Small molecule chondroitin sulfate group effect complex for intervening in arthritic inflammation and use thereof

Abstract

The application discloses a small-molecule chondroitin sulfate group-effect compound for intervening in treatment of arthritis and application, relates to the technical field of arthritis improvement, and aims to solve the problems of single action path of small-molecule chondroitin sulfate obtained by a single degradation method and difficulty in comprehensively responding to a complex pathological process of arthritis. The composition is composed of an oxidative degradation product LMCSO, an acid degradation product LMCSA and an alkali degradation product LMCSB in a weight ratio of 6:3:1, the number-average molecular weights of the three are all less than 10 kDa, and the three are prepared by specific oxidative degradation, acid degradation and alkali degradation methods containing a phase transfer catalyst and microwave assistance. The small-molecule chondroitin sulfate group-effect compound can be used for preparing medicine which is helpful to repairing bone and joint damage and relieving joint pain or stiffness.

Description

Technical Field

[0001] This invention relates to the field of joint inflammation improvement technology. More specifically, this invention relates to a small molecule chondroitin sulfate complex for the interventional treatment of joint inflammation and its application. Background Technology

[0002] Arthritis is a general term for a group of inflammatory or degenerative diseases occurring in and around joints, mainly manifested as joint pain, swelling, stiffness, and limited mobility. In severe cases, it can lead to joint deformities and functional impairment. Common types include osteoarthritis, rheumatoid arthritis, and gouty arthritis. It is influenced by various factors such as age, genetics, obesity, immune abnormalities, metabolic disorders, and trauma, significantly reducing patients' quality of life and imposing a continuous social and medical burden. While current medications for treating joint inflammation have clear efficacy, they generally have certain side effects. Analgesics and anti-inflammatory drugs mainly cause gastrointestinal, renal, and cardiovascular adverse reactions; long-term use of glucocorticoids can easily lead to metabolic disorders and osteoporosis; traditional DMARDs can cause abnormalities in liver and kidney function and blood routine tests; while biologics and JAK inhibitors mainly cause increased risk of infection and immune-related adverse reactions. Therefore, finding biocompatible functional components from natural products for adjunctive improvement of joint inflammation has become one of the directions of related research.

[0003] Chondroitin sulfate (CS) is a sulfated glycosaminoglycan composed of D-glucuronic acid and N-acetyl-D-galactosamine linked by alternating β-1,3 and β-1,4 glycosidic bonds, and is widely found in animal cartilage and other tissues. Studies have shown that chondroitin sulfate has certain anti-inflammatory and cartilage-protective effects, but its natural form has a large molecular weight, typically in the tens of kDa or even higher. High molecular weight chondroitin sulfate has poor solubility in water, and its absorption and utilization rate in the gastrointestinal tract after oral administration is limited, making it difficult to effectively reach the joints to exert its beneficial effects. In addition, the bioavailability of large molecular weight chondroitin sulfate may be limited by enzymatic hydrolysis efficiency during in vivo metabolism. Therefore, obtaining low molecular weight chondroitin sulfate (LMCS), which has a smaller molecular weight, better solubility, and is easily absorbed, is one of the issues of interest in this field.

[0004] Existing technologies for preparing small-molecule chondroitin sulfate mainly include oxidative degradation, acid degradation, and enzymatic degradation. Oxidative degradation typically uses oxidants such as hydrogen peroxide to break glycosidic bonds via free radical reactions catalyzed by metal ions; acid degradation utilizes hydrogen ions to catalyze the hydrolysis of glycosidic bonds; and enzymatic degradation uses specific enzymes for mild degradation. However, each of these methods has limitations in practical applications. For example, while simple oxidative degradation is relatively fast, the free radical reaction process is difficult to control precisely, potentially leading to the loss of sulfate groups or oxidative side reactions, affecting the structural integrity of the product. In acid degradation reactions, strong acid environments and high temperatures easily cause random cleavage of glycosidic bonds, resulting in a wide molecular weight distribution of the product and the possibility of desulfurization or isomerization, reducing product activity. Although enzymatic degradation has high specificity, enzyme preparations are expensive, and the reaction conditions are harsh, which is not conducive to large-scale production.

[0005] In alkaline degradation, traditional alkaline treatments often require high pH and temperature, resulting in vigorous reactions that easily induce β-elimination of chondroitin sulfate, leading to the destruction of the sugar chain structure and the removal of sulfate groups. To address this issue, researchers have attempted to introduce phase-transfer catalysts to improve reaction selectivity, but balancing catalytic efficiency with the degree of degradation remains a challenge. Furthermore, small molecule products obtained from single degradation methods often possess similar chain segment structures and terminal groups, potentially leading to a limited pathway of action in alleviating joint inflammation and failing to comprehensively address the complex pathophysiological processes of joint inflammation, such as inflammatory responses, oxidative stress, and immune cell infiltration.

[0006] In summary, although various methods exist for preparing small-molecule chondroitin sulfate in the prior art, obtaining compositions with relatively stable structures, controllable molecular weights, and suitability for improving joint inflammation remains a problem to be solved in this field. In particular, single degradation methods often fall short of requirements in balancing product yield, structural integrity, and bioactivity. Summary of the Invention

[0007] One object of the present invention is to provide a small molecule chondroitin sulfate complex and its application for the interventional treatment of joint inflammation, so as to at least solve the above-mentioned problems.

[0008] To achieve the objectives and other advantages of this invention, a small-molecule chondroitin sulfate complex for the intervention and treatment of joint inflammation is provided, which is composed of oxidative degradation product LMCSO, acid degradation product LMCSA, and alkaline degradation product LMCSB in a weight ratio of 6:3:1, wherein...

[0009] The number-average molecular weights of LMCSO, LMCSA, and LMCSB are all less than 10 kDa;

[0010] The LMCSO is prepared by a method including the following steps: contacting a chondroitin sulfate solution with an oxidative degradation reagent to carry out an oxidative degradation reaction, wherein the oxidative degradation reagent includes H2O2 and copper acetate, the reaction pH is 5.5-9.0, and the reaction temperature is 40-55℃;

[0011] The LMCSA is prepared by a method including the following steps: contacting a chondroitin sulfate solution with an acid solution to carry out an acid degradation reaction at a reaction temperature of 40-70℃.

[0012] The LMCSB is prepared by a method comprising the following steps: dissolving chondroitin sulfate in water, adding 0.5%-1% (by mass of chondroitin sulfate) of hexadecyltrimethylammonium bromide (CTAB) as a phase transfer catalyst, and stirring until homogeneous; then adding a borate-sodium hydroxide buffer solution to make the final concentration of chondroitin sulfate in the reaction system 15-25 mg / mL, the final concentration of the buffer solution 0.1-0.3 M (based on borate), and the pH value 8.5-9.5; placing the mixture in a microwave reactor for alkaline degradation reaction at a reaction temperature of 40-70℃.

[0013] Preferably, the preparation of LMCSO specifically includes: adding copper acetate to a 7-12 mg / mL chondroitin sulfate solution and mixing well; then, under stirring conditions, slowly adding H2O2 solution dropwise over 15-20 minutes, with a final H2O2 concentration of 60 mM and a final copper acetate concentration of 0.65 ± 0.05 mM; after the H2O2 solution is added, the mixture is first reacted at 45 ± 1 °C for 50-60 minutes, then the temperature is raised to 50 ± 1 °C and the reaction continues for another 50-60 minutes; after the reaction is completed, the pH is adjusted to 9.0, and the mixture is allowed to stand for 30-60 minutes, then centrifuged to remove Cu. 2+ The supernatant was filtered through a 0.22 μm filter membrane and then subjected to tangential flow ultrafiltration to collect the fraction with a relative molecular mass less than 10 kDa. After concentration, dialysis, and freeze-drying, the product was obtained.

[0014] Preferably, the preparation of LMCSA specifically includes: dissolving chondroitin sulfate in water, adding mannitol as a protective agent, with a final concentration of mannitol of 0.05-0.1 M, and then mixing it with HCl solution to achieve a final concentration of chondroitin sulfate solution of 15-25 mg / mL and a final concentration of HCl solution of 0.1-0.5 M; gradually heating the mixture to 65±2℃ at a heating rate of 0.5-1.0℃ / min, and holding it at 45±1℃ and 55±1℃ for 30-45 minutes respectively during the heating process, with a total reaction time of 480-500 minutes; immediately placing the reaction vessel in an ice-water bath after the reaction, lowering the system temperature to below 25℃ within 2 minutes, then adjusting the pH to 7.0 to terminate the reaction, filtering through a 0.22 μm filter membrane, and then using tangential flow ultrafiltration to collect the fraction with a relative molecular mass less than 10 kDa, which is then concentrated, dialyzed, and freeze-dried to obtain the final product.

[0015] Preferably, the preparation of LMCSB further includes: the alkaline degradation reaction is carried out under stirring conditions using a multi-stage stepped temperature control mode: the first stage is heated to 45±1℃ with a power of 300-400W and kept at a constant temperature for 10-15 minutes; the second stage is heated to 55±1℃ with a power of 400-500W and kept at a constant temperature for 15-20 minutes; the third stage is heated to 65±1℃ with a power of 500-600W and kept at a constant temperature for 5-10 minutes; during the reaction, the microwave output power is dynamically adjusted through real-time temperature monitoring; after the reaction is completed, the pH value is adjusted back to 7.0, and the mixture is allowed to stand for 30 minutes to allow CTAB to precipitate, and the precipitate is removed by centrifugation; the supernatant is filtered through a 0.22 μm filter membrane, and then subjected to tangential flow ultrafiltration to collect the portion with a relative molecular mass less than 10 kDa, which is then concentrated, dialyzed, and freeze-dried to obtain the final product.

[0016] The present invention also provides the application of the above-mentioned small molecule chondroitin sulfate complex for the interventional treatment of joint inflammation in the preparation of a medicament for improving joint inflammation.

[0017] Preferably, the drug is used to reduce joint inflammation scores, reduce joint swelling, improve exercise capacity, reduce inflammatory factor levels, inhibit immune cell infiltration, reduce oxidative stress, or regulate organ indices, or one or more of the following:

[0018] The present invention also provides a pharmaceutical composition comprising the above-described small molecule chondroitin sulfate complex for the interventional treatment of joint inflammation and a pharmaceutically acceptable carrier.

[0019] The present invention has at least the following beneficial effects:

[0020] First, by combining small-molecule chondroitin sulfate obtained from three different degradation methods (oxidation, acid, and alkali) in a specific ratio (6:3:1), the limitation of a single degradation product having a single pathway of action is overcome. Due to their different degradation mechanisms, the three products differ in chain segment structure, distribution of sulfate groups, and composition of terminal groups. After combination, they can synergistically improve the complex pathological process of joint inflammation from multiple aspects.

[0021] Secondly, by strictly controlling the concentration of copper acetate catalyst and the hydrogen peroxide dropping rate, precise regulation of the oxidative degradation reaction process was achieved. Employing a two-step temperature-controlled reaction mode (first 45℃ then 50℃) facilitates the stable release of free radicals and avoids excessive sulfate group removal or side reactions caused by overly vigorous local reactions. After the reaction, the pH was adjusted to 9.0 to ensure Cu... 2+ Precipitation removal simplifies the purification process. Combined with tangential flow ultrafiltration technology, it precisely retains components with molecular weights less than 10 kDa, resulting in a more concentrated molecular weight distribution of the product. This helps ensure batch-to-batch stability of the composition and provides a uniform raw material for subsequent drug efficacy.

[0022] Third, by adding mannitol as a protective agent, the active groups of chondroitin sulfate can be stabilized during acidic hydrolysis, helping to reduce the damage of the sulfate ester bonds to the acid. Using programmed temperature rise and staged holding at 45℃ and 55℃ respectively makes the hydrolysis of glycosidic bonds more gradual and orderly, which is beneficial for obtaining products with a narrow molecular weight distribution. Rapid cooling and neutralization after the reaction can promptly terminate the hydrolysis reaction and prevent excessive degradation. This method, while ensuring that the molecular weight is reduced to the desired range, better maintains the basic structural characteristics of the product, providing a small molecule component with good structural integrity for the composition.

[0023] Fourth, the phase-transfer catalyst hexadecyltrimethylammonium bromide was combined with microwave-assisted technology for alkaline degradation reactions. The phase-transfer catalyst promotes mass transfer between the liquid and solid phases, improving degradation efficiency; microwave radiation utilizes both thermal and non-thermal effects to ensure more uniform heating of the reaction system. A multi-stage stepped heating mode combined with real-time temperature feedback control achieves precise temperature control, avoiding the violent elimination reactions caused by localized overheating in traditional alkaline degradation. After the reaction, the catalyst is allowed to precipitate and is easily removed by allowing it to stand. This method enables effective degradation under relatively mild conditions, which is beneficial for obtaining structurally intact small-molecule products.

[0024] Fifth, the small-molecule chondroitin sulfate complex of the present invention, through a multi-pathway and multi-target mechanism of action, helps to comprehensively address the pathological aspects of joint inflammation, including inflammatory responses, immune disorders, and cartilage damage. Compared with single components, this composition has a better overall improvement effect. Preparing this composition into a drug can provide a biocompatible intervention approach derived from natural polysaccharides, expanding the application scope of small-molecule chondroitin sulfate in the field of autoimmune joint diseases.

[0025] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0026] Figure 1 This is a graph showing the change in mouse body weight according to an embodiment of the present invention;

[0027] Figure 2 This is a graph showing the effect of LMCS on the thymus index, liver index, and spleen index of mice according to an embodiment of the present invention.

[0028] Figure 3 This is a graph showing the effect of LMCS according to an embodiment of the present invention on white blood cells, granulocytes, lymphocytes and monocytes in mouse blood routine tests;

[0029] Figure 4 This is a graph showing the effect of LMCS on rotarod time in mice according to an embodiment of the present invention;

[0030] Figure 5 This is a photograph of the diseased area of ​​a mouse on day 56 of one embodiment of the present invention;

[0031] Figure 6 This is a graph showing the effect of LMCS on the thickness of mouse toe joints according to an embodiment of the present invention;

[0032] Figure 7 This is a graph showing the effect of LMCS on mouse joint scores according to an embodiment of the present invention;

[0033] Figure 8 This is a diagram showing the effect of LMCS on HE and TRAP staining of mouse joints according to an embodiment of the present invention;

[0034] Figure 9 This is a graph showing the effect of LMCS on the pathological score of H&E staining of mouse joints according to an embodiment of the present invention.

[0035] Figure 10 This is a graph showing the effect of LMCS on mouse joint immune cells and angiogenesis according to an embodiment of the present invention;

[0036] Figure 11 This is a graph showing the effect of different compounding methods on mouse joint scores in one embodiment of the present invention;

[0037] Figure 12 This is a graph showing the effect of different preparation methods on mouse joint scores in one embodiment of the present invention;

[0038] Figure 13 This is a graph showing the effect of different preparation methods on mouse rotator time in one embodiment of the present invention. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0040] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0041] According to one embodiment of the present invention, a small molecule chondroitin sulfate complex for the intervention and treatment of joint inflammation comprises an oxidative degradation product LMCSO, an acid degradation product LMCSA, and an alkaline degradation product LMCSB, which are mixed in a weight ratio of 6:3:1. The number average molecular weights of LMCSO, LMCSA, and LMCSB are all less than 10 kDa. The preparation method of LMCSO includes: contacting a chondroitin sulfate solution with an oxidative degradation reagent, the oxidative degradation reagent including H2O2 and copper acetate, wherein the reaction pH can be selected within the range of 5.5 to 9.0, for example, pH 6.0, 7.0, or 8.0, and the reaction temperature can be selected within the range of 40°C to 55°C, for example, 42°C, 48°C, or 52°C. The preparation method of LMCSA includes: contacting a chondroitin sulfate solution with an acid solution to perform an acid degradation reaction, wherein the reaction temperature can be selected within the range of 40°C to 70°C, for example, 45°C, 55°C, or 65°C. The preparation method of this LMCSB includes: dissolving chondroitin sulfate in water, adding 0.5% to 1% (by mass of chondroitin sulfate) of hexadecyltrimethylammonium bromide (CTAB) as a phase transfer catalyst, and stirring until homogeneous; then adding a borate-sodium hydroxide buffer solution, such that the final concentration of chondroitin sulfate in the reaction system can be selected in the range of 15 mg / mL to 25 mg / mL, for example, 18 mg / mL, 20 mg / mL, or 22 mg / mL; the final concentration of the buffer solution, calculated as borate, can be selected in the range of 0.1 M to 0.3 M, for example, 0.15 M, 0.2 M, or 0.25 M; and the pH value can be selected in the range of 8.5 to 9.5, for example, 9.0; placing the mixture in a microwave reactor for alkaline degradation reaction, the reaction temperature of which can be selected in the range of 40℃ to 70℃, for example, 45℃, 55℃, or 65℃.

[0042] In this embodiment, by combining the small molecule chondroitin sulfate obtained by the above three different degradation methods in a specific ratio, the composition can act on the pathological links related to arthritis through multiple pathways. Compared with the products obtained by a single degradation method, it can more comprehensively address the complex physiological and pathological processes of arthritis.

[0043] According to another embodiment of the present invention, in the preparation of LMCSO, copper acetate is first added to a chondroitin sulfate solution with a concentration of 7 mg / mL to 12 mg / mL and mixed thoroughly. The concentration of the chondroitin sulfate solution can be specifically selected according to actual needs, for example, 8 mg / mL, 9 mg / mL, 10 mg / mL, or 11 mg / mL. Subsequently, under stirring conditions, hydrogen peroxide (H2O2) solution is slowly added dropwise over a time period of 15 to 20 minutes. The dropping rate can be adjusted appropriately according to the size of the reaction vessel to ensure uniform dispersion of H2O2. The final concentration of H2O2 in the reaction system is controlled at 60 mM, and the final concentration of copper acetate is controlled at 0.65 ± 0.05 mM. This concentration range can be achieved by pre-calculating the required volumes of copper acetate and H2O2 to be added. Local overconcentration should be avoided during the dropping process to reduce the occurrence of side reactions. After the H2O2 solution is added dropwise, the mixture is first kept at a constant temperature of 45±1℃ for 50 to 60 minutes, for example, 55 minutes. Then, the temperature is increased to 50±1℃ and the reaction continues for another 50 to 60 minutes, for example, 55 minutes. This segmented temperature control helps control the release rate of free radicals, ensuring a smooth oxidative degradation reaction and thus obtaining small molecule products with a narrow molecular weight distribution. After the reaction, the pH of the reaction system is adjusted to 9.0, and the mixture is allowed to stand for 30 to 60 minutes, for example, 45 minutes. During the standing process, copper ions (Cu) in the solution... 2+ A precipitate will form, which is then removed by centrifugation to purify the product. The resulting supernatant is first filtered through a 0.22 μm pore size membrane to remove any potentially present small particulate matter. The filtered solution is then processed using a tangential flow ultrafiltration system to collect fractions with a relative molecular mass less than 10 kDa. This ultrafiltration step can utilize an ultrafiltration membrane module with an appropriate molecular weight cutoff, enriching products within the target molecular weight range through cyclic concentration. Finally, the collected fraction is concentrated, dialyzed to remove small molecule impurities, and then freeze-dried to obtain LMCSO lyophilized powder.

[0044] The LMCSO product obtained by the above preparation method has a stable number-average molecular weight below 10 kDa. Furthermore, due to the use of a segmented temperature-controlled oxidative degradation process, the molecular weight distribution of the product is relatively concentrated, and the retention of sulfate groups is high. This preparation process operates under mild conditions with good reproducibility, providing the composition with a homogeneous and relatively structurally intact small-molecule chondroitin sulfate component, which is beneficial for subsequent applications of the composition in research related to improving joint inflammation.

[0045] According to another embodiment of the present invention, in the preparation of LMCSA, chondroitin sulfate is first dissolved in water, and mannitol is added as a protective agent. The final concentration of mannitol in the reaction system can be controlled in the range of 0.05 M to 0.1 M, specifically selected according to the source and purity of chondroitin sulfate, for example, 0.06 M, 0.08 M, or 0.09 M can be used. The addition of mannitol helps to stabilize the sugar chain structure of chondroitin sulfate during subsequent acid degradation, reducing the shedding of sulfate groups. Subsequently, the above solution is mixed with HCl solution to control the final concentration of chondroitin sulfate in the range of 15 mg / mL to 25 mg / mL, for example, 18 mg / mL, 20 mg / mL, or 22 mg / mL can be used; the final concentration of HCl solution is controlled in the range of 0.1 M to 0.5 M, for example, 0.2 M, 0.3 M, or 0.4 M can be used. During mixing, the HCl solution should be added slowly and stirred continuously to avoid excessively high local acid concentrations. The initial temperature of the mixture is typically room temperature, such as 25°C, to prepare for the heating and degradation stage. The mixture is gradually heated to 65±2°C at a rate of 0.5°C / min to 1.0°C / min; for example, a heating rate of 0.6°C / min, 0.8°C / min, or 0.9°C / min can be selected. During the heating process, when the temperature reaches 45±1°C, the system is held at this temperature for 30 to 45 minutes; for example, 35 or 40 minutes can be selected. Then, the temperature is continued to rise until it reaches 55±1°C, and the system is again held at this temperature for 30 to 45 minutes; for example, 35 or 40 minutes can be selected. After completing these two holding stages, the temperature is further increased to 65±2°C and the reaction is maintained, so that the total reaction time of the entire acid degradation process is controlled within the range of 480 to 500 minutes; for example, a reaction time of 490 minutes can be selected. This segmented heating method allows the acid hydrolysis reaction to proceed sequentially at different temperature gradients, which is beneficial for the gradual breaking of glycosidic bonds, resulting in small molecule products with a relatively concentrated molecular weight distribution. After the reaction is complete, the reaction vessel is placed in an ice-water bath, and the system temperature is rapidly reduced to below 25°C within 2 minutes. Rapid cooling effectively inhibits the continued hydrolysis reaction and prevents excessive degradation. Subsequently, the pH of the reaction system is adjusted to 7.0 with an alkaline solution to completely terminate the reaction. The resulting solution is first filtered through a 0.22 μm pore size membrane to remove any insoluble impurities that may be generated. The filtered solution is then processed using a tangential flow ultrafiltration system to collect the fraction with a relative molecular mass less than 10 kDa. This ultrafiltration step can use an ultrafiltration membrane module with an appropriate molecular weight cutoff, enriching the product within the target molecular weight range through cyclic concentration. Finally, the collected fraction is concentrated and dialyzed to remove small molecule salts and protective agents such as mannitol, and then freeze-dried to obtain LMCSA lyophilized powder.

[0046] The LMCSA product obtained by the above preparation method has a stable number-average molecular weight below 10 kDa. Due to the introduction of mannitol as a protective agent during acid degradation and the use of programmed temperature rise and segmented holding processes, the product exhibits a high degree of retention of sulfate groups and a relatively uniform molecular weight distribution. This preparation method can provide the composition with a small-molecule chondroitin sulfate component with good structural integrity, which is beneficial for its synergistic effect in improving joint inflammation-related applications.

[0047] According to another embodiment of the present invention, in the preparation of LMCSB, chondroitin sulfate is first dissolved in water, and then 0.5% to 1% (by mass of chondroitin sulfate) of hexadecyltrimethylammonium bromide (CTAB) is added as a phase transfer catalyst, for example, 0.6%, 0.8%, or 0.9% CTAB can be added. The mixture is stirred thoroughly to disperse the catalyst. The addition of CTAB helps to promote mass transfer between the two phases in the subsequent alkaline degradation reaction, thereby improving the reaction efficiency. Subsequently, a borate-sodium hydroxide buffer solution is added to the above solution to adjust the parameters of the reaction system. The final concentration of chondroitin sulfate is controlled in the range of 15 mg / mL to 25 mg / mL, for example, 18 mg / mL, 20 mg / mL, or 22 mg / mL can be used; the final concentration of the buffer solution, calculated as borate, is controlled in the range of 0.1 M to 0.3 M, for example, 0.15 M, 0.2 M, or 0.25 M can be used; the pH value of the reaction system is controlled in the range of 8.5 to 9.5, for example, pH 9.0 can be used. The buffer solution should be added slowly and with stirring to ensure a uniform and stable pH in the system. The prepared mixture is then placed in a microwave reactor and subjected to alkaline degradation under stirring. The reaction is carried out using a multi-stage stepped temperature control mode: In the first stage, the system is heated to 45±1℃ with a microwave power of 300W to 400W, for example, 320W, 350W or 380W can be selected, and the reaction is held at this temperature for 10 to 15 minutes, for example, 12 minutes or 14 minutes; In the second stage, the system is heated to 55±1℃ with a microwave power of 400W to 500W, for example, 420W, 450W or 480W can be selected, and the reaction is held at this temperature for 15 to 20 minutes, for example, 16 minutes or 18 minutes; In the third stage, the system is heated to 65±1℃ with a microwave power of 500W to 600W, for example, 520W, 550W or 580W can be selected, and the reaction is held at this temperature for 5 to 10 minutes, for example, 6 minutes or 8 minutes. Throughout the reaction, the system temperature was monitored in real-time by a temperature probe, and the microwave output power was dynamically adjusted accordingly to ensure that the temperature remained stable near the set value at each stage, avoiding excessive temperature fluctuations that could affect the product structure. After the reaction, the pH of the reaction system was adjusted to 7.0 using an acid solution to terminate the alkaline degradation reaction. The reaction solution was then allowed to stand for 30 minutes to allow the added phase transfer catalyst CTAB to fully precipitate. After standing, the precipitate was removed by centrifugation. The resulting supernatant was first filtered through a 0.22 μm pore size membrane to remove any remaining small particles. The filtered solution was then processed using a tangential flow ultrafiltration system to collect components with a relative molecular mass less than 10 kDa.This ultrafiltration step can utilize an ultrafiltration membrane module with an appropriate molecular weight cutoff to enrich products within the target molecular weight range through cyclic concentration. Finally, the collected fraction is concentrated and dialyzed to remove small molecule buffer salts and residual CTAB, and then freeze-dried to obtain LMCSB lyophilized powder.

[0048] The LMCSB product obtained by the above preparation method has a stable number-average molecular weight below 10 kDa. This method employs multi-stage stepped microwave heating and real-time temperature feedback control, achieving precise temperature control and avoiding the violent elimination reactions caused by localized overheating or temperature runaway in traditional alkaline degradation processes. This approach helps to effectively reduce molecular weight while maintaining the integrity of the sugar chains and the retention of sulfate groups. The preparation method features mild reaction conditions and a controllable process, providing the composition with a relatively structurally complete and homogeneous small-molecule chondroitin sulfate component.

[0049] Example 1:

[0050] The preparation of a small molecule chondroitin sulfate complex includes the following steps:

[0051] (1) Preparation of small molecule chondroitin sulfate:

[0052] LMCSO: Dissolve an appropriate amount of chondroitin sulfate raw material in deionized water to prepare a chondroitin sulfate solution with a concentration of 10 mg / mL, with a total volume of 1 L. Accurately weigh 26.0 mg of copper acetate and add it to the above chondroitin sulfate solution to achieve a final concentration of 0.65 mM in the reaction system. Stir at 200 rpm for 15 minutes to ensure complete dissolution and homogeneity of the copper acetate. While maintaining stirring at 200 rpm, slowly add 6.8 mL of 30% hydrogen peroxide (H2O2) solution to the above mixture using a constant flow pump at a dropping rate of 0.6 mL / min, ensuring the addition process is completed within 18 minutes. Control the final concentration of H2O2 in the reaction system to 60 mM. After the H2O2 solution is added, place the reaction vessel in a constant temperature water bath, set the temperature to 45℃, and react at this temperature for 55 minutes, continuously stirring at 150 rpm during the reaction. Subsequently, the water bath temperature was adjusted to 50°C, and the reaction was continued at this constant temperature for 55 minutes, with the stirring rate remaining constant. Throughout the reaction process, the pH of the reaction system was maintained within its natural fluctuation range of 5.5 to 9.0 without additional adjustment. After the reaction was complete, the pH of the reaction system was adjusted to 9.0 using 1 M sodium hydroxide solution, and the system was allowed to stand at room temperature for 45 minutes to allow the copper ions (Cu) generated in the reaction system to settle. 2+Allow sufficient precipitation. Transfer the settled reaction solution to a 500 mL centrifuge bottle and centrifuge at 8000 rpm for 20 minutes to remove the copper precipitate. Collect the supernatant. Filter the supernatant under pressure using a 0.22 μm pore size microporous membrane at 0.2 MPa to remove any remaining fine particles. The filtered solution is then fractionated using a tangential flow ultrafiltration system. The ultrafiltration system is equipped with a hollow fiber membrane module with a molecular weight cutoff of 10 kDa and a membrane area of ​​0.1 m². 2 Under operating conditions of 0.15 MPa inlet pressure and 0.05 MPa reflux pressure, circulating ultrafiltration was performed, and the permeate (components with a relative molecular mass less than 10 kDa) was collected until the permeate volume reached 800 mL. The collected permeate was transferred to a rotary evaporator and concentrated under reduced pressure at 40°C in a water bath to approximately one-fifth of its original volume, about 160 mL. The concentrate was placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed against deionized water. The dialysate was replaced every 4 hours for a total of 5 dialysis cycles, totaling 24 hours, to remove small molecule impurities. After dialysis, the liquid in the bag was removed and freeze-dried at -50°C and 10 Pa for 48 hours to obtain LMCSO lyophilized powder, with a yield of 72% calculated by weight. Gel permeation chromatography showed that its weight-average molecular weight was 8.2 kDa, its number-average molecular weight was 6.5 kDa, and its molecular weight distribution coefficient was 1.26.

[0053] LMCSA: Dissolve an appropriate amount of chondroitin sulfate raw material in deionized water to prepare a chondroitin sulfate stock solution with a concentration of 40 mg / mL. Weigh 14.6 g of mannitol, dissolve it in an appropriate amount of deionized water, and prepare a 0.5 M mannitol stock solution. Take 250 mL of the above chondroitin sulfate stock solution, add 80 mL of mannitol stock solution, mix well, and then add deionized water to bring the total volume to 400 mL. Then slowly add 300 mL of 2 M HCl solution to the mixture while stirring at 200 rpm, so that the final total volume of the reaction system is 1 L. Control the final concentration of chondroitin sulfate in the reaction system to be 20 mg / mL, the final concentration of mannitol to be 0.08 M, and the final concentration of hydrochloric acid to be 0.3 M. Transfer the prepared reaction solution to a 2 L three-necked flask and place it in a temperature-controlled oil bath. Turn on the stirrer and set the speed to 150 rpm. A programmed temperature controller was used, with a heating rate set at 0.8℃ / min, starting from room temperature (25℃). When the temperature reached 45℃, a hold-up program was initiated, maintaining the temperature at 45±1℃ for 35 minutes. After the hold-up period, the temperature was increased again at 0.8℃ / min, and when it reached 55℃, the hold-up program was restarted, maintaining the temperature at 55±1℃ for 40 minutes. After the hold-up period, the temperature was increased again at 0.8℃ / min to 65℃, and the reaction continued at 65±2℃, bringing the total reaction time to 490 minutes. The temperature was recorded every 30 minutes during the reaction to ensure that temperature fluctuations remained within the controlled range. After the reaction, the three-necked flask was immediately placed in an ice-water bath, and the system temperature was rapidly reduced to 22℃ within 2 minutes. Then, the pH of the reaction solution was slowly adjusted to 7.0 with 5M sodium hydroxide solution while stirring, until the reaction was completely terminated. The resulting reaction solution was first pressure filtered using a microporous membrane with a pore size of 0.22 μm, at a pressure controlled at 0.2 MPa, to remove insoluble impurities generated during the reaction. The filtered solution was then fractionated using a tangential flow ultrafiltration system. The ultrafiltration system was equipped with a hollow fiber membrane module with a molecular weight cutoff of 10 kDa and a membrane area of ​​0.2 m². 2Under operating conditions of 0.18 MPa inlet pressure and 0.06 MPa reflux pressure, circulating ultrafiltration was performed, and the permeate (components with a relative molecular mass less than 10 kDa) was collected until the permeate volume reached 1.8 L. The collected permeate was transferred to a rotary evaporator and concentrated under reduced pressure at 45°C in a water bath to one-fifth of its original volume, approximately 360 mL. The concentrate was placed in a dialysis bag with a molecular weight cutoff of 500 Da, and dialyzed against deionized water. The dialysate was replaced every 4 hours for a total of 6 replacements, and dialyzed for 24 hours to remove small molecule salts and mannitol. After dialysis, the liquid in the bag was removed and freeze-dried at -55°C and 8 Pa for 52 hours to obtain LMCSA lyophilized powder, with a yield of 61% calculated by weight. Gel permeation chromatography showed that its weight-average molecular weight was 7.6 kDa, its number-average molecular weight was 5.8 kDa, and its molecular weight distribution coefficient was 1.31.

[0054] LMCSB: Dissolve an appropriate amount of chondroitin sulfate raw material in deionized water to prepare a chondroitin sulfate stock solution with a concentration of 50 mg / mL. Weigh 1.6 g of hexadecyltrimethylammonium bromide (CTAB), dissolve it in a small amount of deionized water, and set aside. Prepare borate-sodium hydroxide buffer: Weigh 19.1 g of borax and 8.0 g of sodium hydroxide, dissolve them in deionized water, and bring the volume to 1 L to obtain a buffer solution with a borate concentration of 0.2 M and a pH of 9.0. Place 400 mL of the above chondroitin sulfate stock solution in a 2 L reaction vessel, add CTAB solution to make the CTAB mass account for 0.8% of the chondroitin sulfate mass, and stir at 300 rpm for 20 minutes to fully disperse the CTAB. Then add 500 mL of borate-sodium hydroxide buffer, and add deionized water to make the total volume 1 L, and continue stirring at 200 rpm for 10 minutes. The final concentration of chondroitin sulfate in the reaction system was controlled at 20 mg / mL, the final concentration of borate at 0.2 M, and the pH at 9.0. The prepared reaction solution was transferred to a 2 L container specifically designed for microwave reactors, equipped with a stirrer and a fiber optic temperature probe. The microwave reactor was turned on, and the stirring speed was set to 150 rpm. A multi-stage, stepped temperature control program was employed: In the first stage, the microwave power was set to 350W to raise the system temperature to 45℃. After reaching 45℃, the system was held at this temperature for 12 minutes. During this stage, the microwave power was automatically adjusted between 300W and 400W to maintain temperature stability, based on real-time feedback from the temperature probe. In the second stage, the microwave power was automatically switched to 450W, raising the system temperature to 55℃. After reaching 55℃, the system was held at this temperature for 18 minutes, with the microwave power automatically adjusted between 400W and 500W. In the third stage, the microwave power was automatically switched to 550W, raising the system temperature to 65℃. After reaching 65℃, the system was held at this temperature for 8 minutes, with the microwave power automatically adjusted between 500W and 600W. Throughout the reaction, the temperature probe collected temperature data every 2 seconds. The control system dynamically adjusted the output power based on temperature deviations to ensure that temperature fluctuations in each stage did not exceed ±1℃. After the reaction, the pH of the reaction solution was immediately adjusted to 7.0 with 2M hydrochloric acid solution while stirring at 200 rpm to terminate the alkaline degradation reaction. The reaction solution was allowed to stand at room temperature for 30 minutes to allow the added CTAB to precipitate. The settled reaction solution was then transferred to a 500 mL centrifuge bottle and centrifuged at 10,000 rpm for 25 minutes to remove the CTAB-containing precipitate. The supernatant was collected. The supernatant was then pressure filtered using a 0.22 μm pore size microporous membrane at a pressure controlled at 0.18 MPa to remove any remaining small particulate matter. The filtered solution was then fractionated using a tangential flow ultrafiltration system equipped with a hollow fiber membrane module with a molecular weight cutoff of 10 kDa and a membrane area of ​​0.2 m². 2Circulating ultrafiltration was performed under the operating conditions of an inlet pressure of 0.16 MPa and a reflux pressure of 0.05 MPa. The permeate, i.e., the component with a relative molecular mass less than 10 kDa, was collected until the permeate volume reached 1.6 L. The collected permeate was transferred to a rotary evaporator and concentrated under reduced pressure at 40°C in a water bath to one-fifth of its original volume, approximately 320 mL. The concentrate was placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed against deionized water. The dialysate was replaced every 4 hours for a total of 6 replacements, and dialyzed for 24 hours to remove small molecule buffer salts and residual CTAB. After dialysis, the liquid in the bag was removed and freeze-dried at -60°C and a vacuum of 5 Pa for 56 hours to obtain LMCSB lyophilized powder, with a yield of 58% calculated by weight. Gel permeation chromatography showed that its weight-average molecular weight was 7.1 kDa, its number-average molecular weight was 5.4 kDa, and its molecular weight distribution coefficient was 1.31.

[0055] (2) Preparation of small molecule chondroitin sulfate complex:

[0056] The LMCSO, LMCSA and LMCSB prepared according to the above method were mixed in a weight ratio of 6:3:1 to obtain the small molecule chondroitin sulfate complex LMCS-M.

[0057] Experimental Example 1:

[0058] 1. Experimental Methods

[0059] 1.1 Arthritis mouse model 1) Emulsion preparation process

[0060] Inside a clean bench, use a 1 mL sterile syringe to draw up a collagen solution (2 mg / mL) and transfer it to a pre-cooled glass homogenizer. Add an equal volume of complete Freund's adjuvant (CFA, 10 mg / mL) or incomplete Freund's adjuvant (IFA) to the same homogenizer. Emulsify thoroughly using a syringe reciprocating injection method (≥50 times) until the mixture is homogeneous and milky white. Maintain ice bath conditions throughout (0-4 °C) to avoid thermal denaturation of the collagen. Drop 50 μL of the emulsion onto the surface of deionized water. If the droplet maintains its intact shape in the aqueous phase (no diffusion), it indicates the formation of a stable water-in-oil (W / O) emulsion. If the droplet disperses rapidly, continue emulsification until the desired consistency is achieved.

[0061] 2) Modeling

[0062] Primary immunization (Day 0): Using a 25 μL Hamilton microsyringe (model 701N), inject 0.1 mL of an emulsion (an emulsion of type II collagen and complete Freund's adjuvant) subcutaneously into the base of the mouse's tail. Booster immunization (Day 21): Inject 0.1 mL of an emulsion (an emulsion of type II collagen and incomplete Freund's adjuvant) subcutaneously into the other side of the base of the tail.

[0063] 1.2 Grouping

[0064] The mice that successfully developed the model were randomly divided into 5 groups of 12 mice each, including the model group (physiological saline, 150 mg / kg / day), the LMCS-M intervention group (LMCS-M, 150 mg / kg / day), the LMCSO intervention group (LMCSO, 150 mg / kg / day), the LMCSA intervention group (LMCSA, 150 mg / kg / day), and the LMCSB intervention group (LMCSB, 150 mg / kg / day).

[0065] The control group (physiological saline, 150 mg / kg / day) consisted of 12 normal mice.

[0066] 1.3 Observation of changes in mouse body weight and toe joint thickness

[0067] Starting 21 days after the second immunization, mouse hind limb ankle swelling was systematically monitored 2-3 times per week using digital vernier calipers (Mitutoyo 500-196-30, 0.01mm accuracy). During measurement, a gentle rodent restraint device (Braintree Scientific, RB-MC) was used to keep the mouse's limbs naturally extended, and the measurement site was located at the point of greatest swelling in the tibiotarsal joint space. The initial measurement (T0) was used as the individual baseline, and the single-measurement swelling (ΔT) was obtained by calculating the difference between the current measurement (Tn) and T0. To ensure data reliability, the entire procedure was performed by the same experimenter (within-group coefficient of variation <5%), and each limb was measured three times, with the average taken (interval ≥30 seconds). The entire experiment adhered to non-invasive principles to minimize animal stress.

[0068] 1.4 Clinical score of arthritis in mice

[0069] Starting 21 days after the second immunization, the joints of the limbs of mice were systematically clinically evaluated 2-3 times per week using a modified Arthritis Index scoring system (0-4 points / limb, total score 0-16 points). Specific criteria were as follows: 0 points (no erythema or swelling in the joint); 1 point (erythema with mild swelling in a single finger / toe joint); 2 points (erythema with moderate swelling in the metatarsal or ankle joints, affecting ≥2 joints); 3 points (diffuse erythema from the ankle to the metatarsal joints with severe swelling and limited mobility); 4 points (severe erythema and swelling of the entire foot with joint ankylosis, complete loss of weight-bearing function). Data were collected by two independent observers in a double-blind scoring process (Kappa consistency coefficient >0.90) at a fixed time each day (09:00-11:00 AM) under standardized light conditions (500 lux). The final limb scores were calculated and subjected to repeated measures ANOVA.

[0070] 1.5 Mouse Rotor Test

[0071] Rotary head fatigue testing was conducted using a Ugo Basile 47650 rotary head apparatus. Adaptable mice were pre-trained before the experiment: during the pre-training phase (9:00-11:00 AM daily), a constant rotation speed of 15 rpm (± 0.5 rpm) was set for two consecutive days, with 15 minutes of adaptive training. Mice exhibiting jumping escape (limbs leaving the track surface for >2 seconds) or passive rotation (gliding along the axis for >5 seconds without resuming active gait) behaviors were excluded. Formal testing was conducted on day 54. Ten mice in each group were fasted for 6 hours and placed on a 3 cm diameter rotating track, tested in a linear acceleration mode (5→30 rpm / 5 minutes), with the ambient temperature controlled at 22 ± 1 °C.

[0072] 1.6 Organ Index Measurement

[0073] Twenty-four hours after the last administration, mice were euthanized by CO2 asphyxiation. The thymus, liver, kidneys, and spleen were rapidly and aseptically removed in a clean dissection table. The organs were rinsed in pre-cooled saline to remove residual blood, blotted dry with absorbent paper, and weighed using a precision analytical balance (Sartorius CPA225D, accuracy 0.1 mg). The final body weight of the mice was recorded simultaneously (accurate to 0.1 g). The organ index was calculated as: organ wet weight (mg) / animal body weight (g) × 100%. All procedures were performed at 4 °C on an ice tray to maintain tissue viability. Data were entered independently by two people and cross-validated. Dissection instruments were autoclaved, and the balance was calibrated daily using standard weights to ensure measurement accuracy.

[0074] 1.7 Complete Blood Count (CBC)

[0075] Whole blood samples were collected from mice into pre-cooled EDTA-K2 anticoagulant vacuum blood collection tubes (BD Microtainer).® (1.5 mg EDTA / mL blood), gently invert and mix 8-10 times, then immediately place in a 4 °C biosafety transport box and complete the test within 2 hours. The test was performed using a Sysmex XN-1000V fully automated blood analyzer, focusing on white blood cell parameters: total white blood cell count (WBC), lymphocyte count (LYM), monocyte count (MON), and granulocyte count.

[0076] 1.8 Histopathological analysis of mouse ankle joint tissue

[0077] After euthanasia, the ankle joints of the hind limbs of experimental mice were rapidly amputated and immediately immersed in 4% paraformaldehyde (pH 7.4) for fixation at room temperature for 24 hours. Subsequently, they were transferred to 10% EDTA decalcification solution (pH 7.2) for dynamic decalcification (the endpoint of decalcification was determined by the absence of resistance during needle puncture). After decalcification, the tissues were cleared using a gradient of ethanol and xylene, embedded in paraffin, and then prepared into 4 μm continuous sagittal sections using a Leica RM2255 rotary microtome. After drying, dewaxing, and rehydration, the sections were stained with hematoxylin-eosin (HE) and tartrate-resistant acid phosphatase (TRAP).

[0078] 1.9 Detection of cytokines in mouse synovial tissue

[0079] After the experimental mice were euthanized, synovial tissue from the ankle joint was quickly collected, and 100 mg of tissue was added to 350 μL of pre-cooled lysis buffer (containing 1% protease inhibitor Cocktail, Roche cOmplete). TM After mechanical homogenization on ice (Polytron PT 1200E, 15s × 3 intermittent cycles), lysis was performed for 1 hour (vortexing for 10 seconds every 5 minutes). The supernatant was collected by centrifugation at 16060 × g for 20 minutes at 4 °C. Protein concentration was determined using the BCA method and adjusted to 1 mg / mL. ELISA kits (IL-1β, IL-6, HIF-1α, TNF-α, INF-γ, IL-17) were used, and absorbance was measured at 450 nm using a microplate reader (BioTek Synergy H1) according to the manufacturer's instructions.

[0080] 1.10 Immunofluorescence detection

[0081] For immunofluorescence staining analysis of paraffin-embedded ankle tissue sections, dewaxing was first performed using a xylene gradient. Blocking with PBS containing 5% BSA and 0.3% Triton X-100 for 1 hour at room temperature was used to inhibit non-specific binding. Then, primary antibody working solutions targeting FasL, IL-17, CD30, CD31, and VEGF were added, and the sections were incubated overnight (12-16 hours) at 4 °C in a humidified chamber. The next day, the sections were washed three times (10 minutes each) with TBST (containing 0.05% Tween-20), and then incubated with Alexa Fluor 488 / 594-labeled secondary antibodies (1:1000 dilution) for the corresponding species for 1 hour at room temperature in the dark. After counterstaining the cell nuclei with DAPI (1 μg / mL) for 5 minutes, the sections were mounted with mounting medium containing the anti-fluorescence quencher (ProLong Diamond). Multichannel fluorescence images were acquired using a laser confocal microscope (excitation wavelengths: DAPI 405 nm, FITC 488 nm, TRITC 561 nm), and the fluorescence intensity was quantified using ImageJ.

[0082] 2. Experimental Results

[0083] 2.1 Effects of LMCS on mouse body weight

[0084] Clinical observations show that RA patients often experience limited mobility, emotional stress, and digestive disorders due to joint swelling and pain, leading to weight loss. Therefore, systematically monitoring the dynamic changes in mouse body weight in this animal experiment can serve as an indicator for assessing disease progression, metabolic status, and the effectiveness of interventions.

[0085] according to Figure 1 Dynamic analysis of body weight showed that in the early stages of the experiment (days 0-4), the control group mice experienced a stable weight gain (+0.6 g), while the model group showed a significant negative weight gain (-1.11 g), indicating that metabolic homeostasis was affected early in the induction of arthritis. By the second immunization stage (day 20), the normal group reached a weight of 18.8 g, while the model group decreased to 17.0 g. The intervention groups (LMCS-M, LMCSO, LMCSA, and LMCSB) had weights of 18.2, 18.0, 17.9, and 17.8 g, respectively, indicating that polysaccharide intervention could alleviate weight loss in RA mice. Notably, from the second immunization to the end of gavage (days 20-43), the intervention group's weight showed a "V-shaped" trend (first decreasing then increasing), with no significant difference between the intervention and normal groups by day 43; however, the model group continuously deviated from the recovery trajectory from day 28, ultimately resulting in a significantly lower weight than the intervention group. p<0.05). Comprehensive analysis showed that weight loss in RA mice was associated with disease progression, and that polysaccharide gavage intervention could effectively alleviate the effects of polysaccharide gavage on food intake and weight in arthritis mice. In particular, LMCS-M showed greater advantages in early protection, which may be attributed to its excellent activity in maintaining the intestinal physical and biological barriers.

[0086] 2.2 Effects of LMCS on organ indices in mice

[0087] Rheumatoid arthritis (RA), as a systemic autoimmune disease, is closely related to the functional state of immune organs. Immune organs regulate the body's immune homeostasis: the thymus, as a central immune organ, participates in immune regulation through T cells; the spleen, as the largest peripheral immune organ, participates in the clearance of immune complexes through mediating humoral and cellular immunity; and the liver, as a metabolic-immune interaction hub, regulates immune responses through multiple mechanisms, including innate immune responses, adaptive immune responses, immune tolerance, and metabolic regulation. The weight ratio of the thymus, liver, and spleen to body weight can reflect the overall immune function status of the body to some extent. Figure 2 The results showed that the thymus index and spleen index of the model group mice were significantly higher than those of the normal group. p <0.05 indicates the body's immune activation state, which may be related to abnormal lymphocyte activation. After polysaccharide intervention, the thymus, liver, and spleen indices in all treatment groups were significantly lower than those in the model group ( p <0.05), among which LMCS-M showed the best immunomodulatory effect, with its thymus index decreasing by 15.6% and spleen index decreasing by 18.7% compared with the LMCSB group.

[0088] 2.3 Effects of LMCS on mouse blood routine tests

[0089] A complete blood count (CBC) can reflect the degree of systemic inflammation. The results of a CBC in mice are as follows: Figure 3 As shown, the white blood cell level in the model group mice was approximately twice that of the normal group. p <0.05%, with monocytes and granulocytes increasing by approximately 3.5 and 4 times, respectively. Polysaccharide intervention significantly inhibited the abnormal increase in peripheral blood leukocyte subsets (including granulocytes, lymphocytes, and monocytes) in RA mice. p <0.05), with LMCS-M showing the most significant inhibitory effect, which is attributed to the strong anti-inflammatory activity of LMCS-M.

[0090] 2.4 Analysis of Mouse Motor Function

[0091] Typical clinical manifestations of rheumatoid arthritis (RA) include joint swelling, painful stiffness, and impaired weight-bearing function. The degree of joint motor function impairment and disease-related inflammation levels can be quantitatively assessed by measuring the duration of the rotarod test in RA mice. Figure 4As shown, the normal group mice could maintain movement for 567 ± 37 seconds in the rotarod experiment, while the model group could only maintain it for 86 ± 8 seconds. p <0.05). This result confirms that joint inflammation in the model group mice led to a significant decrease in motor coordination and limited weight-bearing behavior, resulting in a sharp shortening of rotarod time. In the model mice, polysaccharide intervention significantly prolonged the rotarod time in RA mice ( p <0.05%. Among them, the intervention effect of LMCS-M was particularly outstanding, with a rotator time of 330 ± 48 seconds, which was significantly better than other intervention groups ( p <0.05). This result demonstrates the excellent activity of LMCS-M in improving joint movement function and inhibiting synovial inflammation in RA mice.

[0092] 2.5 Observation of mouse joint morphology and appearance

[0093] like Figure 5 As shown, morphological observation of diseased joints allows for a direct assessment of the pathological progression of rheumatoid arthritis (RA). The ankle joints of normal mice showed a normal state, while RA mice exhibited significant pathological swelling. Notably, polysaccharide intervention significantly reduced joint swelling in RA mice, especially with LMCS-M intervention. These results morphologically confirm that small-molecule chondroitin sulfate can improve the joint pathology in arthritic mice.

[0094] 2.6 Analysis of Arthritis Index in Mice

[0095] Inflammatory cell infiltration within the joint cavity and pathological proliferation of the synovial tissue can cause morphological changes in the joint, manifesting as typical clinical symptoms such as redness, swelling, heat, pain, and joint deformity. By using a standardized joint scoring system (0-4 points, based on the degree of redness and swelling and limited mobility) combined with toe joint thickness measurement (accuracy ± 0.1 mm), the intensity and severity of synovial inflammation in RA mice can be monitored. Figure 6 , Figure 7 As shown, the changes in paw joint thickness and joint score curves in RA mice reveal the characteristics of disease progression. After the second immune challenge (D0), RA mice gradually developed typical symptoms such as joint swelling. The normal group maintained a joint score of 0 throughout the observation period (D21-D53), while the model group's score continuously increased from D0, reaching 7.83 ± 0.27 points by the end of D32, consistent with the developmental pattern of chronic inflammation in RA. p<0.05 vs. normal group). The polysaccharide intervention group showed an "upward-remission" change: the scores of each group reached a local peak on D16 and then decreased. The final scores of LMCS-M, LMCSO, LMCSA, and LMCSB were 2.57 ± 0.17, 3.31 ± 0.18 and 3.63 ± 0.17, 3.70 ± 0.20, respectively. Notably, the improvement in arthritis symptoms in the LMCS-M group was approximately 70% higher than that in the model group, significantly higher than other treatment groups ( p <0.05). This indicates that the LMCS-M group can significantly inhibit rheumatoid arthritis symptoms in RA mice, and their activity order is: LMCS-M group > LMCSO > LMCSA ≈ LMCSB. This shows that the structural differences among small molecule chondroitin sulfate determine their activity.

[0096] 2.7 Pathological analysis of mouse joints

[0097] H&E staining can effectively observe the lesions in the joints of RA mice. For example Figure 8 , Figure 9 As shown, the joints of mice in the normal group exhibited intact structures, clear articular surface contours, uniform articular cartilage thickness, and smooth surfaces, with no pathological hyperplasia observed in the synovial tissue. The model group mice, however, showed significant joint pathological changes: blurred joint anatomy and abnormal synovial tissue hyperplasia, with extensive inflammatory cell infiltration in localized areas; erosion of the articular cartilage layer with irregular surface structure, accompanied by bone destruction and neovascularization. The polysaccharide intervention group showed reduced synovial tissue hyperplasia compared to the model group, significantly inhibited immune cell infiltration, and clearly discernible bony structures of the articular surfaces. Pathological scores showed a reduction of approximately 24-55% in the intervention group compared to the model group. p <0.05%, among which, the intervention of LMCS-M was significantly better than other intervention groups ( p <0.05). Abnormal activation of osteoclasts is a major cause of bone erosion in rheumatoid arthritis (RA), and TRAP staining can effectively locate osteoclasts. TRAP staining analysis showed that no obvious osteoclasts were detected in the joint tissues of normal mice, while characteristic purplish-red staining was observed in the joints of RA mice, representing osteoclast formation (TRAP positive). Among them, osteoclast aggregation was most significant in the RA model group (marked by red arrows). After polysaccharide intervention, all polysaccharide groups showed an inhibitory effect on osteoclast formation, with LMCS-M intervention showing the best inhibitory effect. The results of HE and TRAP staining were consistent with the results of motor function and morphological indicators in RA mice.

[0098] 2.8 Effects of LMCS on Joint Immune Cell Content and Angiogenesis in RA Mice

[0099] Studies have shown that immune cells play a crucial role in the development of rheumatoid arthritis (RA). Immunofluorescence staining for FasL, CD31, and IL-17 can reflect the effect of LMCS on the number of Th1, Th2, and Th17 cells in the joints of RA mice. Figure 10 As shown, compared to the normal group, the model group exhibited immune cell aggregation. After polysaccharide intervention, all intervention groups effectively inhibited the abnormal accumulation of immune cells, with the LMCS-M group showing particularly outstanding results.

[0100] In rheumatoid arthritis (RA), angiogenesis not only promotes inflammatory cell infiltration but also exacerbates synovial hyperplasia, cartilage destruction, and bone loss. Vascular endothelial growth factor (VEGF), as a key regulator of angiogenesis, along with CD31 (an endothelial cell-specific transmembrane glycoprotein), can be used to characterize angiogenesis in joints. Figure 10 As shown, the joint tissues of the model group mice exhibited enrichment of VEGF and CD31, with their positive expression intensity upregulated compared to the normal group, indicating angiogenesis. The polysaccharide intervention group showed inhibition of the expression of these markers, with LMCS-M intervention showing the strongest inhibitory effect on angiogenesis.

[0101] 2.9 Effects of LMCS on inflammation-related cytokines in RA mice

[0102] The levels of key pro-inflammatory mediators in the synovial tissue of the ankle joint of RA mice were quantitatively determined using high-sensitivity ELISA. After normalizing total protein concentration using the BCA method, the expression of cytokines such as TNF-α, IL-1β, IL-6, IFN-γ, and IL-17 was systematically evaluated. As shown in Table 1, the levels of pro-inflammatory cytokines in the synovial tissue homogenate of RA model mice were significantly increased, with the concentrations of TNF-α, IL-1β, and IL-6 increasing by approximately 5, 2.9, and 3 times, respectively, compared to the normal group. p <0.05). After intervention with LMCS-M, LMCSO, LMCSA, and LMCSB, the inflammatory microenvironment of the synovium in RA mice was significantly improved, with a reduction of 30-60% (all values ​​not specified). p< 0.05 vs. model group). Notably, the intervention effect of LMCS-M was significantly better than that of other groups ( p <0.05).

[0103] IFN-γ and IL-17 can characterize the infiltration of Th1 / Th17 immune cells in joints, and their quantitative detection is of great significance for characterizing rheumatoid arthritis (RA). Abnormal activation of Th17 cells directly drives synovial inflammation and bone erosion through IL-17 secretion, while IFN-γ secreted by Th1 cells amplifies the autoimmune response by enhancing antigen presentation. In RA patients, the Th1 / Th17 balance is significantly shifted towards Th17, forming an inflammatory microenvironment that leads to destructive joint pathological progression. Therefore, the measurement of IFN-γ and IL-17 can serve as indicators of inflammatory activity in arthritis, used to evaluate disease severity and progression. As shown in Table 1, after intervention with LMCS-M, LMCSO, LMCSA, and LMCSB, IFN-γ and IL-17 levels in the synovium of model mice were significantly decreased (…). p <0.05), especially the effect of LMCS-M intervention, which shows that they have a significant advantage in improving the infiltration of Th1 / Th17 immune cells in the joint. This phenomenon indicates that LMCS-M improves the inflammatory microenvironment of the synovium by regulating the infiltration of Th1 and Th17 immune cells.

[0104] The hypoxic microenvironment of diseased joints in rheumatoid arthritis (RA) can lead to abnormal activation of hypoxia-inducible factor-1α (HIF-1α), promote the secretion of inflammatory factors (such as TNF-α and IL-1β), and drive pathological angiogenesis and abnormal proliferation of fibroblast-like synovial cells (FLS), thus forming a vicious cycle of "hypoxia-inflammation-tissue hyperplasia," which continuously exacerbates joint inflammation and destruction. As shown in Table 1, the HIF-1α content in the ankle synovial tissue of RA model mice was significantly increased to 32.81 ± 3.13 pg / mg total protein, which was approximately 2.8 times higher than that in the normal group (11.51 ± 1.38 pg / mg total protein). p <0.05). After intervention with LMCS-M, LMCSO, LMCSA, and LMCSB, the expression level of HIF-1α decreased to 14.02 ±1.74, 17.77 ±2.99 and 20.97 ± 2.21, 25.54 ±1.63 pg / mg total protein, respectively (all values ​​are 0.05). p< 0.05 vs model group). Notably, the regulatory effect of LMCS-M was significantly better than that of other intervention groups ( p <0.05).

[0105] Table 1. Effects of LMCS on cytokines in mouse ankle synovium

[0106]

[0107] Note: Data results are expressed as mean ± standard deviation (n = 6). Different letters in the same column indicate significant differences between groups (p < 0.05).

[0108] In summary, in a mouse model of arthritis, LMCS-M demonstrated superior overall improvement compared to single-component formulations across multiple dimensions. Specifically, LMCS-M significantly alleviated weight loss in RA mice, reduced thymus, liver, and spleen indices, inhibited abnormal increases in peripheral blood leukocytes (especially granulocytes and monocytes), improved motor coordination, and reduced joint swelling and joint scores. Histopathological analysis further confirmed that LMCS-M effectively inhibited synovial hyperplasia, immune cell infiltration, osteoclast activation, and pathological angiogenesis. Immunofluorescence and ELISA results showed that LMCS-M significantly downregulated the expression of local pro-inflammatory factors (TNF-α, IL-1β, IL-6, IFN-γ, IL-17) and HIF-1α in the joint, and inhibited the positive expression of immune and vascular-related markers such as FasL, CD31, and VEGF. In summary, LMCS-M demonstrates the potential to comprehensively improve the pathological process of rheumatoid arthritis by synergistically regulating inflammation, immunity, and angiogenesis through multiple targets and pathways, and its activity is significantly superior to that of single degradation products.

[0109] Furthermore, during the experimental process, the present invention also used the clinical score of arthritis as an indicator to conduct experiments on other formulations. The preparation methods of LMCSO, LMCSA, and LMCSB were all in accordance with Example 1, and the results are as follows: Figure 11 As shown. According to Figure 11It is evident that different compounding methods exhibit significant differences in their intervention effects on RA mice. The figure shows that, among the pairwise compounding groups, LMCSO-LMCSA (i.e., LMCSO and LMCSA in a 1:1 ratio) was more effective than LMCSO-LMCSB (LMCSO and LMCSB in a 1:1 ratio), and the improvement effect of LMCSO-LMCSA even surpassed that of the equal-ratio ternary compounding group LMCSM1 (1:1:1). This indicates that the combination of LMCSO and LMCSA has a stronger synergistic effect in anti-inflammation and cartilage protection, while the addition of LMCSB, if inappropriately proportioned, may weaken this synergy. However, after further adjustment of the ratio, the effect of LMCSM2 (2:1:1) was further enhanced, surpassing LMCSO-LMCSA, indicating that increasing the proportion of LMCSO helps to enhance overall activity. The 6:3:1 ratio group specified in this invention showed the most outstanding effect among all compounding combinations, significantly superior to any other ratio, including the relatively effective LMCSO-LMCSA and LMCSM2. This result confirms that optimal multi-target synergy can only be achieved and a comprehensive approach to the complex pathological process of arthritis be taken when the three products are combined in a specific ratio of 6:3:1. Therefore, the ratio specified in this invention is the most effective combination.

[0110] Example 2:

[0111] The preparation of the small molecule chondroitin sulfate complex LMCS-M3 followed the same steps as in Example 1, except that multi-stage stepped microwave heating and real-time temperature feedback control were not used in the preparation of LMCSB. Instead, a traditional constant-temperature oil bath heating method was employed for the alkaline degradation reaction. Specifically, the reaction solution prepared in Example 1 was directly placed in a constant-temperature oil bath, the reaction temperature was set to 60°C, and the reaction was carried out at a constant temperature for 60 minutes with a stirring speed of 150 rpm. The product was designated as LMCSB-Y. Its weight-average molecular weight was 8.9 kDa, number-average molecular weight was 6.2 kDa, and molecular weight distribution coefficient was 1.44.

[0112] Example 3:

[0113] The preparation of the small molecule chondroitin sulfate complex LMCS-M4 follows roughly the same steps as in Example 1. The difference is that, in the preparation of LMCSB, a phase transfer catalyst and microwave assistance are not used; instead, a traditional alkaline degradation method is employed.

[0114] The specific steps are as follows: Chondroitin sulfate was dissolved in 0.5 M NaOH solution to prepare a chondroitin sulfate alkaline solution with a final concentration of 20 mg / mL. The mixture was placed in a constant temperature oil bath, and the reaction temperature was set to 60℃. The reaction was carried out at a constant temperature for 60 minutes with a stirring speed of 150 rpm. After the reaction was completed, the pH was adjusted to 7.0 with HCl to terminate the reaction. The reaction solution was filtered through a 0.22 μm filter membrane, and the fraction with a molecular weight less than 10 kDa was collected by tangential flow ultrafiltration. After concentration, dialyzing, and lyophilization, the LMCSB product was obtained, denoted as LMCSB-T. The weight-average molecular weight was 10.2 kDa, the number-average molecular weight was 5.8 kDa, and the molecular weight distribution coefficient was 1.76.

[0115] Example 4:

[0116] The preparation of the small molecule chondroitin sulfate complex LMCS-M5 follows roughly the same steps as in Example 1. The difference is that mannitol is not added as a protective agent when preparing LMCSA, and the programmed temperature rise and segmented temperature holding process is not used. Instead, the traditional isothermal acid degradation method is adopted.

[0117] The specific steps are as follows: Chondroitin sulfate is dissolved in deionized water, and HCl solution is added to make the final concentration of chondroitin sulfate 20 mg / mL and the final concentration of HCl 0.3 M. The mixture is placed in a constant temperature oil bath, the reaction temperature is set to 65℃, and the reaction is carried out at a constant temperature for 8 hours with a stirring speed of 150 rpm. After the reaction is completed, it is immediately cooled to below 25℃ in an ice bath, and the pH is adjusted to 7.0 with NaOH to terminate the reaction. The reaction solution is filtered through a 0.22 μm filter membrane, and the fraction with a molecular weight less than 10 kDa is collected by tangential flow ultrafiltration. After concentration, dialyzing, and freeze-drying, the LMCSA product is obtained, denoted as LMCSA-C. The weight-average molecular weight is 9.3 kDa, the number-average molecular weight is 5.5 kDa, and the molecular weight distribution coefficient is 1.69.

[0118] Experimental Example 2: Effects of different preparation methods on the efficacy of small molecule chondroitin sulfate complex in improving RA

[0119] 1. Experimental Methods

[0120] Referring to the RA mouse modeling and grouping method in Example 1, the successfully modeled mice were randomly divided into the following groups:

[0121] Model group (physiological saline)

[0122] Example 1 Composition Group (LMCS-M, composed of LMCSO, LMCSA, and LMCSB in a ratio of 6:3:1)

[0123] Example 2 Composition Group (LMCS-M3, composed of LMCSO, LMCSA, and LMCSB-Y in a ratio of 6:3:1)

[0124] Example 3 Composition Group (LMCS-M4, composed of LMCSO, LMCSA, and LMCSB-T in a ratio of 6:3:1)

[0125] Example 4 Composition Group (LMCS-M5, composed of LMCSO, LMCSA-C, and LMCSB in a ratio of 6:3:1)

[0126] Each group consisted of 12 mice, with a separate normal control group (saline). Starting 21 days after the second immunization, mice were administered the drug via gavage daily at a dose of 150 mg / kg / day for 21 consecutive days. Observational indicators included joint score, toe joint thickness, and rotarod time.

[0127] 2. Experimental Results

[0128] 2.1 Impact on joint scores

[0129] The results are as follows Figure 12 As shown, the joint scores of mice in the Example 1 composition (LMCS-M) intervention group were significantly lower than those in Example 2, 3, and 4 groups (p<0.05), and closer to the normal group level. Although the Example 2 group (LMCS-M3) was better than the model group, its effect was significantly weaker than that of the Example 1 group, indicating that the LMCSB-Y prepared by traditional oil bath heating had low activity. The Example 3 group (LMCS-M4) showed the worst improvement, with its joint score decreasing by only about 30%, indicating that the LMCSB-T prepared by the traditional NaOH alkaline degradation method suffered severe structural damage and a significant decrease in activity. The Example 4 group (LMCS-M5) showed an effect between that of Example 2 and Example 3, indicating that the lack of mannitol protection and the poor structural integrity of the acid degradation products due to segmented temperature control affected the overall synergistic effect of the composition.

[0130] 2.2 Effect on Rotation Time

[0131] The results of the rotator experiment are as follows Figure 13 As shown, the mice in Example 1 had the longest duration of movement, which was significantly better than those in Example 2, Example 3 and Example 4 (p<0.05).

[0132] In summary, different preparation methods significantly affect the structure and molecular weight distribution of small molecule chondroitin sulfate, thereby influencing its bioactivity in improving arthritis. The LMCSB (multi-stage stepped microwave heating + phase transfer catalyst) and LMCSA (mannitol protection + programmed temperature rise and segmented holding) preparation methods specified in this invention can obtain small molecule chondroitin sulfate with higher activity. When the three are combined in a 6:3:1 ratio, they exhibit the best synergistic effect in improving RA.

[0133] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the small molecule chondroitin sulfate complex for the interventional treatment of arthritis according to the present invention will be readily apparent to those skilled in the art.

[0134] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A small molecule chondroitin sulfate complex for the intervention and treatment of joint inflammation, characterized in that, It is composed of oxidative degradation product LMCSO, acid degradation product LMCSA, and alkaline degradation product LMCSB in a weight ratio of 6:3:1, wherein, The number-average molecular weights of LMCSO, LMCSA, and LMCSB are all less than 10 kDa; The LMCSO is prepared by the following steps: copper acetate is added to a 7-12 mg / mL chondroitin sulfate solution and mixed well. Then, under stirring, H2O2 solution is slowly added dropwise over 15-20 minutes. The final concentration of H2O2 is 60 mM, and the final concentration of copper acetate is 0.65±0.05 mM. After the H2O2 solution is added, the mixture is first reacted at 45±1℃ for 50-60 minutes, and then the temperature is raised to 50±1℃ to continue the reaction for 50-60 minutes. The reaction pH is 5.5-9.

0. The LMCSA is prepared by a method comprising the following steps: dissolving chondroitin sulfate in water, adding mannitol as a protective agent, the final concentration of mannitol being 0.05-0.1 M, and then mixing it with HCl solution to make the final concentration of chondroitin sulfate solution 15-25 mg / mL and the final concentration of HCl solution 0.1-0.5 M; gradually heating the mixture to 65±2℃ at a heating rate of 0.5-1.0℃ / min, and holding it at 45±1℃ and 55±1℃ for 30-45 minutes respectively during the heating process, the total reaction time being 480-500 minutes; The LMCSB is prepared by a method comprising the following steps: dissolving chondroitin sulfate in water, adding 0.5%-1% (by mass of chondroitin sulfate) of hexadecyltrimethylammonium bromide (CTAB) as a phase transfer catalyst, and stirring until homogeneous; then adding a borate-sodium hydroxide buffer solution to make the final concentration of chondroitin sulfate in the reaction system 15-25 mg / mL, the final concentration of the buffer solution 0.1-0.3 M (based on borate), and the pH value 8.5-9.5; placing the mixture in a microwave reactor for alkaline degradation reaction at a reaction temperature of 40-70℃.

2. The small molecule chondroitin sulfate complex for the interventional treatment of joint inflammation as described in claim 1, characterized in that, The preparation of LMCSO specifically includes: after the reaction is complete, adjusting the pH to 9.0, letting it stand for 30-60 minutes, and then centrifuging to remove Cu. 2+ The supernatant was filtered through a 0.22 μm filter membrane and then subjected to tangential flow ultrafiltration to collect the fraction with a relative molecular mass less than 10 kDa. After concentration, dialysis, and freeze-drying, the product was obtained.

3. The small molecule chondroitin sulfate complex for the interventional treatment of joint inflammation as described in claim 1, characterized in that, The preparation of LMCSA specifically includes: after the reaction is completed, the reaction vessel is immediately placed in an ice-water bath, the system temperature is reduced to below 25°C within 2 minutes, the pH is adjusted to 7.0 to terminate the reaction, and after filtration through a 0.22 μm filter membrane, tangential flow ultrafiltration is used to collect the portion with a relative molecular mass less than 10 kDa, which is then concentrated, dialyzed, and freeze-dried to obtain the final product.

4. The small molecule chondroitin sulfate complex for the interventional treatment of joint inflammation as described in claim 1, characterized in that, The preparation of LMCSB specifically includes: the alkaline degradation reaction is carried out under stirring conditions, using a multi-stage stepped temperature control mode: the first stage is heated to 45±1℃ at 300-400W power and kept at a constant temperature for 10-15 minutes; the second stage is heated to 55±1℃ at 400-500W power and kept at a constant temperature for 15-20 minutes; the third stage is heated to 65±1℃ at 500-600W power and kept at a constant temperature for 5-10 minutes; during the reaction, the microwave output power is dynamically adjusted through real-time temperature monitoring; after the reaction is completed, the pH value is adjusted back to 7.0, and the mixture is allowed to stand for 30 minutes to allow CTAB to precipitate, which is then removed by centrifugation; the supernatant is filtered through a 0.22 μm filter membrane, and then subjected to tangential flow ultrafiltration to collect the fraction with a relative molecular mass less than 10 kDa, which is then concentrated, dialyzed, and freeze-dried to obtain the final product.

5. The use of the small molecule chondroitin sulfate complex for the interventional treatment of joint inflammation as described in any one of claims 1-4 in the preparation of a medicament for improving arthritis.

6. The application as described in claim 5, characterized in that, The drug is used to reduce joint inflammation scores, reduce joint swelling, improve exercise capacity, reduce inflammatory factor levels, inhibit immune cell infiltration, reduce oxidative stress, or regulate one or more of the following organ indices:

7. A pharmaceutical composition, characterized in that, It comprises the small molecule chondroitin sulfate complex for the interventional treatment of joint inflammation as described in any one of claims 1-4 and a pharmaceutically acceptable carrier.

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