Low-molecular-weight chondroitin sulfate composition as well as preparation method and application thereof

Abstract

The invention relates to a low-molecular-weight chondroitin sulfate composition as well as a preparation method and application thereof, and aims to solve the problems that a chondroitin sulfate product obtained by a single degradation mode is limited in function and difficult to synergistically regulate intestinal barriers. The preparation method comprises the following steps: firstly, preparing LMCSO through oxidative degradation; secondly, preparing LMCSH through hydrothermal degradation; and finally, the LMCSD is prepared through deamination degradation. Finally, the obtained LMCSO, LMCSH and LMCSD are compounded according to the weight ratio of 5: 3: 2, and the low-molecular-weight chondroitin sulfate composition LMCSs-C is obtained. The composition LMCSs-C prepared by the invention can synergistically improve the intestinal barrier function through multiple action mechanisms, not only shows an obvious intestinal barrier repair effect in vitro and animal models, but also has good biocompatibility and safety, and shows a wide application prospect in intestinal health maintenance and related disease intervention.

Description

Technical Field

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

[0002] The intestinal barrier refers to the comprehensive defense mechanism of the intestinal mucosal system to prevent harmful factors from entering other tissues, organs or blood circulation of the body. Impairment of the intestinal barrier can directly lead to the entry of pro-inflammatory substances across the epithelial barrier, activating the inflammatory response; and inflammation in turn further weakens the barrier function by disrupting tight junctions and other ways, resulting in intestinal injury. 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 secreted factors, which participates in pathogen recognition and immune regulation; and the biological barrier mainly composed of intestinal flora and their metabolites, which plays an important role in maintaining the 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 contains D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) linked by a β-(1→3) bond, and its molecular weight is usually 20 to 70 kDa. Studies have shown that chondroitin sulfate has multiple biological activities such as antioxidant, anti-inflammatory, immune regulation, and flora regulation, indicating that it may have the potential to enhance the intestinal barrier, but this direction has not been fully concerned. At present, low-molecular-weight chondroitin sulfate (LMCS) obtained by degrading chondroitin sulfate shows better bioavailability and biological activity. However, due to different degradation mechanisms, low-molecular-weight chondroitin sulfates prepared by different methods have structural differences, which may lead to differences in their effects on the intestinal barrier (physical, immune, biological barriers). Therefore, studying the effects of low-molecular-weight chondroitin sulfates with different structures on intestinal barrier function and preparing a composition with the best enhancement effect through compounding is of great significance for maintaining intestinal health. Summary of the Invention

[0004] The object 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 functions of the physical barrier, immune barrier and biological barrier, and improve the application effect of low-molecular-weight chondroitin sulfate in enhancing intestinal barrier function.

[0005] To achieve the objectives and other advantages of the present invention, a method for preparing a low molecular weight chondroitin sulfate composition is provided, including: adding H2O2 and ascorbic acid with final concentrations of 20 - 30 mM respectively 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 freeze-drying the obtained product after purification through a 0.5 kDa ultrafiltration membrane to obtain an oxidative degradation product LMCSO of chondroitin sulfate; adjusting the pH of a 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 freeze-drying the obtained product after purification through a 0.5 kDa ultrafiltration membrane to obtain a hydrothermal degradation product LMCSH of chondroitin sulfate; mixing an 80 mg / mL chondroitin sulfate solution with hydrazine hydrate containing 2.5% - 3.5% hydrazine sulfate in a volume ratio of 1:1, carrying out a deacetylation reaction under nitrogen protection at 85°C - 95°C. At the 8th hour of the deacetylation reaction, sampling is carried out and the reaction is immediately quenched to below -20°C in dry ice to terminate the reaction. Then the sample is redissolved with deionized water, centrifuged at 4°C and 10,000 rpm for 10 minutes using a 3 kDa ultrafiltration centrifugal tube. Then the content of N-acetylgalactosamine in the sample is measured using high performance anion exchange chromatography - pulsed amperometric detection method and the degree of deacetylation DDA% is calculated. If DDA% ≥ 95%, the reaction is immediately terminated. Otherwise, the reaction continues and the content of N-acetylgalactosamine is sampled and detected every hour and the degree of deacetylation DDA% is calculated until DDA% ≥ 95%. Sampling, detection and calculation of the degree of deacetylation DDA% are completed within 60 minutes. Then the mixture after the deacetylation reaction is subjected to alcohol precipitation and dialysis to obtain a deacetylated product. The deacetylated product is reacted with a nitrite solution at 4 - 6°C for 30 minutes, the reaction is terminated by adjusting the pH to 8.0, and then a NaBH4 solution is added for reduction at 45°C - 55°C for 1.5 - 2.5 hours. The molar ratio of the deacetylated product, 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. Then the obtained product is freeze-dried after purification through a 0.5 kDa ultrafiltration membrane to obtain a deaminated degradation product LMCSD of chondroitin sulfate; compounding LMCSO, LMCSH and LMCSD according to 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 evenly, and then an H2O2 solution is added dropwise under continuous stirring; after the addition of the H2O2 solution is completed, the mixture is reacted at 35 ± 1°C for 10 - 15 minutes, and then heated to 38 ± 1°C and continued to react for 10 - 15 minutes; the final concentrations of H2O2 and ascorbic acid are both 25 ± 0.5 mM.

[0007] Preferably, in the step of preparing LMCSH, the reaction process is controlled by programmed temperature. First, the temperature is raised to 118 ± 1 °C at a rate of 120 ± 5 °C / min; then, the reaction is carried out at a constant temperature of 118 ± 1 °C for 60 ± 5 minutes; then, the temperature is lowered to 100 ± 2 °C at a rate of 15 ± 3 °C / min; then, the reaction is carried out at a constant temperature of 100 ± 2 °C 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 lowered to below 25 °C within 2 minutes.

[0008] Preferably, in the step of preparing LMCSD, when using high performance anion exchange chromatography-pulsed amperometric detection to determine the content of N-acetylgalactosamine in the sample, 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 and dialysis is as follows: cool the mixture after the deacetylation reaction to room temperature, slowly add 4 times the volume of pre-cooled anhydrous ethanol at 4 °C with stirring, and let it stand for precipitation at 4 °C for 2 hours; collect the precipitate, dissolve the precipitate in deionized water, put it into a dialysis bag with a cut-off molecular weight of 10 kDa, and dialyze it in deionized water at 4 °C for 24 hours, changing the water no less than 6 times during this period.

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

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

[0012] Preferably, the above pharmaceutical composition is used for the treatment of diseases related to intestinal barrier dysfunction, including inflammatory bowel disease, irritable bowel syndrome or intestinal flora dysregulation.

[0013] The present invention also provides an application of the above low molecular weight chondroitin sulfate composition in the preparation of health products for enhancing intestinal barrier function.

[0014] Preferably, the enhancement of intestinal barrier function includes: up-regulating the gene expression of tight junction proteins ZO-1, Occludin and Claudin-1; promoting the generation of intestinal short-chain fatty acids SCFAs; reducing the abundance of pathogenic Escherichia-Shigella.

[0015] The present invention has at least the following beneficial effects: 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, degree of deacetylation) by precisely controlling the condition parameters (concentration, pH, temperature, time, molar ratio) of the three degradation processes of oxidation, hydrothermal, and deamination cracking, and the purification means (0.5 kDa ultrafiltration); by real-time monitoring the degree of deacetylation (DDA%) during the deamination cracking process and precisely controlling the reaction end point, the accuracy and batch-to-batch consistency of the LMCSD structure are ensured; then, the three LMCS products are compounded in an optimized ratio of 5:3:2, enabling the composition to effectively integrate the unique biological activity advantages of the three single-component LMCSs: LMCSO enhances the mucus barrier, LMCSD promotes penetration and anti-inflammation, and LMCSH stimulates GLP-1 secretion, generating a significant synergistic effect, comprehensively and synergistically enhancing the functions of the intestinal physical barrier, immune barrier, and biological barrier, and providing a composition with a clear structure and controllable activity for efficiently maintaining intestinal health.

[0016] Second, the present invention effectively avoids the violent or uncontrollable oxidation reaction caused by excessive local H2O2 concentration by stipulating that ascorbic acid and chondroitin sulfate solution are first mixed and then H2O2 solution is added dropwise under continuous stirring, improving the reaction uniformity and safety; adopting staged precise temperature control (starting the reaction at a lower temperature first and then slightly increasing the temperature to continue the reaction) helps to achieve the oxidative cleavage of chondroitin sulfate molecules more mildly and controllably, reducing the generation of excessive degradation or by-products; controlling 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 homogeneity of the LMCSO product, making it more stable and efficient in enhancing the function of the intestinal mucus physical barrier.

[0017] Third, the present invention realizes the fine control of the hydrothermal depolymerization process by setting specific heating rates, reaction temperatures (including a high-temperature section and a slightly lower constant-temperature section), and cooling rates, and finally requires rapid cooling in an ice-water bath; dynamic temperature management can more effectively regulate the hydrolysis rate and degree of β-(1→4) glycosidic bonds, avoiding the problem of too wide molecular weight distribution or uncontrollable structural damage caused by excessive temperature fluctuations; rapid cooling can terminate the reaction in time and lock the target molecular structure; the prepared LMCSH product has a more concentrated molecular weight and a more expected structure, and has better and more stable activities in stimulating the secretion of GLP-1 by intestinal endocrine cells, exerting anti-inflammatory and regulating metabolic functions.

[0018] Fourth, by defining 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 quantify the content of N-acetylgalactosamine in the reaction system, thereby precisely calculating the degree of deacetylation (DDA%), so as to realize the real-time monitoring of the deacetylation reaction process and ensure that DDA% ≥ 95%, ultimately ensuring the high structural consistency and biological activity of the LMCSD product.

[0019] Fifth, by controlling the sulfate content (15.0 - 16.5%) and uronic acid content (28.0 - 29.0%) within a specific range, the present invention ensures that the composition has appropriate electronegativity and molecular polarity, making the quality of the composition controllable and the function stable.

[0020] Sixth, the low molecular weight chondroitin sulfate composition of the present invention avoids the therapeutic limitations of single components. Through the multi-target action mechanism of LMCSs-C, it realizes the synergistic intervention of intestinal barrier dysfunction: up-regulating the expression of ZO-1 / Occludin / Claudin-1 to repair the physical barrier; promoting the proliferation of SCFA-producing bacteria such as Bifidobacterium to improve the chemical barrier; inhibiting the colonization of Escherichia-Shigella to consolidate the immune barrier, and is particularly suitable for treating diseases related to intestinal barrier dysfunction and enhancing intestinal barrier function. Moreover, due to the uniform molecular weight and no toxic residues, it has good oral bioavailability and safety.

[0021] Other advantages, objectives and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0022] Figure 1 is a three-dimensional CLSM image of FITC-labeled low molecular weight chondroitin sulfate in the mucus layer; Figure 2 is the content of low molecular weight chondroitin sulfate in the chamber at a fixed permeation time point; Figure 3 is the permeation result of FD4 and phenol red in the in vitro small intestine mucus gel layer; Figure 4 is the result of the effect of LMCS treatment on the transepithelial electrical resistance of Caco-2 cell monolayers treated with DSS; Figure 5 is the result of the effect of LMCS treatment on the IL-8 secretion level of Caco-2 cells; Figure 6Shows the results of the effect of LMCS treatment on the relative mRNA expression levels of tight junction proteins ZO-1, Claudin-1, and Occludin in Caco-2 cells; Figure 7 Are the apparent permeability coefficients of CS and LMCS on Caco-2 cell monolayers; Figure 8 Shows the results of the effect of LMCS treatment on the secretion levels of NO, IL-1β, and IL-6 in RAW264.7 cells; Figure 9 Shows the results of the effect of LMCS treatment on the GLP-1 secretion level in STC-1 cells; Figure 10 Is the analysis of intestinal flora species differences based on the LDA score bar chart; Figure 11 Are the short-chain fatty acid concentrations after 24 h of in vitro fermentation in the CS, LMCSO, LMCSD, and LMCSH groups; Figure 12 Shows the results of the effect of LMCS and LMCSs-C treatment on the length and pathological score of the mouse colon; Figure 13 Is the staining result of mouse colon tissue sections; Figure 14 Shows the results of the effect of LMCSs-C with different ratios on the length and pathological score of the mouse colon; Figure 15 Shows the results of the effect of LMCSs-C prepared by different methods on the length and pathological score of the mouse colon. Detailed implementation mode

[0023] The following further elaborates on the present invention in conjunction with examples and drawings, so that those skilled in the art can implement it with reference to the text of the specification.

[0024] It should be understood that terms such as "having", "including", and "comprising" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0025] It should be noted that the experimental methods described in the following implementation examples are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0026] According to an embodiment of the present invention, when preparing the oxidative degradation product LMCSO, the concentration of the chondroitin sulfate solution is 10 mg / mL. The final concentrations of H2O2 and ascorbic acid can be selected as 20 mM, 25 mM or 30 mM. The target pH adjustment is 5.5 ± 0.2, and actually 5.3, 5.5 or 5.7 can be selected. The reaction temperature can be selected as 30°C, 35°C or 40°C, and the time can be selected as 20 minutes, 30 minutes or 40 minutes. The ultrafiltration equipment can select a spiral ultrafiltration membrane package with a molecular weight cut-off of 0.5 kDa, and the freeze dryer can select an ordinary 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, under the condition of maintaining 35°C with a constant temperature magnetic stirrer, 30% H2O2 solution is slowly added dropwise, and the dropping time is controlled within 5 minutes. After the dropping is completed, the pH is adjusted to 5.5 ± 0.2 with 0.1 M HCl, and the reaction continues for 30 minutes. The reaction solution is purified through a 0.5 kDa ultrafiltration membrane package under a pressure of 0.2 MPa, and the permeate is collected and freeze-dried to obtain the LMCSO product.

[0027] When preparing the hydrothermal degradation product LMCSH, the concentration of the chondroitin sulfate solution is 10 mg / mL. The target pH adjustment is 4.0 ± 0.2, and actually it can be controlled within 3.8 - 4.2. The reaction temperature can be selected as 100°C, 110°C or 120°C, and the time can be selected as 2 hours, 2.5 hours or 3 hours. The reaction vessel can select a high-pressure reaction kettle with a polytetrafluoroethylene lining, and the temperature control system can select a programmable temperature oven. Specifically, prepare a 10 mg / mL aqueous solution of chondroitin sulfate, and adjust the pH to 4.0 ± 0.2 with glacial acetic acid. Transfer it to the high-pressure reaction kettle and seal it, and place it in the programmable temperature oven: heat it to 118°C at a rate of 120°C / min and keep it at a constant temperature for 60 minutes; then cool it to 100°C at a rate of 15°C / min and keep it at a constant temperature for 60 minutes. Immediately immerse the reaction kettle in an ice-water bath after the reaction ends, and cool it to below 25°C within 2 minutes. Take the reaction solution, purify it by ultrafiltration with 0.5 kDa, and then freeze-dry it to obtain the LMCSH powder.

[0028] When preparing the deaminated degradation product LMCSD, the concentration of chondroitin sulfate is 80 mg / mL. The content of hydrazine sulfate in hydrazine hydrate can be selected as 2.5%, 3.0% or 3.5%. The deacetylation temperature can be selected as 85 °C, 90 °C or 95 °C, and the reaction time is terminated with the threshold value of DDA%≥95%. Alcohol precipitation uses 4 volumes of absolute ethanol, and dialysis can select a dialysis bag with a molecular weight cut-off of 10 kDa. Specifically, an 80 mg / mL chondroitin sulfate solution can be mixed with hydrazine hydrate containing 3.0% hydrazine sulfate in a volume ratio of 1:1, filled with nitrogen and sealed in a pressure-resistant glass reactor. React in an oil bath at 90 °C, take 2 mL of samples every 60 minutes and quickly cool them with dry ice at -80 °C. After reconstitution, filter through a 0.22 μm filter membrane, and use an ion chromatography system to detect the residual amount of N-acetylgalactosamine. When DDA%≥95%, cool the reaction solution and add 4 volumes of absolute ethanol pre-cooled at -20 °C, and let it stand and precipitate at 4 °C for 2 hours. Centrifuge to collect the precipitate, dissolve it in water, put it into a 10 kDa dialysis bag and dialyze at 4 °C for 24 hours. Mix the dialysis solution and 0.5 M NaNO2 solution in a molar ratio of free amino group:NaNO2 = 1:1.2, react at 5 °C for 30 minutes, and adjust the pH to 8.0 to terminate. Then add 0.1 M NaBH4 solution and reduce it at 50 °C for 2 hours, and obtain LMCSD through ultrafiltration and freeze-drying with a 0.5 kDa filter. The weight ratio of LMCSO, LMCSH, and LMCSD is 5:3:2. A V-type dry powder mixer can be selected as the mixing equipment. Specifically, weigh the freeze-dried LMCSO (50 g), LMCSH (30 g), and LMCSD (20 g) respectively and place them in the hopper of the mixer. Set the rotation speed at 20 rpm and mix for 30 minutes, sieve through a 60-mesh sieve and then package to obtain the LMCSs-C composition. According to another embodiment of the present invention, ascorbic acid is preferentially added to the chondroitin sulfate solution, and the dropping rate of the H2O2 solution can be selected as 0.5 mL / min, 1 mL / min or 1.5 mL / min. A four-blade paddle stirrer can be selected as the stirring device, which is assembled at the central axis position of the reaction vessel. An online pH meter probe can be selected for pH monitoring, which is installed at a depth of 1 / 3 from the liquid surface on the side wall of the reactor. Specifically, add a 10 mg / mL chondroitin sulfate solution to a three-necked flask, and maintain a constant temperature water bath at 35 °C. Turn on the stirrer (rotation speed 300 rpm), add ascorbic acid powder to a final concentration of 25 mM, and stir for 5 minutes until dissolved. Dropwise add 30% H2O2 solution to the lower part of the liquid surface at a rate of 1 mL / min through a constant flow pump. The temperature in the first stage can be selected as 34°C, 35°C or 36°C, and the time can be selected as 10 minutes, 12 minutes or 15 minutes; the temperature in the second stage can be selected as 37°C, 38°C or 39°C, and the time can be selected as 10 minutes, 12 minutes or 15 minutes. The temperature control device can be a programmable constant temperature water bath, and the temperature sensor is placed at the geometric center of the reaction solution. Specifically, after the addition of H2O2 is completed, the reaction is maintained at 35°C for 12 minutes. Then, the temperature is raised to 38°C at a rate of 1°C / min and the reaction continues for 12 minutes. During the reaction, the pH value fluctuation is controlled within 5.4 - 5.6, and immediately after the end, it is cooled in an ice bath to below 10°C. FTIR detection shows that the product with two-stage temperature control has a higher peak intensity at 1070 cm⁻¹. The final concentrations of H2O2 and ascorbic acid can be selected as 24.5 mM, 25.0 mM or 25.5 mM. The metering device can be a precision pipette or an electronic balance (accuracy 0.1 mg). Specifically, weigh 0.176 g of ascorbic acid powder and accurately weigh it to ±0.001 g with a microbalance. Measure 0.113 mL of 30% H2O2 solution, and control the error within ±1% using a calibrated micro syringe. After addition, titrate by iodometry to verify that the actual concentration is in the range of 25 ± 0.5 mM. According to another embodiment of the present invention, the heating rate can be selected as 115°C / min, 120°C / min or 125°C / min. The target temperature can be selected as 116°C, 118°C or 120°C. The heating device can be a programmable oven, and the reaction vessel can be a stainless steel high-pressure reactor, which is assembled at 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, fill the chondroitin sulfate solution with pH 4.0 into the high-pressure reactor and seal it. Set the oven to heat from 25°C to 118°C at a rate of 120°C / min, and monitor the temperature of the inner wall of the reactor in real time. When it reaches 118°C, automatically switch to the constant temperature mode. The temperature record shows that the actual heating time is 58 ± 2 seconds. The constant temperature can be selected as 116°C, 118°C or 120°C, and the time can be selected as 55 minutes, 60 minutes or 65 minutes. The pressure control valve can be a spring safety valve, which is assembled at the exhaust port of the kettle cover. The thermocouple probe can be inserted into the reaction solution to a depth of 1 / 2. Specifically, maintain a constant temperature of 118°C for 60 minutes, and record the temperature of the reaction solution every 10 minutes. Control the internal pressure to be stable at 0.25 ± 0.02 MPa through the pressure valve. The cooling rate can be selected as 12°C / min, 15°C / min or 18°C / min. The target temperature can be selected as 98°C, 100°C or 102°C. The cooling system can be an air-cooling module, which is assembled in the rear air duct of the oven. Specifically, start air cooling at the end of the high-temperature section and cool to 100°C at a rate of 15°C / min. Adjust the air volume during the cooling process to maintain a linear cooling. The absorbance of melanoidins detected by ultraviolet spectroscopy ≤ 0.05. The low temperature constant temperature can be selected from 98°C, 100°C or 102°C, and the time can be selected from 55 minutes, 60 minutes or 65 minutes. The quenching device can be a stainless steel ice water bath, which is assembled on the operating table. 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. Start stirring at 500 rpm, and the system temperature drops below 25°C within 2 minutes. Product detection shows that the particle size polydispersity index PDI <0.2. According to another embodiment of the present invention, the chromatographic column type can be CarboPac PA10, PA20 or PA30. The column temperature control range can be selected from 30-40°C, and can actually be set to 35°C. The chromatographic column can be installed in the constant temperature chamber of the ion chromatograph, and the sample injector can be an automatic sample injector, which is assembled 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 taken for injection. A CarboPac PA20 chromatographic column (3×150 mm) is selected, and the column temperature is maintained at 35°C. The detector can be a pulsed amperometric detector with a gold working electrode. The flow rate can be selected as 0.4 mL / min, 0.5 mL / min or 0.6 mL / min. The infusion pump can use a double-plunger series pump, and a pulse damper can be installed at the pump outlet, and the installation position is 15-20 cm away from the chromatographic column inlet. Specifically, the flow rate is set to 0.5 mL / min through the chromatographic workstation. The eluent is a NaOH solution prepared with ultrapure water, which is delivered to the system through an online degasser. The actual flow rate error is calibrated with a flow meter before operation. Three-needle continuous injection shows that the retention time deviation is ≤0.1 minutes. 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. The gradient mixer can use a dual-channel proportional valve, which is installed between the infusion pump and the injection valve. Specifically, set the gradient program: maintain 2 mM NaOH for 0-5 minutes; linearly increase to 100 mM for 5-20 minutes; maintain 100 mM for 20-30 minutes. The gradient linear correlation coefficient sets the threshold R 2 ≥0.995. Use a mixed sample of galactosamine and N-acetylgalactosamine to verify that the resolution is ≥1.5. According to another embodiment of the present invention, the ethanol addition multiple can be selected as 3 times, 4 times or 5 times the volume. The pre-cooling temperature of ethanol can be selected as 0°C, 2°C or 4°C. The standing time can be selected as 1 hour, 2 hours or 3 hours. The precipitation container can be a conical centrifuge bottle and placed on the internal shelf of a 4°C freezer. The stirring device can be a glass rod for manual stirring or a magnetic stirrer. Specifically, cool the reaction solution after the deacetylation to below 25°C. Measure 4 times the volume of absolute ethanol and pre-cool it to 4°C. Slowly inject the ethanol under stirring at 300 rpm, and the injection duration is not less than 5 minutes. After stopping the stirring, move it to a 4°C freezer and let it stand for 2 hours. Take the supernatant for hydrazine residue detection, and the detected residue amount meets the safety threshold. The dialysis bag cut-off molecular weight can be selected as 8 kDa, 10 kDa or 12 kDa. The dialysis time can be selected as 20 hours, 24 hours or 28 hours. The set threshold for the number of water changes is not less than 6 times. The dialysis system can be a magnetic stirring water bath tank, assembled inside a 4°C freezer; the dialysis bag fixing frame is installed in the center of the tank body. Specifically, collect the alcohol-precipitated precipitate, dissolve it with deionized water. Inject it into a dialysis bag with a cut-off molecular weight of 10 kDa and clamp both ends. Place it in deionized water at 4°C and start magnetic stirring at 200 rpm. Replace the dialysis solution every 4 hours, with a cumulative of 6 water changes and a total duration of 24 hours. The conductivity detection of the dialysis solution shows that the ion residue meets the standard.

[0029] The alcohol precipitation operation should be carried out prior to dialysis. The volume of deionized water for dissolving the precipitate is controlled to be 1 - 2 times that of the precipitate. The liquid loading volume of the dialysis bag does not exceed 80% of its volume. After the standing of alcohol precipitation is completed, centrifuge at 5000 rpm for 10 minutes at 4°C to collect the precipitate. 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 mouth, exclude the air bubbles and then clamp it. The detection of the final product shows that the recovery rate of the target component meets the expectation. The sequence of alcohol precipitation first and then dialysis ensures the recovery of macromolecular active components. The three work together to reduce the loss of free amino oxidation, control the residual hydrazine content below the safety limit, and meet the requirements of oral preparations.

[0030] Example 1: Prepare a low-molecular-weight chondroitin sulfate composition, which comprises the following steps: (1) Preparation of low-molecular-weight chondroitin sulfate (LMCS): Oxidative degradation method (LMCSO), hydrothermal degradation method (LMCSH) and deamination cleavage method (LMCSD).

[0031] LMCSO: Sequentially add H2O2 and ascorbic acid with a final concentration of 25 mM each to a 10 mg / mL chondroitin sulfate solution, adjust the pH to 5.5, react at 35°C for 30 minutes, and purify the obtained product through a 0.5 kDa ultrafiltration membrane and then lyophilize it to obtain the oxidative degradation product LMCSO of chondroitin sulfate.

[0032] LMCSH: The chondroitin sulfate solution at 10 mg / mL was adjusted to pH 4.0 with glacial acetic acid, reacted at 110 °C for 2.5 h, and the resulting product was purified through a 0.5 kDa ultrafiltration membrane and then freeze-dried to obtain the hydrothermally degraded product LMCSH of chondroitin sulfate.

[0033] LMCSD: The chondroitin sulfate solution at 80 mg / mL was mixed with hydrazine hydrate containing 3.0% hydrazine sulfate in a volume ratio of 1:1. Under nitrogen protection, deacetylation reaction was carried out at 90 °C. At the 8th hour of the deacetylation reaction, a sample was taken and immediately quenched in dry ice to below -20 °C to terminate the reaction. Then the sample was redissolved with deionized water, centrifuged at 4 °C and 10000 rpm for 10 minutes using a 3 kDa ultrafiltration centrifugal tube. Then the content of N-acetylgalactosamine in the sample was determined by high performance anion exchange chromatography-pulsed amperometric detection method and the degree of deacetylation DDA% was calculated. If DDA% ≥ 95%, the reaction was immediately terminated. Otherwise, the reaction was continued and the content of N-acetylgalactosamine was sampled and detected every hour and the degree of deacetylation DDA% was calculated until DDA% ≥ 95%. Sampling, detection and calculation of the degree of deacetylation DDA% were completed within 60 min. Then the mixture after the deacetylation reaction was cooled to room temperature, and 4 times the volume of pre-cooled anhydrous ethanol at 4 °C was slowly added with stirring. It was left to stand and precipitate at 4 °C for 2 h. After standing, the precipitate was collected by centrifugation at 5000 rpm for 10 minutes at 4 °C. The precipitate was dissolved in 1.5 times the volume of deionized water, filled into a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyzed in deionized water at 4 °C for 24 h, during which the dialysis solution was changed every 4 h to obtain the deacetylated product. Then the deacetylated product was reacted with sodium nitrite solution at 4 °C for 30 min, and the reaction was terminated by adjusting the pH to 8.0. Then NaBH4 solution was added and reduced at 50 °C for 2 h. The molar ratio of the deacetylated product, sodium nitrite solution and 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. Then the resulting product was purified through a 0.5 kDa ultrafiltration membrane and then freeze-dried to obtain the deaminated degraded product LMCSD of chondroitin sulfate.

[0034] (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.

[0035] Experimental Example 1: Structural characterization of low molecular weight chondroitin sulfate Experimental method: (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 solutions and sample solutions (0.1 mg / mL) with different concentrations were respectively added to test tubes, shaken well, reacted in boiling water for 10 minutes, cooled in an ice - water bath, then 0.1 mL of carbazole reagent was added, shaken well, reacted in boiling water for 15 minutes, and cooled to room temperature in an ice - water bath. The absorbance values of the standard solutions and sample solutions were measured at 530 nm, and a standard curve was plotted. There were 3 parallels in each group, and water was used instead of the sample as a blank control to obtain the uronic acid content of the sample.

[0036] (2)Determination of sulfate content: The sulfate content in chondroitin sulfate was determined according to the gelatin turbidimetry method. The sample was dissolved in 1 M hydrochloric acid solution to prepare a 1 mg / mL sample solution, acid - hydrolyzed in a sealed state at 105 °C for 2 hours, cooled to room temperature, and fixed - volume to 10 mL with 1 M hydrochloric acid. 0.2 mL of the standard solution and the sample solution were respectively added to 3.8 mL of 1% trichloroacetic acid and 1.0 mL of barium chloride - gelatin solution, shaken well, and left standing for 15 minutes. The absorbance value of the reaction solution was measured at 360 nm, and a standard curve was plotted. There were 3 parallels in each group, and 1 M hydrochloric acid was used instead of the sample as a blank control to obtain the sulfate content of the sample.

[0037] (3)Molecular weight determination: Mobile phase: Prepare a sodium sulfate solution with a concentration of 1 M, filter out impurities through a 0.22 μm ultrafiltration membrane, and ultrasonically treat 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 the mobile phase, then filter through a 0.22 μm ultrafiltration membrane. The molecular weight of chondroitin sulfate was determined by high - performance size - exclusion chromatography coupled with multi - angle laser light scattering and refractive index detectors (HPSEC - MALLS - RI). Set the refractive index increment dn / dc to 0.135 to determine the molecular weight of chondroitin sulfate. Chromatographic conditions: TSK gel G5000PWxl (7.8×300 mm) was used 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; the column temperature was 30 °C.

[0038] (4)Fourier transform infrared spectroscopy (FT - IR) and nuclear magnetic resonance spectroscopy (NMR) analysis: The infrared absorption spectrum of the sample was determined by a Fourier transform infrared spectrometer. The sample and KBr were ground and mixed in a mortar, and then pressed into a test thin slice containing 0.5% of the sample. In the wavenumber range of 4000 - 400 cm -1 Data collection was carried out at a resolution of 4 cm -1 and 64 scan times.

[0039] (5)Nuclear magnetic resonance spectrum: Determined by an AVANCE III HD 500 MHz nuclear magnetic resonance spectrometer. The sample solution was prepared using D2O as the solvent. One-dimensional / two-dimensional nuclear magnetic resonance spectra were collected, including 1 1H spectrum, 13 13C spectrum, 1 1H- 1 1H correlation spectroscopy (COSY), 1 1H- 13 13C heteronuclear single quantum coherence spectroscopy (HSQC) and heteronuclear multiple bond coherence spectroscopy (HMBC).

[0040] (6)Mass spectrometry analysis: Chondroitin sulfate was enzymatically digested with chondroitinase ABC at 37 °C for 10 hours. The dialyzed sample was analyzed using a triple quadrupole tandem mass spectrometer (Agilent 6460 QqQMS) 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 was in the negative ion mode, the drying gas temperature was 350 °C, the drying gas flow rate was 8 L / min, the nebulizer pressure was 15 psi, the capillary voltage was 4000 V, the fragmentation voltage was 200 V, and the m / z range was 100 - 2000.

[0041] Experimental results: The determination results of uronic acid content, sulfate content, and molecular weight are shown in the following table.

[0042] Note: Mean ± SD of three determinations, different letters in the same column indicate significant differences ( p <0.05).

[0043] As can be seen from the above table, the molecular weights and uronic acid contents of the three low molecular weight chondroitin sulfates are significantly lower than those of chondroitin sulfate CS, and the sulfate contents of LMCSD and LMCSH are significantly lower than those of chondroitin sulfate CS.

[0044] The Fourier transform infrared spectroscopy results show the potential changes in chemical bonds and functional groups of low molecular weight chondroitin sulfate during different degradation reactions. The NMR and UHPLC-MS results indicate that oxidative degradation preferentially breaks the β-(1→3) bond of CS, resulting in the oxidation of GlcA to form an adipic acid structure; deamination degradation specifically cleaves the β-(1→4) bond of CS, generating oligosaccharides containing anTal-ol termini, and some structures lose acetyl and sulfate groups; hydrothermal degradation hydrolyzes the β-(1→4) bond of CS, accompanied by the destruction of sulfate groups, generating oligosaccharides with a complete CS structure.

[0045] Experimental Example 2: Activity analysis of low molecular weight chondroitin sulfate Experimental methods: (1)Preparation of porcine intestinal mucus: Fresh porcine intestines were obtained from a local slaughterhouse and rinsed successively with deionized water and 10 mM phosphate buffer (pH 6.7). Mucus was collected by gently scraping the mucosal surface. To prevent degradation, a protease inhibitor (MedChemExpress, Shanghai, China) was added at a ratio of 100:1 (v / v). Subsequently, the samples were stored at -80 °C for later use.

[0046] (2)Permeation experiment of the mucus layer: Low molecular weight chondroitin sulfate was labeled with FITC. For the in vitro mucus layer permeation experiment, a Transwell 24-well plate (pore size 0.4 μm) was used. 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). 1.2 mL of PBS buffer was added to the lower chamber. The Transwell was incubated overnight in a 37 °C incubator, and the permeation ability was evaluated by detecting the content of low molecular weight chondroitin sulfate in the lower chamber. Subsequently, it was fixed with a 75:25 (v / v) ethanol / acetic acid mixture for 60 minutes, stained with Alexa Fluor 488-labeled wheat germ agglutinin (WGA), and then a large-range Z-axis scanning imaging of the mucus gel was performed using a Leica confocal laser scanning microscope in Germany.

[0047] (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 a 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) respectively. 1.2 mL of PBS buffer was added to the lower chamber. After the Transwell was incubated 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.

[0048] (4)Construction of the Caco-2 / RAW264.7 co-culture model: Caco-2 cells were seeded at a density of 4×10 4 cells / well in Transwell inserts (0.4 μm PET membrane, Corning, USA) and continuously cultured for 21 days. A Millicell-ERS2 volt-ohm meter (Millipore, USA) was used to monitor the transepithelial electrical resistance (TEER), and Caco-2 cells with a TEER value >500 Ω·cm 2cells were used for the experiment. RAW264.7 cells (1×10 5 cells / well) were seeded on the bottom of a 24-well plate. After incubation overnight with 0.5 mg / mL of the sample in the co-culture system, 10 μg / L TNF-α was then added and the cells were stimulated for 24 hours, and 3% DSS was added and the cells were treated for 1 hour. The integrity of the Caco-2 monolayer was evaluated by the change in TEER value after DSS treatment. The basolateral medium was collected, and the levels of nitric oxide (NO), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected using a kit (China Youpin Biotechnology Co., Ltd.). To evaluate the transport efficiency of the polysaccharide in the Caco-2 cell monolayer model, 1 mg / mL of the sample was added to the apical side (AP), and HBSS buffer was added to the basolateral side (BL). After incubation for 2 hours, the basolateral medium was collected, and the sample concentration was detected using a bovine-derived CS ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., China), and the apparent permeability coefficient (Papp) of CS and LMCS was calculated.

[0049] (5) q-PCR analysis: Caco-2 cells were cultured as described above. After collecting the basolateral medium, the level of IL-8 was detected using a kit (China Youpin Biotechnology Co., Ltd.). Total RNA of Caco-2 cells was extracted using TRIzol reagent (Life Technologies, USA), and after reverse transcription to synthesize cDNA, real-time fluorescence quantitative RT-PCR was performed using SYBR Green Premix Pro Taq HS reagent (Aikerui Biotech Co., Ltd., China).

[0050] (6) In vitro GLP-1 secretion detection: STC-1 cells were seeded in a 96-well plate at a density of 1×10 5 cells per well and incubated. After the cells were cultured overnight in a medium containing 0.5 mg / mL of the sample, the medium was collected, and the secretion level of glucagon-like peptide-1 (GLP-1) was detected using an ELISA kit (China Youpin Biotechnology Co., Ltd.).

[0051] (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 use of antibiotics in the past 3 months). After equal mixing of the fecal samples of each volunteer, 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, then centrifuged at 500 rpm for 10 minutes, and immediately stored in an anaerobic jar after centrifugation. The basal medium was prepared as shown in the following table. Finally, chondroitin sulfate and its degradation products were selected as 10% (w / v) carbon sources, and the carbon source-free medium was used as a blank control (BLK). 1 mL of the fecal suspension was added to each medium group (CS, LMCSO, LMCSH, LMCSD, BLK), and the mixture was cultured in an anaerobic environment at 37 °C, with each experiment repeated three times. Fermentation samples were collected at 0, 6, 12, and 24 hours and immediately frozen at -80 °C. The supernatant of the fermentation samples was divided into two parts: one part was used to measure the total carbohydrate content and pH; the other part was subjected to ethanol precipitation, dialysis, and freeze-drying to obtain samples at different fermentation time points (such as CS-0 h, CS-6 h, CS-12 h, CS-24 h), and then the physicochemical properties of the fermented polysaccharides were analyzed.

[0052] (8)Analysis of intestinal flora: After 24 h of in vitro fermentation, DNA was extracted from the samples of fructooligosaccharide (FOS), CS, LMCSO, LMCSH, LMCSD, and the blank group (BLK) using the OMEGA Soil DNA Kit (Omega Bio-Tek, Norcross, USA). All the extracted DNA samples were analyzed by Shanghai New Life Biotechnology Co., Ltd. Species annotation was performed using a pre-trained Naive Bayes classifier, aligning with the SILVA138 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). The relative abundances of microbial compositions at the phylum to species levels were visualized using Krona charts (https: / / github.com / marbl / Krona / wiki).

[0053] (9) Determination of short-chain fatty acids (SCFAs): The pH value of 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, and after acidification and homogenization with 50 μL of 20% phosphoric acid, 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 group of experiments was repeated three times.

[0054] (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, 100 μL of acetonitrile aqueous solution was added and vortexed, and the supernatant was collected for mass spectrometry analysis after centrifugation. Metabolites were detected using a Vanquish UHPLC system coupled to a high-resolution Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Chromatographic separation was performed using an ACQUIYUPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm, Waters, Ireland).

[0055] Experimental results: The penetration behavior of FITC-labeled low molecular weight chondroitin sulfate in the mucus layer was observed by three-dimensional CLSM scanning, and its penetration ability was analyzed by Transwell method. Compared with CS, low molecular weight chondroitin sulfate was more evenly distributed in the mucus layer and significantly diffused to the lower layer, among which LMCSH had a more obvious diffusion effect ( Figure 1 In the figure, the red part is Alexa Fluor 488-WGA labeled mucin, and the green part 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 a fixed penetration time point. 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 molecular 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 penetration results of FD4 in the in vitro small intestinal mucus gel layer, and (b) shows the penetration results of phenol red in the in vitro small intestinal mucus gel layer) The polysaccharide significantly reduced the permeability of the mucus layer to small molecules (phenol red) and macromolecules (FD4) ( p <0.05), demonstrating that low molecular weight chondroitin sulfate can enhance the ability of the mucus layer barrier. Among them, LMCSO has the strongest effect on enhancing the mucus barrier.

[0056] Changes in transepithelial electrical resistance (TEER) can quantify the improvement effect of low molecular weight chondroitin sulfate on DSS-induced epithelial cell barrier damage. Treatment with low molecular weight chondroitin sulfate can effectively alleviate the intestinal epithelial cell barrier disruption caused by DSS ( p <0.05) ( Figure 4 ). Compared with the model group, low molecular weight chondroitin sulfate reduced the IL-8 level by about 14 - 20% ( Figure 5 ), and low molecular weight chondroitin sulfate can effectively and significantly up-regulate the mRNA expression levels of tight junction proteins (ZO-1, Claudin-1, and Occludin) ( Figure 6 , (a) shows the results of the effect of LMCS treatment on the relative mRNA expression level of tight junction protein ZO-1 in Caco-2 cells, (b) shows the results of the effect of LMCS treatment on the relative mRNA expression level of tight junction protein Claudin-1 in Caco-2 cells, (c) shows the results of the effect of LMCS treatment on the relative mRNA expression level of tight junction protein Occludin in Caco-2 cells), which is consistent with the TEER results. This indicates that low molecular weight chondroitin sulfate can effectively improve the tight junction protein disruption 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 cross the Caco-2 monolayer to reach RAW264.7 cells, and LMCSD has the strongest penetration ability ( Figure 7 ). Adding low molecular weight chondroitin sulfate can effectively inhibit the production of IL-1β and IL-6 induced by DSS and TNF-α, and the level in the LMCSO group is the lowest ( Figure 8 , (a) shows the results of the effect of LMCS treatment on the NO secretion level of RAW264.7 cells, (b) shows the results of the effect of LMCS treatment on the IL-1β secretion level of RAW264.7 cells, (c) shows the results of the effect of LMCS treatment on the IL-6 secretion level of RAW264.7 cells). Compared with the control group, polysaccharide treatment promoted the secretion of GLP-1 by STC-1 cells ( p <0.05), and the secretion level after LMCSH treatment was significantly higher than that of other treatment groups ( p <0.05) ( Figure 9 ).

[0057] Low molecular weight chondroitin sulfate promotes the production of short-chain fatty acids (SCFAs), metabolites such as GlcNAc-6P, lactic acid, and progesterone to protect the intestinal barrier by reducing the abundance of Escherichia-Shigella Figure 10 ), among which LMCSO has the best effect. Figure 11 ). Among them, LMCSO has the best effect.

[0058] The above experimental results show that LMCSO has the strongest activity in enhancing the mucus barrier, promoting the generation of beneficial intestinal metabolites and inhibiting the proliferation of harmful bacteria; LMCSD can effectively penetrate epithelial cells and reach the body due to its excellent penetration ability, and shows excellent efficacy comparable to LMCSO in anti-inflammatory and strengthening the mucus barrier; while LMCSH can significantly promote the secretion of glucagon-like peptide-1 (GLP-1) by enteroendocrine cells, thereby playing an anti-inflammatory role.

[0059] Experimental Example 3: Compound of low molecular weight chondroitin sulfate LMCSO, LMCSD and LMCSH were compounded in a ratio of 5:3:2, and the intestinal barrier function enhancement of the composition was verified by animal experiments.

[0060] Experimental method: (1) Animal model verification: After 5 days of adaptive feeding, 48 mice were randomly divided into 6 groups, with 8 mice in each group, namely: control group, model group, LMCSO group, LMCSD group, LMCSH group, and LMCSs-C group. The control group freely drank distilled water throughout the experimental period (8 days), and the other groups were fed 3% DSS for the first 4 days and then changed to ordinary distilled water for the next 4 days. All mice were gavaged once a day. The control group and the model group were given 200 μL of PBS, and the interventions were 200 μL of PBS containing the corresponding water extract (the content of the water extract was 2 mg / mL). The experimental mice were sacrificed after 8 days.

[0061] (2) Collection of colon tissue: After the experiment was terminated, the experimental and control group mice 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 laid flat on sterile filter paper to avoid stretching. The colon length was accurately measured using a vernier caliper and recorded.

[0062] (3) Histopathological analysis of mouse colon tissues: After the experimental mice were sacrificed, the hind limb ankle joints were quickly excised and immediately immersed in 4% paraformaldehyde (pH 7.4) for fixation at room temperature for 24 hours. Subsequently, they were transferred to 10% EDTA decalcifying solution (pH 7.2) for dynamic decalcification (the end point of decalcification was determined by no resistance to acupuncture). After decalcification was completed, the tissues were treated with gradient ethanol and xylene for clearing, and then embedded in paraffin. 4-μm continuous sagittal sections were prepared using a Leica RM2255 rotary microtome. After baking, dewaxing, and rehydration, hematoxylin-eosin (HE) staining was performed.

[0063] Experimental results: Colon length is a biological indicator of the severity of intestinal inflammation in mice. Figure 12 ((a) shows the results of the effects of LMCS and LMCSs-C treatments on mouse colon length, and (b) shows the results of the effects of LMCS and LMCSs-C treatments on mouse colon pathological scores) demonstrate the quantitative analysis of colon length and pathological scores of mice in different experimental groups. The morphological results showed that compared with the blank group, the colon of mice in the DSS model group was significantly shortened, while the colon length in the polysaccharide intervention group was significantly restored compared with the model group ( p <0.05). Quantitative data analysis showed that the colon length of mice in the model group was significantly reduced by 54% compared with the healthy control group ( p <0.05). After intervention with LMCSs-C, the degree of colon length shortening was the smallest, and compared with other LMCSs, the colon length was closest to that of the healthy blank group. After pathological scoring, the LMCSs-C group had the best effect in improving colon inflammation compared with other groups, and its score was closest to the level of the healthy blank group. Pathomorphological evaluation of colon tissues was performed by H&E staining ( Figure 13 , the right image (40×) of each group in the figure is an enlarged view of the area outlined by the left (10×) frame). The results showed that the colon of mice in the model group presented typical inflammatory pathological changes, including destruction of crypt structure, thickening and edema of the mucosal layer, focal ulcer formation, and infiltration of a large number of neutrophils and lymphocytes. However, the colon of mice in the LMCSs-S group did not show severe inflammatory pathological changes.

[0064] In summary, LMCSs-C, as a low molecular weight chondroitin sulfate composition, can synergistically improve intestinal barrier function through multiple mechanisms of action: on the one hand, the LMCSO component composed of oxidative degradation products can significantly enhance the mucus barrier and inhibit pathogen colonization; on the other hand, the deamination cleavage product LMCSD, with its excellent epithelial penetration ability, can not only act deeply on the submucosal cells, but also play a dual role of anti-inflammatory and mucus barrier strengthening; in addition, the hydrothermal degradation product LMCSH can effectively stimulate enteroendocrine cells to secrete glucagon-like peptide-1 (GLP-1), further regulate inflammatory response, and maintain barrier homeostasis. This composition not only shows obvious intestinal barrier repair effects in vitro and in animal models, but also has good biocompatibility and safety, showing broad application prospects in intestinal health maintenance and related disease intervention.

[0065] Furthermore, the present invention also conducted experiments on other ratios during the experiment, and the results are as follows Figure 14 ((a) shows the effect of different ratios of LMCSs-C treatment on the colon length of mice, (b) shows the effect of different ratios of LMCSs-C treatment 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.

[0066] Embodiment 2: 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 through 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, the mixed solution 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 program temperature control, first heated to 118±1°C at a rate of 120°C / min; then reacted at a constant temperature of 118±1°C for 60 minutes; then cooled to 100±2°C at a rate of 15°C / min; and then reacted at a constant temperature of 100±2°C for 60 minutes; after the reaction, the reaction vessel was immediately placed in an ice water bath, and the system temperature was reduced to below 25°C within 2 minutes.

[0067] Embodiment 3: To prepare a low molecular weight chondroitin sulfate composition, the general steps are the same as in Example 1, except that when preparing LMCSD, during the deacetylation reaction, the degree of deacetylation is not measured, and the deacetylation reaction is directly carried out at 90 °C for 12 h.

[0068] Experimental Example 4: The low molecular weight chondroitin sulfate compositions prepared in Example 1, Example 2 and Example 3 were subjected to animal experiments (refer to Experimental Example 3), and the results are as Figure 15 ((a) shows the results of the effect of LMCSs-C treated by different preparation methods on the colon length of mice, and (b) shows the results of the effect of LMCSs-C treated by different preparation methods on the colon pathology score of mice). The results show that the composition in Example 2 (LMCSs-C2) has a significantly better effect than that in Example 1 (LMCSs-C), and is comparable to the healthy blank group. The composition in Example 1 has a significantly better effect than that in Example 3 (LMCSs-C3). This shows that when preparing low molecular weight chondroitin sulfate, the addition method of ascorbic acid and H2O2, as well as the control of temperature and degree of deacetylation during the reaction, all have significant effects on the activity and stability of low molecular weight chondroitin sulfate, and thus affect the effect of low molecular weight chondroitin sulfate composition in improving intestinal barrier function.

[0069] The equipment quantity and processing scale described here are used to simplify the description of the present invention. The applications, modifications and variations of a low molecular weight chondroitin sulfate composition, its preparation method and application of the present invention are obvious to those skilled in the art.

[0070] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details and the illustrated and described examples here.

Claims

1. A preparation method of a low-molecular-weight chondroitin sulfate composition, characterized in that Comprising: Mix the oxidative degradation product LMCSO of chondroitin sulfate, the hydrothermal degradation product LMCSH of chondroitin sulfate, and the deamination degradation product LMCSD of chondroitin sulfate according to a weight ratio of 5:3:2 to obtain the low-molecular-weight chondroitin sulfate composition LMCSs-C; wherein, The preparation method of LMCSD is as follows: Mix the chondroitin sulfate solution with the hydrazine hydrate solution containing hydrazine sulfate, and carry out a deacetylation reaction at 85°C - 95°C under nitrogen protection. At the 8th hour of the reaction, take a sample, measure the content of N-acetylgalactosamine in the sample and calculate the degree of deacetylation DDA%. If DDA% ≥ 95%, immediately terminate the reaction. Otherwise, continue the reaction and take samples for detection every hour until DDA% ≥ 95%. Sampling, detection, and calculation of the degree of deacetylation are completed within 60 minutes. After the deacetylation reaction is completed, the mixed solution is subjected to alcohol precipitation and dialysis to obtain the deacetylated product. React the deacetylated product with the nitrite solution at 4 - 6°C for 30 minutes, adjust the pH to alkaline to terminate the reaction, and then add the NaBH4 solution to reduce at 45°C - 55°C for 1.5 - 2.5 hours. The molar ratio of the deacetylated product, 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 an ultrafiltration membrane and then freeze-dried to obtain it.

2. The preparation method of the low-molecular-weight chondroitin sulfate composition according to claim 1, characterized in that, The preparation method of LMCSO is as follows: Add H2O2 and ascorbic acid with a final concentration of 20 - 30 mM to the 10 mg / mL chondroitin sulfate solution, adjust the pH to 5.5 ± 0.2, react at 30°C - 40°C for 20 - 40 minutes, and the obtained product is purified by a 0.5 kDa ultrafiltration membrane and then freeze-dried to obtain it.

3. The preparation method of the low molecular weight chondroitin sulfate composition according to claim 2, characterized in that, In the step of preparing LMCSO, first add ascorbic acid to the chondroitin sulfate solution and mix well, and then dropwise add the H2O2 solution under continuous stirring. After the addition of the H2O2 solution is completed, react the mixed solution at 35 ± 1°C for 10 - 15 minutes, and then raise the temperature to 38 ± 1°C and continue to react for 10 - 15 minutes. The final concentrations of H2O2 and ascorbic acid are both 25 ± 0.5 mM.

4. The preparation method of the low-molecular-weight chondroitin sulfate composition according to claim 1, characterized in that, The preparation method of LMCSH is as follows: Adjust the pH of the 10 mg / mL chondroitin sulfate solution to 4.0 ± 0.2 with acetic acid, react at 100°C - 120°C for 2 - 3 hours, and the obtained product is purified by a 0.5 kDa ultrafiltration membrane and then freeze-dried to obtain it; wherein, the reaction process uses program temperature control. First, heat up to 118 ± 1°C at a rate of 120 ± 5°C / min; then keep the temperature constant at 118 ± 1°C for 60 ± 5 minutes; then cool down to 100 ± 2°C at a rate of 15 ± 3°C / min; then keep the temperature constant at 100 ± 2°C for 60 ± 5 minutes; after the reaction is completed, immediately place the reaction vessel in an ice-water bath and lower the system temperature below 25°C within 2 minutes.

5. The preparation method of the low molecular weight chondroitin sulfate composition according to claim 1, characterized in that, In the step of preparing LMCSD, the content of N-acetylgalactosamine in the sample was determined by high performance anion exchange chromatography-pulsed amperometric detection, and the detection conditions were: chromatographic column CarboPac PA20, flow rate 0.5 mL / min, NaOH gradient 2-100 mM; the specific method of alcohol precipitation and dialysis was: after the deacetylation reaction was completed, the mixture was cooled to room temperature, and 4 times the volume of pre-cooled anhydrous ethanol at 4°C was slowly added under stirring, and allowed to stand at 4°C for 2 hours to precipitate; the precipitate was collected, dissolved in deionized water, loaded into a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyzed against deionized water at 4°C for 24 hours, with the water changed at least 6 times during the period.

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

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

8. The pharmaceutical composition according to claim 7, wherein, It is used for treating diseases related to intestinal barrier dysfunction, including inflammatory bowel disease, irritable bowel syndrome or intestinal flora dysregulation.

9. Use of the low molecular weight chondroitin sulfate composition according to claim 6 in the preparation of a health product for enhancing intestinal barrier function.

10. The application according to claim 9, characterized in that, The enhancement of intestinal barrier function includes: up-regulating the gene expression of tight junction proteins ZO-1, Occludin and Claudin-1; promoting the production of intestinal short-chain fatty acids SCFAs; reducing the abundance of pathogenic Escherichia-Shigella.

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