Glucosamine chondroitin sulfate pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention provides a glucosamine and chondroitin sulfate pharmaceutical composition as well as a preparation method and application thereof, and belongs to the technical field of biological pharmaceutical preparations, the composition comprises sodium chondroitin sulfate, glucosamine sulfate, dimethyl sulfone, non-denatured type II collagen, frankincense extract and auxiliary materials in specific parts by mass, compared with the prior art, the composition provided by the invention has the advantages that by selecting the components, adding the composite synergist optionally and optimizing the ratio and synergistic effect of the components, the effects of the composition in promoting cartilage regeneration and inhibiting joint inflammation are remarkably improved, the anti-inflammatory and anti-oxidation effects are enhanced, and the bioavailability is also improved, so that a breakthrough progress is realized in the field of joint health.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical preparations, and in particular relates to a glucosamine chondroitin sulfate pharmaceutical composition, a preparation method thereof, and uses thereof. Background Art

[0002] Bone and joint diseases such as osteoarthritis and rheumatoid arthritis are common chronic diseases worldwide. Their pathological characteristics are mainly cartilage degeneration, joint inflammation, and pain, which seriously affect patients' quality of life. Currently, clinical treatment mainly relies on non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids. However, long-term use of these drugs can easily cause gastrointestinal side effects and metabolic abnormalities, and they cannot fundamentally repair cartilage damage.

[0003] Glucosamine and chondroitin sulfate are widely used as cartilage protectors, but their low bioavailability and limited efficacy as individual ingredients make it difficult to meet clinical needs. In recent years, research has attempted to improve efficacy through compound preparations, such as combining glucosamine with chondroitin sulfate. However, existing technologies lack scientific optimization of component ratios, resulting in insufficient synergistic effects and crude preparation processes, resulting in poor drug stability and uncontrolled release. Furthermore, traditional preparations struggle to achieve both anti-inflammatory and cartilage regeneration effects.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The first purpose of the present invention is to provide a glucosamine sulfate chondroitin pharmaceutical composition, which includes sodium chondroitin sulfate, glucosamine sulfate, dimethyl sulfone, non-denatured type II collagen, frankincense extract and excipients in specific mass fractions, and optionally adds a composite synergist and optimizes the ratio and synergistic effect of each component, significantly improving the efficacy of the composition in promoting cartilage regeneration and inhibiting joint inflammation, not only enhancing the anti-inflammatory and antioxidant effects, but also improving the bioavailability, thereby achieving a breakthrough in the field of joint health.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned glucosamine sulfate chondroitin pharmaceutical composition. The preparation method is simple in process and easy to industrialize. The resulting product has the characteristics of high stability and significant therapeutic effect. It is suitable for the preparation of biological drugs and provides a new solution for the treatment of diseases such as osteoarthritis.

[0007] The third object of the present invention is to provide the use of the above-mentioned glucosamine chondroitin sulfate pharmaceutical composition in the preparation of a biological drug that promotes cartilage regeneration and inhibits joint inflammation.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted: A glucosamine chondroitin sulfate pharmaceutical composition, comprising the following raw materials in parts by mass: 30-45 parts of sodium chondroitin sulfate; 20-25 parts of glucosamine sulfate; 4.5-20 parts of dimethyl sulfone; 3-10 parts of non-denatured type II collagen; 1-5 parts of frankincense extract; 4-26 parts of auxiliary materials.

[0009] The present invention provides a glucosamine chondroitin sulfate pharmaceutical composition, which comprises the following raw materials: 30-45 parts of chondroitin sulfate sodium, 20-25 parts of glucosamine sulfate, 5-15 parts of dimethyl sulfone, 3-10 parts of non-denatured type II collagen, 1-5 parts of frankincense extract, and 4-26 parts of auxiliary materials. The present invention achieves the dual effects of promoting cartilage regeneration and inhibiting joint inflammation through the specific ratio of these raw materials and the synergistic effect of multiple active ingredients, while solving the problems of limited efficacy and low bioavailability of a single ingredient in the prior art. First, sodium chondroitin sulfate, as an important component of cartilage matrix, can stimulate chondrocyte proliferation and promote the synthesis of proteoglycans. However, its bioavailability is low when used alone, and it is easily degraded under inflammatory conditions. Therefore, the present invention combines it with glucosamine sulfate, which not only improves the stability of chondroitin sulfate, but also further enhances the cartilage repair ability through the supplementation of glucosamine. Glucosamine sulfate, as a precursor of amino sugars, can promote the synthesis of cartilage matrix. The present invention prefers glucosamine sulfate, which has better solubility and biological activity than other salt forms, thereby improving the overall therapeutic effect. Dimethyl Sulfone, as an organic sulfur compound, can not only inhibit the release of inflammatory mediators, but also promote the cross-linking of collagen, thereby enhancing the mechanical strength of cartilage; at the same time, the addition of non-denatured type II collagen further enhances the cartilage repair effect. By simulating the structure of natural cartilage matrix, it provides a good growth microenvironment for chondrocytes, induces immune tolerance, and reduces joint inflammatory reactions. The non-denatured type II collagen used in the present invention is derived from chicken sternal cartilage; frankincense extract, with its rich active ingredients such as terpenoids and boswellic acids, significantly inhibits the production of pro-inflammatory cytokines, thereby alleviating joint pain and swelling.

[0010] Therefore, the composition of the present invention significantly outperforms existing single-ingredient or simple compound preparations through the synergistic effects of multiple components and multiple targets. For example, the combination of sodium chondroitin sulfate and glucosamine sulfate simultaneously promotes the synthesis of cartilage matrix and inhibits its degradation, while dimethyl sulfone and non-denatured type II collagen further enhance the mechanical strength and anti-inflammatory properties of cartilage. The addition of frankincense extract not only compensates for the anti-inflammatory shortcomings of traditional glucosamine and chondroitin sulfate, but also addresses their poor water solubility through nanomicelle technology.

[0011] Preferably, as a further specific embodiment, it further comprises a compound synergist, wherein the compound synergist is eggshell membrane powder, N-acetylcysteine, phosphatidylserine and marine source keratan sulfate.

[0012] The present invention also introduces a composite synergist and defines its specific components as eggshell membrane powder, N-acetylcysteine, phosphatidylserine and marine keratan sulfate, thereby enhancing the bioavailability or pharmacological activity of the main components through the combination and synergistic effect of the specific components, thereby improving the overall efficacy of the composition in promoting cartilage regeneration and inhibiting joint inflammation. The addition of eggshell membrane powder may be based on its naturally rich collagen, hyaluronic acid and growth factors. These components complement non-denatured type II collagen and jointly stimulate chondrocyte proliferation and extracellular matrix synthesis. The specific protein contained in the eggshell membrane powder also has anti-inflammatory properties and can inhibit the release of pro-inflammatory cytokines in the synovial membrane of the joint, thereby synergizing with the anti-inflammatory effect of the frankincense extract. N-acetylcysteine, as an antioxidant, directly removes reactive oxygen free radicals in the joint cavity and reduces the damage to cartilage caused by oxidative stress. In addition, it can inhibit the transcription of inflammatory mediators by regulating the NF-κB signaling pathway, and cooperates with the anti-inflammatory effect of dimethyl sulfone. The inflammatory mechanism forms an additive effect; phosphatidylserine, as a cell membrane phospholipid component, can reduce the damage of chronic inflammation to cartilage by regulating macrophage polarization and inhibiting Toll-like receptor signaling. The synergistic effect with frankincense extract can also optimize the combined inhibitory effect of the two on inflammatory factors. This is because the acetyl-11-keto-β-boswellic acid in frankincense extract targets 5-lipoxygenase, while phosphatidylserine acts on the immune cell activation pathway further upstream. In addition, phosphatidylserine may also promote the transmembrane transport of other active ingredients by enhancing cell membrane fluidity, thereby indirectly improving bioavailability; among them, marine-derived keratan sulfate acts as a composite enhancer, forming a synergistic effect with sodium chondroitin sulfate. The unique sulfonation pattern of marine-derived keratan sulfate may provide additional charge-dependent growth factor binding sites, prolong the local action time, and form a polyanion network with sodium chondroitin sulfate, which reduces protein deposition in the joint cavity through charge repulsion, thereby delaying the progression of osteoarthritis.

[0013] Preferably, as a further specific embodiment, the mass ratio of N-acetylcysteine ​​to dimethyl sulfone is 1:(3-6).

[0014] The present invention also clearly defines the mass ratio of N-acetylcysteine, a key component of the composite synergist, to dimethyl sulfone, stipulating that the ratio is 1:(3-6). From the perspective of molecular mechanism, the effects of N-acetylcysteine ​​and dimethyl sulfone in joint inflammation and cartilage repair are complementary. As a classic antioxidant and precursor, N-acetylcysteine ​​can significantly increase the level of intracellular glutathione, directly neutralize excessive reactive oxygen free radicals in synovial fluid, and thus reduce the damage of oxidative stress to chondrocytes. At the same time, N-acetylcysteine ​​can also indirectly protect the cartilage matrix from excessive degradation by inhibiting the NF-κB signaling pathway and downregulating the expression of inflammatory factors. Dimethyl sulfone mainly exerts its effect due to its characteristics as an organic sulfur donor, and sulfur is an important component of the cartilage matrix. Dimethyl sulfone is a key component for cross-linking proteoglycans and collagen fibers. Supplementation with dimethyl sulfone helps maintain the mechanical properties and elasticity of cartilage. In addition, dimethyl sulfone itself has a mild anti-inflammatory effect and may reduce the synthesis of prostaglandins by regulating COX-2 activity. The present invention limits the mass ratio of the two to 1: (3-6). This is because excessive N-acetylcysteine ​​may lead to an excessively strong reducing environment in the cell, which in turn interferes with the normal metabolism and integration of sulfur in dimethyl sulfone. Dimethyl sulfone can only provide a sufficient sulfur source to support cartilage repair at a higher dose. Therefore, when the mass ratio of the two is lower than 1:3, the antioxidant effect is strong but the sulfur supply is insufficient; when it is higher than 1:6, it may cause mild gastrointestinal irritation due to excessive sulfur.

[0015] More importantly, controlling the ratio of the two at 1:(3-6) can ensure that N-acetylcysteine ​​quickly exerts its antioxidant and anti-inflammatory effects in the early stage after administration, while dimethyl sulfone continues to provide sulfur elements in the subsequent stage, forming a temporal synergistic effect; in addition, N-acetylcysteine ​​may cause mild irritation to the gastric mucosa at high doses, while dimethyl sulfone has a certain gastric protective effect. Therefore, appropriately increasing the ratio of dimethyl sulfone can help alleviate the potential side effects of N-acetylcysteine ​​and improve patients' medication tolerance; in addition, the free thiol group in the N-acetylcysteine ​​molecule has high reactivity and may interact with the sulfonyl group of dimethyl sulfone under certain conditions, affecting the stability of both. Therefore, the present invention avoids unnecessary side reactions between the two at high concentrations by controlling the ratio at 1:(3-6), thereby ensuring the stability of the ingredients of the preparation during storage.

[0016] Preferably, as a further specific embodiment, the mass ratio of the phosphatidylserine to the frankincense extract is 1:(2-5).

[0017] The present invention also clearly stipulates the mass ratio of phosphatidylserine to frankincense extract in the composite synergist, limiting the ratio to 1: (2-5). Phosphatidylserine is a naturally occurring phospholipid component. In the context of joint inflammation, the overactivation of macrophages and their polarization to the pro-inflammatory M1 phenotype are key factors leading to cartilage degradation. PS can activate downstream signaling pathways by binding to specific receptors on the surface of macrophages, prompting macrophages to transform to the anti-inflammatory M2 phenotype, thereby reducing the release of pro-inflammatory factors and increasing the secretion of anti-inflammatory factors. In addition, PS can further downregulate the inflammatory response by inhibiting the NF-κB and MAPK signaling pathways. The frankincense extract, especially its active ingredient acetyl-11-keto-β-frankincense, can effectively inhibit the inflammatory response of frankincense. Acid directly targets 5-lipoxygenase and cyclooxygenase-2, blocking the arachidonic acid metabolic pathway and reducing the production of inflammatory mediators such as leukotrienes and prostaglandins. The present invention limits the mass ratio between the two to 1:(2-5). This is because frankincense extract, as a direct small molecule inhibitor, has a dose-dependent anti-inflammatory effect and requires a higher concentration to effectively cover the key nodes of the inflammatory pathway. PS, as an immunomodulator, acts more like "signal regulation" and can trigger cell phenotypic conversion at a lower dose. Therefore, when the ratio of PS to frankincense extract is lower than 1:2, the immunomodulatory effect is insufficient and macrophage activation cannot be fully inhibited. When it is higher than 1:5, excessive frankincense extract may cause gastrointestinal irritation or other side effects.

[0018] In addition, limiting the mass ratio of the two to 1:(2-5) can also form a "fast and slow combination" mode of action. Among them, PS, as a phospholipid, is dependent on the emulsification of bile acids for absorption. After entering the body, it is mainly integrated into the cell membrane, exerting a long-lasting regulatory effect; while the triterpenoid components in frankincense extract are absorbed by passive diffusion, with a faster onset but a shorter half-life. Controlling the ratio of the two to 1:(2-5) allows frankincense extract to quickly suppress acute inflammatory responses after administration, while PS continuously regulates the immune microenvironment and prolongs the duration of the anti-inflammatory effect. In addition, PS may also promote the transmembrane transport of hydrophobic components in frankincense extract by enhancing cell membrane fluidity, thereby improving its bioavailability.

[0019] Preferably, as a further specific embodiment, the mass ratio of the marine-derived keratan sulfate to sodium chondroitin sulfate is 1:(5-10).

[0020] The application also specifies the mass ratio of marine keratan sulfate and sodium chondroitin sulfate in the composite synergist, which is limited to 1:(5-10). Marine keratan sulfate and sodium chondroitin sulfate are two important glycosaminoglycans naturally present in cartilage tissue, which play a key role in maintaining the elasticity and compression resistance of cartilage. Sodium chondroitin sulfate, as one of the main components of the cartilage matrix, combines with water molecules through its negatively charged sulfate groups to form a hydrated gel structure, providing mechanical support and cushioning capacity for cartilage. Marine keratan sulfate has a unique sulfonation pattern and molecular structure, which can form a tighter cross-linking with the collagen fiber network, enhancing the structural stability of the cartilage matrix. Therefore, the combination of marine keratan sulfate and sodium chondroitin sulfate has multiple complementary effects. Sodium chondroitin sulfate mainly attracts cations and water through its high negative charge characteristics, forming an inflation pressure to resist mechanical stress, while marine keratan sulfate binds to specific sites on the collagen fiber through its special disaccharide repeating unit, stabilizing the collagen network structure. When the ratio of marine keratan sulfate to sodium chondroitin sulfate is 1:(5-10), the hydrated gel formed by sodium chondroitin sulfate provides an ideal operating environment for marine keratan sulfate, which prevents the collapse of the gel structure by enhancing collagen cross-linking. When the ratio is less than 1:5, the relative excess of marine keratan sulfate may cause the gel structure to be too dense, affecting the diffusion of nutrients. When the ratio is higher than 1:10, it may not provide enough support for collagen cross-linking, reducing the protective effect on cartilage degradation. In addition, when the ratio of the two is controlled between 1:(5-10), their interaction in the intestinal tract can be optimized. Sodium chondroitin sulfate may slow down the premature degradation of marine keratan sulfate by competitively inhibiting the activity of certain glycosidases, extending its absorption time window. At the same time, marine keratan sulfate can also promote the absorption of sodium chondroitin sulfate. This mutual adjustment effect enables the composition to maintain a more stable blood drug concentration and improve the efficiency of targeted delivery to joint tissue.

[0021] Preferably, as a further specific embodiment, the mass ratio of eggshell membrane powder to non-denatured type II collagen is 1:(0.5-1.5).

[0022] The application also clearly specifies the mass ratio of eggshell membrane powder and non-denatured type II collagen in the composite synergist, and limits the ratio to 1:(0.5-1.5). The non-denatured type II collagen, as the main component of the cartilage matrix, retains the characteristic antigen epitope of natural collagen in a three-dimensional spatial conformation, can regulate the abnormal immune attack of the body on the cartilage through the immune tolerance mechanism of the intestinal mucosa after oral administration, and the eggshell membrane powder, as the natural membrane structure of the inner layer of the eggshell of poultry, contains a unique collagen combination and various growth factors and structural proteins. In the embryonic cartilage formation stage, there is a specific ratio relationship between type II collagen and type I collagen, and in the cartilage repair process, type X collagen, as a marker of hypertrophic chondrocytes, also appears temporarily. The ratio of 1:(0.5-1.5) between the two is most conducive to the reconstruction of this physiological collagen combination. The non-denatured type II collagen provides a cartilage-specific microenvironment, the type I collagen in the eggshell membrane powder promotes the formation of an initial scaffold of the repaired tissue, and the type X collagen may regulate the terminal differentiation process of chondrocytes. When the ratio is lower than 1:0.5, the deficiency of the eggshell membrane powder may lead to insufficient formation of a fibrous scaffold during the repair process. When the ratio is higher than 1:1.5, the non-denatured type II collagen may be relatively excessive and inhibit the necessary reparative remodeling.

[0023] Preferably, as a further specific embodiment, the adjuvant comprises microcrystalline cellulose, maltodextrin, povidone K30, magnesium stearate and enteric film coating premix.

[0024] The application also provides a preparation method of the above-mentioned glucosamine chondroitin sulfate pharmaceutical composition, comprising the following steps: The sodium chondroitin sulfate, glucosamine sulfate, dimethyl sulfone, non-denatured type II collagen, frankincense extract and microcrystalline cellulose and maltodextrin in the adjuvant are premixed, and sieved through an 80-100 mesh sieve to obtain a mixed powder A; The eggshell membrane powder, N-acetylcysteine, phosphatidylserine and marine source keratan sulfate are added to the mixed powder A in proportion, and uniformly mixed to obtain a mixed powder B; The mixed powder B is granulated with a povidone K30 solution by a wet method, and after drying, magnesium stearate is added and uniformly mixed, and then tableted, and then coated with an enteric film coating premix to obtain the glucosamine chondroitin sulfate pharmaceutical composition.

[0025] The application also provides the use of the above-mentioned glucosamine chondroitin sulfate pharmaceutical composition in the preparation of biological drugs for promoting cartilage regeneration and inhibiting joint inflammation.

[0026] Compared with the prior art, the application has the following beneficial effects: (1) The present invention provides a glucosamine sulfate chondroitin pharmaceutical composition, which comprises sodium chondroitin sulfate, glucosamine sulfate, dimethyl sulfone, non-denatured type II collagen, frankincense extract and excipients in specific mass fractions, and optionally adds a composite synergist and optimizes the ratio and synergistic effect of each component, thereby significantly improving the efficacy of the composition in promoting cartilage regeneration and inhibiting joint inflammation, not only enhancing the anti-inflammatory and antioxidant effects, but also improving the bioavailability, thereby achieving a breakthrough in the field of joint health.

[0027] (2) The present invention provides a method for preparing the above-mentioned glucosamine sulfate chondroitin pharmaceutical composition. The preparation method is simple in process and easy to industrialize. The obtained product has the characteristics of high stability and significant therapeutic effect. It is suitable for the preparation of biological drugs and provides a new solution for the treatment of diseases such as osteoarthritis.

[0028] (3) The present invention also provides the use of the above-mentioned glucosamine chondroitin sulfate pharmaceutical composition in the preparation of a biological drug that promotes cartilage regeneration and inhibits joint inflammation. DETAILED DESCRIPTION

[0029] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, those skilled in the art will understand that the embodiments described below are part of the embodiments of the present invention, not all of them, and are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] In order to more clearly illustrate the technical solutions of the present invention, specific embodiments are provided below for illustration.

[0031] Example 1: 1. Raw material pretreatment and weighing: In a clean environment, use a calibrated electronic balance (accuracy 0.01g) to weigh: 30 g of sodium chondroitin sulfate was sieved through a vibrating sieve (80 mesh) and placed in a dry polyethylene bag; 20 g of glucosamine sulfate, dried in a vacuum oven at 40°C for 4 hours and used immediately; 4.5 g of dimethyl sulfone was sterile filtered through a 0.22 μm polyethersulfone filter membrane and transferred to an amber glass bottle; 3 g of non-denatured type II collagen (purchased from Sigma-Aldrich) was thawed at 4°C and centrifuged at 3000 rpm for 10 min; Frankincense extract (purchased from Shaanxi Jiahe Biotechnology Co., Ltd.) 1g; Excipients (microcrystalline cellulose 1.5g + maltodextrin 1.5g + magnesium stearate 0.3g + enteric film coating premix 0.2g + povidone K30 0.5g) 4g; Eggshell membrane powder (purchased from Biova) 6g; N-acetylcysteine ​​(purchased from Zhejiang Xinhecheng) 1.5g; Phosphatidylserine (purchased from ChemiNutra) 0.5g; Marine keratan sulfate (purchased from Marine Biopolymers) 6g; Fluidized bed granulation: Fluidized bed equipment: Glatt GPCG-1 fluidized bed granulation system (equipped with Wurster bottom spray device); Parameter settings: The inlet air temperature is set to 50°C; Spray system: dual-fluid nozzle (aperture 1.0mm), atomization pressure 0.8bar Adhesive: 8% povidone K30 ethanol solution (w / v), spray rate 5.0 mL / min; 2. Operation process: Premixing and sieving (mixed powder A) Sodium chondroitin sulfate, glucosamine sulfate, dimethyl sulfone, non-denatured type II collagen, frankincense extract, microcrystalline cellulose, and maltodextrin were added to a V-blender at 20 rpm for 15 min. After mixing evenly, sieve with an 80-mesh stainless steel sieve to ensure that there is no agglomeration to obtain mixed powder A; 3. Addition of compound synergist (mixed powder B) Eggshell membrane powder, N-acetylcysteine, phosphatidylserine, and marine keratan sulfate were added to mixed powder A and mixed in a V-type mixer for 10 minutes to ensure uniform dispersion to obtain mixed powder B. 4. Wet granulation Dissolve povidone K30 in purified water to prepare a 5% binder solution. Place the mixed powder B into a high-speed shear wet granulator, slowly add the binder solution, and granulate until uniform wet granules are formed. 5. Drying and granulation The wet granules are placed in a fluidized bed dryer and dried to a moisture content of ≤5%. After drying, the granules are sieved through a 20-mesh screen to remove large particles or fine powder. 6. Tablet pressing Magnesium stearate was added to the dry granules, mixed for 5 min, and then compressed into tablets using a rotary tablet press; 7. Coating Enteric film coating premix is ​​used to prepare a 10% coating solution, which is spray-coated in a high-efficiency coating pan to obtain the finished enteric-coated tablets.

[0032] Example 2: 1. Raw material pretreatment and weighing: In a clean environment, use a calibrated electronic balance (accuracy 0.01g) to weigh: 45 g of sodium chondroitin sulfate was sieved through a vibrating sieve (80 mesh) and placed in a dry polyethylene bag; 25 g of glucosamine sulfate, dried in a vacuum oven at 40°C for 4 hours and used immediately; 20 g of dimethyl sulfone was sterile filtered through a 0.22 μm polyethersulfone filter membrane and transferred to an amber glass bottle; 10 g of non-denatured type II collagen (purchased from Sigma-Aldrich) was thawed at 4°C and centrifuged at 3000 rpm for 10 min; Frankincense extract (purchased from Shaanxi Jiahe Biotechnology Co., Ltd.) 5g; Excipients (microcrystalline cellulose 10g + maltodextrin 10g + magnesium stearate 2g + enteric film coating premix 1g + povidone K30 3g) 26g; Eggshell membrane powder (purchased from Biova) 6.67 g; N-acetylcysteine ​​(purchased from Zhejiang Xinhecheng) 3.33 g; Phosphatidylserine (purchased from ChemiNutra) 1g; Marine keratan sulfate (purchased from Marine Biopolymers) 4.5 g; Fluidized bed granulation: Fluidized bed equipment: Glatt GPCG-1 fluidized bed granulation system (equipped with Wurster bottom spray device); Parameter settings: The inlet air temperature is set to 50°C; Spray system: dual-fluid nozzle (aperture 1.0mm), atomization pressure 0.8bar Adhesive: 8% povidone K30 ethanol solution (w / v), spray rate 5.0 mL / min; 2. Operation process: Premixing and sieving (mixed powder A) Sodium chondroitin sulfate, glucosamine sulfate, dimethyl sulfone, non-denatured type II collagen, frankincense extract, microcrystalline cellulose, and maltodextrin were added to a V-blender at 30 rpm for 15 min. After mixing evenly, sieve with a 100-mesh stainless steel sieve to ensure that there is no agglomeration to obtain mixed powder A; 3. Addition of compound synergist (mixed powder B) Eggshell membrane powder, N-acetylcysteine, phosphatidylserine, and marine keratan sulfate were added to mixed powder A and mixed in a V-type mixer for 10 minutes to ensure uniform dispersion to obtain mixed powder B. 4. Wet granulation Dissolve povidone K30 in purified water to prepare a 5% binder solution. Place the mixed powder B into a high-speed shear wet granulator, slowly add the binder solution, and granulate until uniform wet granules are formed. 5. Drying and granulation The wet granules are placed in a fluidized bed dryer and dried to a moisture content of ≤5%. After drying, the granules are sieved through a 20-mesh screen to remove large particles or fine powder. 6. Tablet pressing Magnesium stearate was added to the dry granules, mixed for 5 min, and then compressed into tablets using a rotary tablet press; 7. Coating: Use enteric film coating premix to prepare 10% coating solution, spray coating in a high-efficiency coating pan to obtain the finished enteric-coated tablets.

[0033] Example 3: 1. Raw material pretreatment and weighing: In a clean environment, use a calibrated electronic balance (accuracy 0.01g) to weigh: 37.5 g of sodium chondroitin sulfate was sieved through a vibrating sieve (80 mesh) and placed in a dry polyethylene bag; Glucosamine sulfate 22.5 g, dried in a vacuum oven at 40°C for 4 hours and used immediately; 12.25 g of dimethyl sulfone was sterile filtered through a 0.22 μm polyethersulfone filter membrane and transferred to an amber glass bottle; 6.5 g of non-denatured type II collagen (purchased from Sigma-Aldrich) was thawed at 4°C and centrifuged at 3000 rpm for 10 min; Frankincense extract (purchased from Shaanxi Jiahe Biotechnology Co., Ltd.) 3g; Excipients (microcrystalline cellulose 6g + maltodextrin 6g + magnesium stearate 1g + enteric film coating premix 0.5g + povidone K30 1.5g) 15g; Eggshell membrane powder (purchased from Biova) 6.5 g; N-acetylcysteine ​​(purchased from Zhejiang Xinhecheng) 2.45g; Phosphatidylserine (purchased from ChemiNutra) 1g; Marine keratan sulfate (purchased from Marine Biopolymers) 5g; Fluidized bed granulation: Fluidized bed equipment: Glatt GPCG-1 fluidized bed granulation system (equipped with Wurster bottom spray device); Parameter settings: The inlet air temperature is set to 50°C; Spray system: dual-fluid nozzle (aperture 1.0mm), atomization pressure 0.8bar Adhesive: 8% povidone K30 ethanol solution (w / v), spray rate 5.0 mL / min; 2. Operation process: Premixing and sieving (mixed powder A) Sodium chondroitin sulfate, glucosamine sulfate, dimethyl sulfone, non-denatured type II collagen, frankincense extract, microcrystalline cellulose, and maltodextrin were added to a V-blender at 25 rpm for 15 min. After mixing evenly, sieve with a 90-mesh stainless steel sieve to ensure that there is no agglomeration to obtain mixed powder A; 3. Addition of compound synergist (mixed powder B) Eggshell membrane powder, N-acetylcysteine, phosphatidylserine, and marine keratan sulfate were added to mixed powder A and mixed in a V-type mixer for 10 minutes to ensure uniform dispersion to obtain mixed powder B. 4. Wet granulation Dissolve povidone K30 in purified water to prepare a 5% binder solution. Place the mixed powder B into a high-speed shear wet granulator, slowly add the binder solution, and granulate until uniform wet granules are formed. 5. Drying and granulation The wet granules are placed in a fluidized bed dryer and dried to a moisture content of ≤5%. After drying, the granules are sieved through a 20-mesh screen to remove large particles or fine powder. 6. Tablet pressing Magnesium stearate was added to the dry granules, mixed for 5 min, and then compressed into tablets using a rotary tablet press; 7. Coating: Use enteric film coating premix to prepare 10% coating solution, spray coating in a high-efficiency coating pan to obtain the finished enteric-coated tablets.

[0034] Comparative Example 1: The specific implementation steps are the same as those in Example 3, except that N-acetylcysteine ​​is not used.

[0035] Comparative Example 2: The specific implementation steps are the same as those in Example 3, except that phosphatidylserine is not used.

[0036] Comparative Example 3: The specific implementation steps are the same as those in Example 3, except that marine keratan sulfate is not used.

[0037] Comparative Example 4: The specific implementation steps are the same as those in Example 3, except that no composite synergist is used.

[0038] Experimental Example 1 Verification of in vitro chondrocyte and anti-inflammatory effects of glucosamine chondroitin sulfate pharmaceutical composition 1. Purpose of the experiment The effects of the pharmaceutical compositions of Examples 1-3 and Comparative Examples 1-4 of the present invention on inhibiting chondrocyte proliferation and inflammatory factors were evaluated through in vitro experiments to verify the scientific nature of their synergistic effects and optimized ratios; 2. Experimental Materials and Methods 2.1 Experimental Materials Cell line: human chondrocytes (HC-a cell line, purchased from ATCC); Culture medium: DMEM / F12+10% FBS+1% penicillin / streptomycin; Inflammation inducer: IL-1β (10 ng / mL); Detection reagents: CCK-8 kit (cell proliferation assay); ELISA kit (detection of TNF-α, IL-6, and COL2A1); Experimental group: Examples 1-3: Compositions prepared according to Examples 1-3; Comparative Examples 1-4: compositions with the proportions adjusted according to Comparative Examples 1-4; Control group: single chondroitin sulfate sodium (40 μg / mL) + glucosamine sulfate (25 μg / mL); 2.2 Experimental methods 2.2.1 Chondrocyte proliferation assay HC-a cells were seeded in 96-well plates (5 × 10 3 cells / well), cultured for 24 h; Drugs of each experimental group and control group were added respectively, and the culture was continued for 48 hours; CCK-8 reagent (10 μL) was added to each well, and the absorbance at 450 nm (OD value) was measured after 2 h of incubation to calculate the cell proliferation rate (%); 2.2.2 Evaluation of anti-inflammatory effects Inflammation was induced by pretreatment of cells with IL-1β (10 ng / mL) for 24 h; Drugs from each experimental group were added and cultured for 24 h; The supernatant was collected, and the concentrations of TNF-α and IL-6 were detected by ELISA; At the same time, the secretion of COL2A1 (type II collagen) was detected to evaluate the ability of cartilage matrix synthesis; 2.2.3 Data Statistics Six replicate wells were set up in each group, and the data were expressed as mean ± standard deviation; One-way analysis of variance (ANOVA) was used to compare the differences between groups, and the significance level was set at p < 0.05; The final test results are shown in Table 1 below: Table 1 Test results Group Cell proliferation rate (%) TNF-α inhibition rate (%) IL-6 inhibition rate (%) COL2A1 secretion amount (ng / mL) Example 1 128.7 65.3 58.9 82.6 Example 2 132.4 68.7 62.4 86.3 Example 3 142.6 73.8 69.7 94.2 Comparative Example 1 116.8 51.4 47.2 71.3 Comparative Example 2 121.3 55.7 52.6 76.8 Comparative Example 3 119.5 53.9 50.1 74.2 Comparative Example 4 109.2 45.8 42.3 66.4 Control group 105.9 40.1 37.5 65.7 From the above experimental results, it can be seen that the glucosamine sulfate chondroitin pharmaceutical composition provided by the present invention exhibits remarkable and excellent dual effects of promoting chondrocyte proliferation and inhibiting inflammatory factors in in vitro experiments, and its effect is significantly better than that of traditional single ingredients or simple compound preparations. Among them, Example 3 is the optimal ratio scheme, and its various indicators are significantly better than those of other groups. The experimental data of Comparative Examples 1 to 4 reveal the irreplaceability of each key component in the glucosamine sulfate chondroitin pharmaceutical composition of the present invention from multiple dimensions. Among them, Comparative Example 1 lacks N-acetylcysteine, and its cell proliferation rate is 116.8%, which is 25.8 percentage points lower than that of Example 3, the TNF-α inhibition rate is 51.4%, the IL-6 inhibition rate is 47.2%, and the COL2A1 secretion amount is 71.3 ng / mL, all indicators showed a significant decline. This phenomenon profoundly reflects the core role of N-acetylcysteine ​​in maintaining cellular redox balance. This is because N-acetylcysteine, as a glutathione precursor, can not only directly remove reactive oxygen free radicals in the joint cavity and reduce the damage of oxidative stress to chondrocytes, but also inhibit the transcription of inflammatory mediators by regulating the NF-κB signaling pathway. Its absence leads to the obstruction of sulfur metabolism in dimethyl sulfone, which makes the composition lose its early antioxidant protection and weakens the subsequent support of sulfur to the cartilage matrix. It is more noteworthy that when dimethyl sulfone acts alone in Comparative Example 1, due to the lack of synergy with N-acetylcysteine, its anti-inflammatory effect can only be achieved through limited COX-2 inhibition, and a complete oxidative stress-inflammation cascade inhibition network cannot be formed, which is directly reflected in the experimental result that the TNF-α inhibition rate is 22.4 percentage points lower than that in Example 3; in terms of cartilage matrix synthesis, the aggravation of oxidative stress leads to the inhibition of chondrocyte synthesis function, and the secretion of COL2A1 is reduced by 22.9 ng / mL, which fully proves that N-acetylcysteine ​​indirectly maintains the physiological mechanism of anabolic activity of chondrocytes by protecting them from oxidative damage.

[0039] The experimental design of Comparative Example 2 deliberately excluded the immune regulatory component phosphatidylserine. The cell proliferation rate of 121.3% in the data showed a slight increase compared with that of Comparative Example 1, but it was still 21.3 percentage points lower than that of Example 3. Although the TNF-α inhibition rate of 55.7% and the IL-6 inhibition rate of 52.6% were better than those of the traditional control group, there was still a significant gap compared with Example 3. This is because phosphatidylserine has a unique mechanism of action in regulating the immune microenvironment - it mainly promotes the transformation of pro-inflammatory M1 type to anti-inflammatory M2 type by binding to macrophage surface receptors, rather than directly acting on chondrocytes. Therefore, its absence has a relatively small effect on the direct proliferation ability of cells. The results showed that the lack of phosphatidylserine significantly reduced the bioavailability of frankincense extract, as it enhances cell membrane fluidity and promotes the transmembrane transport of hydrophobic boswellic acids. This disruption of this synergistic mechanism reduced the actual efficacy of the frankincense extract in the combination, indirectly leading to a 17.4 ng / mL decrease in COL2A1 secretion. These results confirm the multiple functions of phosphatidylserine as an "immunomodulatory hub"—both directly regulating macrophage polarization to establish an anti-inflammatory microenvironment and optimizing the cellular uptake of other active ingredients through physicochemical interactions.

[0040] Due to the absence of marine keratan sulfate, Comparative Example 3 exhibited the most dramatic fluctuations in COL2A1 secretion. This phenomenon accurately reflects the special role of keratan sulfate in maintaining the structural stability of the cartilage matrix. This is because the unique sulfonation pattern of marine keratan sulfate enables tight cross-linking with specific sites on collagen fibers. When this component is missing, although sodium chondroitin sulfate can still form a basic hydrated gel, it lacks sufficient collagen network support, resulting in a loose matrix structure and reduced mechanical properties. Experimental data show that this structural defect feedback inhibits the synthetic activity of chondrocytes, manifested as a significant decrease in COL2A1 secretion. In terms of inflammation inhibition, the TNF-α inhibition rate of 53.9% and the IL-6 inhibition rate of 50.1% in Comparative Example 3, while superior to the traditional preparation, are still significantly lower than those in Example 3. This suggests that the polyanionic network formed by keratan sulfate and sodium chondroitin sulfate not only has structural functions but also reduces the deposition of inflammatory mediators in the joint cavity through charge repulsion. Its absence allows the local inflammatory microenvironment to persist.

[0041] Comparative Example 4, the most extreme control group, completely lacked the composite synergist (eggshell membrane powder, N-acetylcysteine, phosphatidylserine, and marine keratan sulfate). Its experimental results showed a "cliff-like decline": the cell proliferation rate of 109.2% was only slightly higher than that of the traditional control group, the TNF-α inhibition rate of 45.8% did not even reach half inhibition, and the COL2A1 secretion of 66.4 ng / mL almost fell back to the basal level. This result strongly demonstrates the core position of the composite synergist system in the present invention - they are not simply auxiliary ingredients, but rather key nodes that constitute a multi-target therapeutic network.

[0042] Therefore, it can be seen that the composite synergist and the main components of the present invention form a precisely regulated functional network, in which N-acetylcysteine ​​dominates oxidative stress regulation, phosphatidylserine is responsible for the balance of the immune microenvironment, marine-derived keratan sulfate ensures matrix structure stability, and eggshell membrane powder provides growth support. These components, through a strict ratio relationship with the main components such as sodium chondroitin sulfate and glucosamine sulfate, form a multi-target mechanism of action. The absence or imbalance of any single component will lead to functional defects in this network, manifested as a significant weakening of the dual efficacy of promoting cartilage regeneration and inhibiting inflammation.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A glucosamine chondroitin sulfate pharmaceutical composition, characterized in that: Calculated by mass, it includes the following raw materials: 30-45 parts of sodium chondroitin sulfate; 20-25 parts of glucosamine sulfate; 4.5-20 parts of dimethyl sulfone; 3-10 parts of non-denatured type II collagen; 1-5 parts of frankincense extract; 4-26 parts of auxiliary materials.

2. The glucosamine sulfate chondroitin pharmaceutical composition according to claim 1, characterized in that The invention also comprises a composite synergist, which is eggshell membrane powder, N-acetylcysteine, phosphatidylserine and marine source keratan sulfate.

3. The glucosamine sulfate chondroitin pharmaceutical composition according to claim 2, characterized in that The mass ratio of the N-acetylcysteine ​​to dimethyl sulfone is 1:(3-6).

4. The glucosamine sulfate chondroitin pharmaceutical composition according to claim 2, characterized in that The mass ratio of the phosphatidylserine to the frankincense extract is 1:(2-5).

5. The glucosamine sulfate chondroitin pharmaceutical composition according to claim 2, characterized in that The mass ratio of the marine-derived keratan sulfate to sodium chondroitin sulfate is 1:(5-10).

6. The glucosamine sulfate chondroitin pharmaceutical composition according to claim 2, characterized in that The mass ratio of the eggshell membrane powder to the non-denatured type II collagen is 1:(0.5-1.5).

7. The glucosamine sulfate chondroitin pharmaceutical composition according to claim 1, characterized in that The auxiliary materials include microcrystalline cellulose, maltodextrin, povidone K30, magnesium stearate and enteric film coating premix.

8. A method for preparing the glucosamine chondroitin sulfate pharmaceutical composition according to any one of claims 1 to 7, characterized in that: The following steps are involved: Sodium chondroitin sulfate, glucosamine sulfate, dimethyl sulfone, non-denatured type II collagen, frankincense extract, and microcrystalline cellulose and maltodextrin in the auxiliary materials are premixed, and the mixture is passed through an 80-100 mesh sieve to obtain mixed powder A; Add eggshell membrane powder, N-acetylcysteine, phosphatidylserine and marine keratan sulfate to mixed powder A in proportion, mix well, and obtain mixed powder B; The mixed powder B and the povidone K30 solution are wet-granulated, dried, and magnesium stearate is added and mixed evenly, followed by tableting, and then coated with an enteric film coating premix to obtain the tablet.

9. Use of the glucosamine chondroitin sulfate pharmaceutical composition according to any one of claims 1 to 7 in the preparation of a biopharmaceutical for promoting cartilage regeneration and inhibiting joint inflammation.