 to terminate the reaction, and then NaBH4 solution was added to reduce it at 50 ° C for 2 hours. The molar ratio of the deacetylated product to the sodium nitrite solution and the NaBH4 solution was 1:1.2:2. The molar number of the deacetylated product was calculated based on the molar number of free amino groups in the deacetylated product. The obtained product was then purified by a 0.5 kDa ultrafiltration membrane and freeze-dried to obtain the deamination degradation product of chondroitin sulfate LMCSD.

[0062] (2) Preparation of low molecular weight chondroitin sulfate composition: A low molecular weight chondroitin sulfate composition (LMCSs-C) was prepared according to the ratio of LMCSO: LMCSD: LMCSH = 5:3:2.

[0063] Experimental Example 1: Structural Characterization of Low Molecular Weight Chondroitin Sulfate

[0064] Experimental methods:

[0065] (1) Determination of uronic acid content: The uronic acid content in chondroitin sulfate was determined according to the sulfuric acid-carbazole method. At room temperature, 2.5 mL of borax-concentrated sulfuric acid solution was added to a glass tube. 0.5 mL of standard solution and sample solution (0.1 mg / mL) of different concentrations were added to the test tubes, shaken well, reacted in boiling water for 10 minutes, and then cooled in an ice water bath. 0.1 mL of carbazole reagent was added, shaken well, reacted in boiling water for 15 minutes, and then cooled to room temperature in an ice water bath. The absorbance of the standard solution and sample solution was measured at 530 nm and a standard curve was drawn. Three parallels were performed in each group. Water was used instead of the sample as a blank control to obtain the uronic acid content of the sample.

[0066] (2) Determination of sulfate content: The sulfate content in chondroitin sulfate was determined by gelatin turbidimetry. The sample was dissolved in 1 M hydrochloric acid solution to prepare a 1 mg / mL sample solution. The solution was sealed and acid-hydrolyzed at 105°C for 2 hours. The solution was cooled to room temperature and the volume was adjusted to 10 mL with 1 M hydrochloric acid. 0.2 mL of the standard solution and sample solution were added to 3.8 mL of 1% trichloroacetic acid and 1.0 mL of barium chloride-gelatin solution respectively. The solution was shaken and allowed to stand for 15 minutes. The absorbance of the reaction solution was measured at 360 nm and a standard curve was drawn. Three parallels were performed in each group. 1 M hydrochloric acid was used instead of the sample as a blank control to obtain the sulfate content of the sample.

[0067] (3) Molecular weight determination: Mobile phase: Prepare a 1 M sodium sulfate solution, pass it through a 0.22 μm ultrafiltration membrane to remove impurities, and ultrasonicate it for 30 minutes to remove bubbles in the mobile phase to obtain the mobile phase; Sample solution: Take 10 mg of chondroitin sulfate sample and dissolve it in 10 mL of mobile phase, and pass it through a 0.22 μm ultrafiltration membrane. The molecular weight of chondroitin sulfate was determined by liquid chromatography coupled with an 18-angle laser scattering spectrometer and a differential detector (HPSEC-MALLS-RI). The refractive index increment dn / dc was set to 0.135 to determine the molecular weight of chondroitin sulfate. Chromatographic conditions: TSK gel G5000PWxl (7.8×300 mm) as the analytical column; the mobile phase was 1 M sodium sulfate solution; the flow rate was 0.5 mL / min; the injection volume was 100 μL; and the column temperature was 30 °C.

[0068] (4) Infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR) analysis: The infrared absorption spectrum of the sample was measured by Fourier transform infrared spectrometer. The sample was ground and mixed with KBr in a mortar and pressed into a test sheet containing 0.5% sample. -1 Wave number range is 4 cm -1Data acquisition was performed with a resolution of 1000 nm and 64 scans.

[0069] (5) Nuclear magnetic resonance spectrum: measured by AVANCE III HD 500MHz NMR instrument. The sample solution was prepared using D2O as solvent. One-dimensional and two-dimensional NMR spectra were collected respectively, including 1 H spectrum, 13 C spectrum, 1 H- 1 H correlation spectroscopy (COSY), 1 H- 13 C heteronuclear single quantum coherence spectroscopy (HSQC) and heteronuclear multiple bond coherence spectroscopy (HMBC).

[0070] (6) Mass spectrometry analysis: Chondroitin sulfate was enzymatically digested using chondroitinase ABC at 37°C for 10 hours. The dialyzed samples were analyzed using an Agilent 6460 QqQMS triple quadrupole mass spectrometer equipped with an ultra-high performance liquid chromatograph (Agilent 1290 infinity II UHPLC) and an electrospray ionization source (ESI). The conditions were as follows: ESI in negative ion mode, drying gas temperature at 350°C, drying gas flow rate at 8 L / min, nebulizer pressure at 15 psi, capillary voltage at 4000 V, fragmentor voltage at 200 V, and m / z range from 100 to 2000.

[0071] Experimental results:

[0072] The results of uronic acid content, sulfate content and molecular weight determination are shown in the table below.

[0073]

[0074] Note: Mean ± SD of three measurements, different letters in the same column indicate significant differences ( p <0.05).

[0075] As can be seen from the above table, the molecular weight and uronic acid content of the three low molecular weight chondroitin sulfates are significantly lower than that of chondroitin sulfate CS, and the sulfate content of LMCSD and LMCSH is significantly lower than that of chondroitin sulfate CS.

[0076] Fourier transform infrared spectroscopy revealed potential changes in chemical bonds and functional groups during the different degradation reactions of low-molecular-weight chondroitin sulfate. NMR and UHPLC-MS results showed that oxidative degradation preferentially destroyed the β-(1→3) bond of CS, leading to the oxidation of GlcA to adipic acid. Deamination degradation specifically cleaved the β-(1→4) bond of CS, generating oligosaccharides containing anTal-ol termini, some of which lost acetyl and sulfate groups. Hydrothermal degradation hydrolyzed the β-(1→4) bond of CS, accompanied by the destruction of sulfate groups, to generate oligosaccharides with intact CS structures.

[0077] Experimental Example 2: Activity Analysis of Low Molecular Weight Chondroitin Sulfate

[0078] Experimental methods:

[0079] (1) Preparation of porcine intestinal mucus: Fresh porcine intestines were obtained from a local slaughterhouse and rinsed with deionized water and 10 mM phosphate buffer (pH 6.7) in sequence. The mucus was collected by gently scraping the mucosal surface. To prevent degradation, protease inhibitors (MedChemExpress, Shanghai, China) were added at a ratio of 100:1 (v / v). The samples were then frozen and stored at -80°C until use.

[0080] (2) Mucus layer permeation experiment: Low molecular weight chondroitin sulfate was labeled with FITC. The in vitro mucus layer permeation experiment was performed using a Transwell 24-well plate (pore size 0.4 μm). 0.16 mL of porcine intestinal mucus was added to the upper chamber, followed by 0.04 mL of FITC-LMCS solution (2 mg / mL), and 1.2 mL of PBS buffer was added to the lower chamber. The Transwell was placed in a 37°C incubator and incubated overnight. The permeability was evaluated by detecting the content of low molecular weight chondroitin sulfate in the lower chamber. Subsequently, the mucus gel was fixed with a 75:25 (volume ratio) ethanol / acetic acid mixture for 60 minutes. After staining with wheat germ agglutinin (WGA) labeled with Alexa Fluor 488, a large-scale Z-axis scanning image of the mucus gel was performed using a German Leica confocal laser scanning microscope.

[0081] (3) Effect of low molecular weight chondroitin sulfate on mucus permeability: Phenol red (354.38 Da) and fluorescein isothiocyanate-dextran 4 kDa (FD4, ≈4000 Da) were used as small molecule and large molecule markers, respectively, to evaluate the effect of low molecular weight chondroitin sulfate on mucus permeability. Porcine intestinal mucus (0.6 mg / mL) was mixed with polysaccharide solution (10 mg / mL) and incubated at 37°C for 2 hours. 0.16 mL of the mucus / polysaccharide mixture was added to the upper chamber of the Transwell, followed by 0.04 mL of phenol red (10 mM) or FD4 (1.25 mM), and 1.2 mL of PBS buffer was added to the lower chamber. After incubating the Transwell at 37°C for 1.5 hours, the concentrations of phenol red and FD4 in the lower chamber were measured using a spectrophotometer and a fluorescence spectrophotometer, respectively.

[0082] (4) Construction of Caco-2 / RAW264.7 co-culture model: Caco-2 cells were cultured at a rate of 4×10 4 Cells were seeded at a density of 100 cells / well in Transwell chambers (0.4 μm PET membrane, Corning, USA) and cultured for 21 days. Transepithelial electrical resistance (TEER) was monitored using a Millicell-ERS2 voltammetry-ohmmeter (Millipore, USA). Cells with a TEER value > 500 Ω·cm were screened. 2 RAW264.7 cells (1×10 5 Caco-2 monolayer integrity was assessed by changes in TEER after DSS treatment. Basolateral culture medium was collected and assayed for nitric oxide (NO), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels using kits (Excellent Biotechnology Co., Ltd., China). To assess the transport efficiency of polysaccharides in the Caco-2 cell monolayer model, 1 mg / mL sample was added to the apical side (AP) and HBSS buffer was added to the basolateral side (BL). After a 2-hour incubation, the basolateral culture medium was collected and assayed for sample concentration using a bovine CS ELISA kit (Shanghai ELISA Biotechnology Co., Ltd., China). The apparent permeability coefficients (Papp) of CS and LMCS were calculated.

[0083] (5) q-PCR analysis: Caco-2 cells were cultured as described above. After collecting the basolateral culture medium, IL-8 levels were measured using a kit (Excellent Biotechnology Co., Ltd., China). Total RNA from Caco-2 cells was extracted using TRIzol reagent (Life Technologies, USA), reverse transcribed into cDNA, and then subjected to real-time fluorescence quantitative RT-PCR using SYBR Green Premix Pro TaqHS reagent (Aicore Biotechnology, China).

[0084] (6) In vitro GLP-1 secretion assay: STC-1 cells were plated at 1×10 5 Cells were seeded at a density of 100 cells / well in 96-well plates and incubated overnight in a medium containing 0.5 mg / mL of sample. The culture medium was then collected and the level of glucagon-like peptide-1 (GLP-1) secretion was measured using an ELISA kit (China Excellent Biotechnology Co., Ltd.).

[0085] (7) In vitro simulated fermentation: Fresh fecal samples were collected from 6 healthy volunteers (3 females and 3 males, aged 23-28 years, with no history of intestinal diseases and no antibiotic use in the past 3 months). After equal amounts of fecal samples from each volunteer were mixed, they were diluted with 0.1 M sterile phosphate buffer to a 10% (w / v) fecal slurry, homogenized with a handheld homogenizer for 3 minutes, and then centrifuged at 500 rpm for 10 minutes. After centrifugation, the fecal suspension was immediately stored in an anaerobic tank. The basal culture medium configuration is shown in the table below. Chondroitin sulfate and its degradation products were finally selected as the 10% (w / v) carbon source, and a carbon-free culture medium was used as a blank control (BLK). 1 mL of fecal suspension was added to each culture medium group (CS, LMCSO, LMCSH, LMCSD, BLK), and the mixture was incubated in an anaerobic environment at 37°C. Each experiment was repeated three times. Fermentation samples were collected at 0, 6, 12, and 24 hours and quickly frozen at -80°C. The supernatant of the fermentation sample was divided into two parts: one was used to determine the total carbohydrate content and pH; the other was ethanol precipitated, dialyzed, and freeze-dried to obtain samples at different fermentation time points (such as CS-0 h, CS-6 h, CS-12 h, and CS-24 h), and the physicochemical properties of the fermented polysaccharides were subsequently analyzed.

[0086]

[0087] (8) Gut microbiota analysis: After 24 h of in vitro fermentation, DNA was extracted from oligofructose (FOS), CS, LMCSO, LMCSH, LMCSD, and blank (BLK) samples using the OMEGA Soil DNA Kit (Omega Bio-Tek, Norcross, USA). All extracted DNA samples were subsequently analyzed by Shanghai Zhongke New Life Biotechnology Co., Ltd. Species annotation was performed using a pre-trained naive Bayes classifier and compared to the SILVA 138 reference database (Silva 138 [https: / / www.arb-silva.de / ] was used for 16S analysis, and UNITE [https: / / unite.ut.ee / ] was used for ITS analysis). Krona charts (https: / / github.com / marbl / Krona / wiki) were used to visualize the relative abundance of microbial composition at the phylum to species level.

[0088] (9) Determination of short-chain fatty acids (SCFAs): The pH value of the samples at different fermentation time points was measured using a pH meter. An appropriate amount of sample was placed in a 2 mL centrifuge tube, acidified and homogenized by adding 50 μL of 20% phosphoric acid, and then 500 μM 4-methylvaleric acid was used as an internal standard. The mixture was shaken and centrifuged for 20 minutes. The supernatant was transferred to an injection bottle for gas chromatography-mass spectrometry (GC-MS) analysis. The samples were separated using an Agilent 7890B gas chromatography system and a DB-FFAP capillary column (30 m × 250 μm × 0.25 μm), and mass spectrometry detection was performed by an Agilent 5977B MSD mass spectrometer. Each experiment was repeated three times.

[0089] (10) Non-targeted metabolomics analysis: Non-targeted metabolomics analysis was performed by Shanghai Zhongke Xinsheng Biotechnology Co., Ltd. After pretreatment, the supernatant was vacuum dried, vortexed with 100 μL of acetonitrile-water solution, centrifuged, and the supernatant was collected for mass spectrometry analysis. Metabolite detection was performed using a Vanquish UHPLC system coupled with a high-resolution Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Chromatographic separation was performed using an ACQUIY UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm, Waters, Ireland).

[0090] Experimental results:

[0091] The permeation behavior of FITC-labeled low molecular weight chondroitin sulfate in the mucus layer was observed by three-dimensional CLSM scanning, and its permeability was analyzed by Transwell method. Compared with CS, low molecular weight chondroitin sulfate was more evenly distributed in the mucus layer and significantly diffused into the lower layer, among which LMCSH had a more obvious diffusion effect ( Figure 1 In the figure, red is Alexa Fluor 488-WGA labeled mucin, and green is FITC labeled CS and LMCS). The ability of low molecular weight chondroitin sulfate to penetrate the mucus layer was evaluated by measuring the content of low molecular weight chondroitin sulfate in the chamber at fixed penetration time points. The results showed that ( Figure 2 , different letters in the figure indicate significant differences between groups ( p <0.05), the same below), the cumulative permeability of low molecular weight chondroitin sulfate, especially LMCSH, was significantly higher than that of CS ( p <0.05). To evaluate the effect of polysaccharides on the molecular permeability of the small intestinal mucus layer, phenol red (molecular weight = 354.38 Da) and FD4 (average molecular weight = 4000 Da) were used as representatives of small and large hydrophilic substances in the in vitro polysaccharide / mucus layer model and loaded into the upper chamber. The results showed that ( Figure 3 , (a) shows the results of FD4's penetration into the intestinal mucus gel layer in vitro, (b) shows the results of phenol red's penetration into the intestinal mucus gel layer in vitro) Polysaccharides significantly reduced the permeability of the mucus layer to small molecules (phenol red) and large molecules (FD4) ( p <0.05), demonstrating that low molecular weight chondroitin sulfate can enhance the mucus barrier. Among them, LMCSO has the strongest effect in enhancing the mucus barrier.

[0092] Changes in transepithelial electrical resistance (TEER) can quantify the improvement effect of low molecular weight chondroitin sulfate on DSS-induced epithelial cell barrier damage. Low molecular weight chondroitin sulfate treatment can effectively alleviate DSS-induced intestinal epithelial cell barrier damage ( p <0.05) ( Figure 4 Compared with the model group, low molecular weight chondroitin sulfate reduced IL-8 levels by approximately 14-20% ( Figure 5 ), low molecular weight chondroitin sulfate can effectively and significantly upregulate the expression levels of mRNAs of tight junction proteins (ZO-1, Claudin-1 and Occludin) ( Figure 6, (a) shows the effect of LMCS treatment on the relative expression level of tight junction protein ZO-1 mRNA in Caco-2 cells, (b) shows the effect of LMCS treatment on the relative expression level of tight junction protein Claudin-1 mRNA in Caco-2 cells, (c) shows the effect of LMCS treatment on the relative expression level of tight junction protein Occludin mRNA in Caco-2 cells), which is consistent with the results of TEER. This shows that low molecular weight chondroitin sulfate can effectively improve the damage of tight junction proteins caused by DSS. The apparent permeability coefficient (Papp value) of low molecular weight chondroitin sulfate on the Caco-2 cell monolayer reflects the ability of the sample to pass through the Caco-2 monolayer to reach RAW264.7 cells, among which LMCSD has the strongest permeability ( Figure 7 The addition of low molecular weight chondroitin sulfate effectively inhibited the production of IL-1β and IL-6 induced by DSS and TNF-α, with the LMCSO group showing the lowest level ( Figure 8 , (a) shows the effect of LMCS treatment on NO secretion level of RAW264.7 cells, (b) shows the effect of LMCS treatment on IL-1β secretion level of RAW264.7 cells, (c) shows the effect of LMCS treatment on IL-6 secretion level of RAW264.7 cells). Compared with the control group, polysaccharide treatment promoted the secretion of GLP-1 in STC-1 cells ( p <0.05, and the secretion level after LMCSH treatment was significantly higher than that in other treatment groups ( p <0.05) ( Figure 9 ).

[0093] Low molecular weight chondroitin sulfate reduces the abundance of Escherichia-Shigella spp. Figure 10 ), promoting short-chain fatty acids SCFAs ( Figure 11 ) and GlcNAc-6P, lactic acid, progesterone and other metabolites produce protective intestinal barrier, among which LMCSO has the best effect.

[0094] The above experimental results show that LMCSO is the most active in strengthening the mucus barrier, promoting the production of beneficial intestinal metabolites and inhibiting the proliferation of harmful bacteria; LMCSD, due to its excellent penetrating ability, can effectively pass through epithelial cells into the body, and shows excellent efficacy comparable to LMCSO in anti-inflammatory and mucus barrier strengthening; and LMCSH can significantly promote the secretion of glucagon-like peptide-1 (GLP-1) by enteroendocrine cells, thereby exerting an anti-inflammatory effect.

[0095] Experimental Example 3: Low molecular weight chondroitin sulfate compound

[0096] LMCSO, LMCSD and LMCSH were compounded in a ratio of 5:3:2, and animal experiments were conducted to verify that the composition enhanced the intestinal barrier function.

[0097] Experimental methods:

[0098] (1) Animal model validation: After 5 days of adaptive feeding, 48 mice were randomly divided into 6 groups, each with 8 mice: control group, model group, LMCSO group, LMCSD group, LMCSH group, and LMCSs-C group. The control group had free access to distilled water throughout the experimental period (8 days). The other groups were fed 3% DSS for the first 4 days and then ordinary distilled water for the next 4 days. All mice were gavaged once a day. The control and model groups received 200 μL PBS, while the intervention groups received 200 μL PBS containing the corresponding aqueous extract (water extract content 2 mg / mL). The experimental mice were killed after 8 days.

[0099] (2) Colon tissue collection: After the experiment was terminated, the mice in the experimental and control groups were euthanized. The abdominal cavity was dissected layer by layer along the midline of the abdomen, and the colon tissue (from the ileocecal valve to the end of the rectum) was completely separated and spread flat on sterile filter paper to avoid traction. The colon length was accurately measured using a vernier caliper and recorded.

[0100] (3) Histopathological analysis of mouse colon: After the experimental mice were sacrificed, the hind limb ankle joints were quickly removed and immediately immersed in 4% paraformaldehyde (pH 7.4) for fixation at room temperature for 24 hours. The tissues were then transferred to 10% EDTA decalcification solution (pH 7.2) for dynamic decalcification (the end point of decalcification was determined by the absence of resistance when punctured). After decalcification, the tissues were transparentized with gradient ethanol and xylene, embedded in wax, and prepared using a Leica RM2255 rotary microtome with 4 μm serial sagittal sections. After drying, dewaxing, and rehydration, the sections were stained with hematoxylin-eosin (HE).

[0101] Experimental results:

[0102] Colon length is a biomarker of intestinal inflammation severity in mice. Figure 12 ((a) shows the effect of LMCS and LMCSs-C treatment on the length of the mouse colon, (b) shows the effect of LMCS and LMCSs-C treatment on the pathological score of the mouse colon) shows the quantitative analysis of the length of the mouse colon and the pathological score of the mouse colon in different experimental groups. The morphological results showed that the colon of the DSS model group was significantly shortened compared with the blank group, while the colon length of the polysaccharide intervention group was significantly restored compared with the model group ( p <0.05). Quantitative data analysis showed that the colon length of the model group mice was significantly reduced by 54% compared with the healthy control group ( p<0.05), while the LMCSs-C intervention showed the smallest reduction in colon length, and compared with other LMCSs, the colon length was closest to the healthy control group. Pathological scoring showed that the LMCSs-C group had the best effect on improving colon inflammation compared with other groups, with the score closest to the healthy control group. Colonic tissue was evaluated for pathological morphology using H&E staining ( Figure 13 The right image (40×) of each group in the figure is an enlarged view of the boxed area on the left (10×). The results showed that the colons of mice in the model group exhibited typical inflammatory pathological changes, including crypt structural destruction, mucosal edema and thickening, focal ulceration, and extensive neutrophil and lymphocyte infiltration. However, the colons of mice in the LMCSs-S group did not exhibit severe inflammatory pathological changes.

[0103] In summary, LMCSs-C, a low-molecular-weight chondroitin sulfate composition, synergistically improves intestinal barrier function through multiple mechanisms of action: First, the LMCSO component, composed of oxidative degradation products, significantly strengthens the mucus barrier and inhibits pathogen colonization. Second, the deamination cleavage product, LMCSD, possesses excellent epithelial penetration, penetrating deeply into submucosal cells and exerting dual anti-inflammatory and mucus barrier-strengthening effects. Furthermore, the hydrothermal degradation product, LMCSH, effectively stimulates enteroendocrine cells to secrete glucagon-like peptide-1 (GLP-1), further modulating the inflammatory response and maintaining barrier homeostasis. This composition not only demonstrates significant intestinal barrier repair effects in vitro and in animal models, but also exhibits excellent biocompatibility and safety, demonstrating broad application prospects in maintaining intestinal health and intervening in related diseases.

[0104] Furthermore, the present invention also conducted experiments on other ratios during the experimental process, and the results are as follows Figure 14 ((a) shows the effect of LMCSs-C treatment with different ratios on the colon length of mice, (b) shows the effect of LMCSs-C treatment with different ratios on the colon pathology score of mice) The results showed that the effect of LMCS-C (ratio of LMCSO:LMCSD:LMCSH=5:3:2) was significantly better than other ratios.

[0105] Example 2:

[0106] The low molecular weight chondroitin sulfate composition was prepared by the same steps as in Example 1, except that when preparing LMCSO, a 10 mg / mL chondroitin sulfate solution was placed in a constant temperature water bath maintained at 35°C, the stirrer was turned on (speed 300 rpm), ascorbic acid powder was added to a final concentration of 25 mM, stirred for 5 minutes until dissolved, and a 30% H2O2 solution was added dropwise to the bottom of the liquid at a rate of 1 mL / min using a constant flow pump to a final concentration of 25 mM, after the addition of H2O2 solution was completed, the pH was adjusted to 5.5, and the mixture was maintained at 35°C for 15 minutes, and then heated to 38°C at a rate of 1°C / min and continued to react for 15 minutes; when preparing LMCSH, the reaction process adopted programmed temperature control, first heating to 118±1°C at a rate of 120°C / min; then reacting at a constant temperature at 118±1°C for 60 minutes; then cooling to 100±2°C at a rate of 15°C / min; and then reacting at a constant temperature at 100±2°C for 60 minutes; after the reaction was completed, the reaction vessel was immediately placed in an ice water bath, and the system temperature was reduced to below 25°C within 2 minutes.

[0107] Example 3:

[0108] The low molecular weight chondroitin sulfate composition was prepared by the same steps as in Example 1, except that during the deacetylation reaction in the preparation of LMCSD, the degree of deacetylation was not measured and the deacetylation reaction was directly carried out at 90° C. for 12 h.

[0109] Experimental Example 4:

[0110] The low molecular weight chondroitin sulfate compositions prepared in Examples 1, 2 and 3 were subjected to animal experiments (refer to Experimental Example 3). The results are as follows: Figure 15 ((a) shows the effect of LMCSs-C treatment with different preparation methods on the colon length of mice, (b) shows the effect of LMCSs-C treatment with different preparation methods on the colon pathology score of mice) The results show that the composition in Example 2 (LMCSs-C2) is significantly better than that in Example 1 (LMCSs-C) and is equivalent to the healthy blank group. The composition in Example 1 is significantly better than that in Example 3 (LMCSs-C3). This indicates that when preparing low molecular weight chondroitin sulfate, the addition method of ascorbic acid and H2O2 and the control of temperature and deacetylation degree during the reaction process have a significant effect on the activity and stability of low molecular weight chondroitin sulfate, thereby affecting the effect of the low molecular weight chondroitin sulfate composition in improving intestinal barrier function.

[0111] The number of equipment and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the low molecular weight chondroitin sulfate composition and its preparation and use will be readily apparent to those skilled in the art.

[0112] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing a low molecular weight chondroitin sulfate composition, characterized in that: include: The oxidative degradation product of chondroitin sulfate LMCSO, the hydrothermal degradation product of chondroitin sulfate LMCSH and the deamination degradation product of chondroitin sulfate LMCSD are compounded in a weight ratio of 5:3:2 to obtain a low molecular weight chondroitin sulfate composition LMCSs-C; wherein, The preparation method of LMCSD is: Mix the chondroitin sulfate solution with the hydrazine hydrate solution containing hydrazine sulfate, and carry out deacetylation reaction at 85℃-95℃ under nitrogen protection. After the reaction is carried out for 8 hours, take a sample, determine the content of N-acetylgalactosamine in the sample and calculate the degree of deacetylation DDA%. If DDA% If the deacetylation rate is ≥95%, the reaction is terminated immediately. Otherwise, the reaction is continued and samples are taken every hour for detection until DDA% is ≥95%. Sampling, detection and calculation of the degree of deacetylation are completed within 60 minutes. The mixed solution after the deacetylation reaction is dialyzed with alcohol precipitation to obtain a deacetylated product. The deacetylated product is reacted with a nitrite solution at 4-6°C for 30 minutes, the pH is adjusted to alkaline to terminate the reaction, and then a NaBH4 solution is added for reduction at 45-55°C for 1.5-2.5 hours. The molar ratio of the deacetylated product to the nitrite solution and the NaBH4 solution is 1:1.1-1.3:1.8-2.

2. The molar number of the deacetylated product is calculated based on the molar number of free amino groups in the deacetylated product. The product is purified by ultrafiltration membrane and freeze-dried to obtain; The preparation method of LMCSH is as follows: a 10 mg / mL chondroitin sulfate solution is adjusted to a pH of 4.0±0.2 with acetic acid, reacted at 100℃-120℃ for 2-3 hours, and the resulting product is purified through a 0.5 kDa ultrafiltration membrane and then freeze-dried to obtain the product; wherein, the reaction process adopts programmed temperature control, first heating to 118±1℃ at a rate of 120±5℃ / min; then reacting at a constant temperature of 118±1℃ for 60±5 minutes; then cooling to 100±2℃ at a rate of 15±3℃ / min; then reacting at a constant temperature of 100±2℃ for 60±5 minutes; after the reaction is completed, the reaction vessel is immediately placed in an ice water bath, and the system temperature is reduced to below 25℃ within 2 minutes.

2. The method for preparing the low molecular weight chondroitin sulfate composition according to claim 1, wherein The preparation method of LMCSO is as follows: H2O2 and ascorbic acid are added to a 10 mg / mL chondroitin sulfate solution at a final concentration of 20-30 mM, the pH is adjusted to 5.5±0.2, the reaction is carried out at 30°C-40°C for 20-40 minutes, and the resulting product is purified by a 0.5 kDa ultrafiltration membrane and then freeze-dried to obtain the product.

3. The method for preparing the low molecular weight chondroitin sulfate composition according to claim 2, wherein: In the step of preparing LMCSO, ascorbic acid is first added to the chondroitin sulfate solution and mixed, and then the H2O2 solution is added dropwise under continuous stirring; after the H2O2 solution is added dropwise, the mixture is reacted at 35±1°C for 10-15 minutes, and then the temperature is raised to 38±1°C and the reaction is continued for 10-15 minutes; the final concentrations of the H2O2 and ascorbic acid are both 25±0.5 mM.

4. The method for preparing the low molecular weight chondroitin sulfate composition according to claim 1, wherein: In the step of preparing LMCSD, the N-acetylgalactosamine content in the sample was determined using high-performance anion exchange chromatography-pulsed amperometric detection. The detection conditions were: chromatographic column CarboPac PA20, flow rate 0.5 mL / min, and NaOH gradient 2-100 mM. The specific method of alcohol precipitation dialysis was as follows: the mixed solution after the deacetylation reaction was cooled to room temperature, and 4 volumes of anhydrous ethanol precooled to 4°C were slowly added with stirring, and the mixture was allowed to stand at 4°C for 2 hours. The precipitate was collected, dissolved in deionized water, placed in a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyzed in deionized water at 4°C for 24 hours, during which the water was changed at least 6 times.

5. A low molecular weight chondroitin sulfate composition, characterized in that: The low molecular weight chondroitin sulfate composition is prepared by the preparation method according to any one of claims 1 to 4, wherein the sulfate content in the low molecular weight chondroitin sulfate composition is 15.0-16.5%, and the uronic acid content is 28.0-29.0%.

6. A pharmaceutical composition, characterized in that The method comprises the low molecular weight chondroitin sulfate composition according to claim 5 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein It is used to treat diseases related to intestinal barrier dysfunction, including inflammatory bowel disease, irritable bowel syndrome or intestinal flora imbalance.

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