Preparation method of compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health

Abstract

The application discloses a composite soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health and a preparation method thereof. First, oyster calcium is uniformly mixed with mono- and di-glycerol fatty acid esters and heated to obtain pretreated calcium powder; beeswax is dissolved in ethyl acetate to prepare a beeswax solution, the pretreated calcium powder is added to prepare a suspension, anhydrous ethanol is added dropwise, and the beeswax is precipitated through a reverse solvent with the calcium powder as a core, and then the modified oyster calcium is prepared through cooling and solidification, solvent removal by suction filtration and vacuum drying. The modified calcium powder and colostrum basic protein are dispersed in perilla seed oil, and phosphorus shrimp oil is compounded and homogenized to obtain a content, and then a capsule shell is matched to press a pill to obtain a finished product. The oyster calcium is subjected to surface hydrophobic modification, the problem of easy agglomeration and sedimentation of the oyster calcium in an oil phase is solved, a solid oil interface is blocked to promote an oxidation reaction, the storage stability of alpha-linolenic acid, DHA and EPA is improved, calcium ion slow release absorption is realized, active oil and protein are synergized to jointly take effect, and osteoarthritis inflammation is effectively relieved, swelling is subsided, and cartilage structure is protected.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health. Background Technology

[0002] With an aging population and changing lifestyles, osteoarthritis has become an increasingly common disease, severely impacting people's quality of life. One of the main symptoms of osteoarthritis is joint pain, causing significant distress to patients. Currently, oral nonsteroidal anti-inflammatory drugs (NSAIDs), primarily glucosamine sulfate, are used. The former's main purposes are anti-inflammatory, analgesic, and anti-swelling effects; the latter is an important component of articular cartilage, playing a role in repairing damage, promoting cartilage matrix formation, and also providing analgesia and anti-inflammation. However, the gastrointestinal, liver, and kidney damage caused by these two substances is well-known in clinical practice, therefore they cannot be taken long-term.

[0003] Antarctic krill is a small crustacean, reaching up to 6 cm in length and with a lifespan of 5-6 years. It is the world's largest single species in terms of biomass. Antarctic krill oil is a complex functional lipid product extracted from Antarctic krill, possessing multiple benefits including lipid-lowering, anti-inflammatory, and improved learning and cognitive abilities. The DHA, EPA, and astaxanthin in krill oil have powerful anti-inflammatory effects, reducing the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, and significantly alleviating osteoarthritis inflammation.

[0004] Basic proteins generally refer to proteins with a pH greater than 7 under isoelectric point conditions. Colostrum basic proteins can stimulate chondrocytes to synthesize mucin, a key component in maintaining cartilage elasticity and thickness. Supplementing with basic proteins can slow down cartilage degeneration and reduce inflammation and pain caused by cartilage wear. Krill oil works in conjunction with basic proteins; the anti-inflammatory environment of krill oil can enhance the sensitivity of basic proteins to osteoblasts, while the osteogenic effect of basic proteins further depends on the DHA provided by krill oil to maintain cell membrane fluidity.

[0005] Oyster calcium is a calcium supplement extracted from oyster shells. Its main components are calcium carbonate and amino acid chelated calcium, which have high bioavailability. Oyster calcium can supplement the body's calcium, providing sufficient calcium raw materials for subchondral bone repair and maintaining bone metabolic balance in the pathological state of osteoarthritis, and improving abnormal subchondral bone remodeling. As a key second messenger in nerve signal transmission, calcium ions can participate in regulating the generation and transmission of peripheral pain signals, moderately reducing pain sensitivity, and indirectly improving joint pain, soreness, and other discomfort caused by osteoarthritis.

[0006] Perilla seed oil is a natural oil extracted from the mature seeds of the perilla plant (Perilla frutescens), a member of the Lamiaceae family. Perilla seed oil contains over 90% unsaturated fatty acids, primarily alpha-linolenic acid, oleic acid, and linoleic acid. The purity of alpha-linolenic acid exceeds 60%. The alpha-linolenic acid and omega-3 fatty acids in perilla seed oil can inhibit the production of inflammatory mediators and reduce inflammatory responses.

[0007] In existing oyster calcium-containing soft capsule systems, the particle density of oyster calcium is significantly higher than that of the oil matrix. Furthermore, oyster calcium exhibits strong surface polarity and high hydrophilicity, resulting in poor thermodynamic compatibility with weakly polar oils. During ingredient homogenization and storage, oyster calcium is highly prone to particle flocculation and agglomeration, rapid sedimentation and stratification, leading to uneven solid-liquid dispersion of the contents. This results in large fluctuations in fill volume and excessive fill volume variation during continuous soft capsule filling, reducing the product's yield. More importantly, the polar surface of unmodified oyster calcium readily adsorbs trace amounts of water vapor in the system, constructing numerous oil-water micro-interfaces at the powder-oil two-phase interface. The oil-water interface is a preferred reaction site for lipid auto-oxidation. Calcium ions accumulate in this interfacial region, altering interfacial polarity, enriching and promoting the decomposition of hydroperoxide, an intermediate product of lipid oxidation, thus advancing the chain oxidation process of oil free radicals. The adsorbed moisture in the powder and the interfacial calcium ions synergistically construct a highly oxidative microenvironment, continuously accelerating the oxidative degradation of polyunsaturated fatty acids such as α-linolenic acid in perilla seed oil and DHA and EPA in krill oil. This results in rancid and rancid odors in the oils, a decrease in the physiological activity of functional unsaturated fatty acids, and ultimately weakens the overall effect of the product in relieving joint inflammation, improving joint swelling, and protecting articular cartilage. Summary of the Invention

[0008] This invention aims to provide a compound soft capsule for relieving osteoarthritis, joint swelling, and maintaining cartilage health. This invention achieves three breakthrough effects through surface modification of oyster calcium. First, the modified oyster calcium can be suspended uniformly in the oil phase for a long time, solving the problems of filling accuracy and production yield. Second, by improving the distribution of calcium in the oil phase, harmful interfacial reactions are reduced, and the storage stability of polyunsaturated fatty acids such as alpha-linolenic acid, DHA, and EPA is improved, ensuring the product's efficacy over its shelf life. Third, the surface modification of oyster calcium regulates the release behavior of calcium in the gastrointestinal tract, improving calcium bioavailability, thereby enhancing the efficacy of the compound soft capsule in relieving osteoarthritis, joint swelling, and maintaining cartilage health.

[0009] A method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling, and maintaining cartilage health includes the following steps: Step 1: Mix 280-320 parts by weight of oyster calcium with 3-6 parts by weight of mono- and diglycerides of fatty acids, then heat to 100-110℃ and maintain for 5 minutes to obtain a pretreated powder; Step 2: Add 25-30 parts by weight of molten beeswax to 600-800 parts by weight of ethyl acetate preheated to 40-50℃, and stir evenly under sealed conditions to obtain a beeswax solution; Step 3: Add the pretreated powder to the beeswax solution, and stir at high speed to obtain a suspension. 4. Under stirring conditions, slowly add 300-400 parts by weight of anhydrous ethanol to the suspension, homogenize under high pressure, and then cool to -20℃ until completely solidified. During the cooling process, continue stirring to prevent caking. Filter to remove the solvent and vacuum dry to obtain oyster calcium modified powder; Step 5. Add the oyster calcium modified powder and 10 parts by weight of colostrum basic protein to 260-295 parts by weight of perilla seed oil, mix and stir evenly, then add 80-90 parts by weight of krill oil, homogenize under high pressure to obtain the contents; Step 6. Prepare capsule shells, compress into pellets, and obtain composite soft capsules that relieve osteoarthritis, joint swelling and maintain cartilage health.

[0010] The content of monoglycerides in monoglycerides and diglycerides is 40-55%, and the content of diglycerides is 30-45%.

[0011] Monoglycerides contain two hydroxyl groups, which anchor the active hydroxyl sites on the surface of oyster calcium through hydrogen bonds, achieving full coverage adsorption of the powder and avoiding local exposure of hydrophilic sites. Diglycerides, on the other hand, have only one hydroxyl group, which fills the gaps between monoglyceride molecules and arranges two hydrophobic carbon chains of fatty acids outward, improving the overall hydrophobicity of the powder and reducing the oil absorption value. The concentration range of monoglycerides (40-55%) and diglycerides (30-45%) is limited to balance anchoring strength and hydrophobic effect; deviations from this range may result in weak coating or insufficient hydrophobicity.

[0012] Melting and holding at 100-110℃ for 5 minutes promotes the melting and spreading of esters, hydrogen bonding and shaping, weakens the strong polarity of oyster calcium surface, provides a compatible interface for subsequent beeswax coating, and prevents beeswax coating layer delamination and uneven coating.

[0013] Furthermore, a method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling, and maintaining cartilage health includes the following steps: Step 1: Mix 300 parts by weight of oyster calcium with 3-6 parts by weight of mono- and diglycerides of fatty acids, and heat to 100-110°C for 5 minutes to obtain a pretreated powder; Step 2: Add 28 parts by weight of molten beeswax to 600-800 parts by weight of ethyl acetate preheated to 40-50°C, and stir evenly under sealed conditions to obtain a beeswax solution; Step 3: Add the pretreated powder to the beeswax solution, and stir at high speed to obtain a suspension. Step 4: Under stirring conditions, slowly add 300-400 parts by weight of anhydrous ethanol to the suspension, homogenize under high pressure, and then cool to -20℃ until completely solidified. During the cooling process, continue stirring to prevent caking. Filter to remove the solvent and vacuum dry to obtain oyster calcium modified powder. Step 5: Add the oyster calcium modified powder and 10 parts by weight of colostrum basic protein to 277 parts by weight of perilla seed oil, mix and stir evenly, then add 85 parts by weight of krill oil, homogenize under high pressure to obtain the contents. Step 6: Prepare capsule shells, compress into pellets, and obtain composite soft capsules that relieve osteoarthritis, joint swelling and maintain cartilage health.

[0014] The oyster calcium was passed through an 80-mesh sieve.

[0015] This invention involves dry-mixing and heating oyster shell calcium with mono- and diglyceride fatty acid ester powders. Under high-temperature conditions, the molten hydrophobic monoglycerides spread and wet the powder surface, with their polar ends facing the inorganic surface (anchored through surface hydroxyl / carbonate / adsorption layer interactions) and the fatty acid chains facing outwards. This reduces surface energy, imparts initial hydrophobicity and better dispersibility, providing a continuous substrate for the uniform coating of beeswax in the solvent system. Simultaneously, the non-polar fatty acid chains extend outwards, significantly reducing the surface energy of the oyster shell calcium and preventing its aggregation in non-polar solvents. Ethyl acetate preheated to 40-50°C effectively dissolves the molten beeswax, forming a homogeneous beeswax solution. The mono- and diglyceride fatty acid esters help oyster shell calcium suspend in the beeswax solution, preventing aggregation. Slowly adding anhydrous ethanol to the suspension disrupts the solubility stability of beeswax, causing it to precipitate as flocculent or flaky particles around the oyster shell calcium core. The freshly extracted beeswax is not fully crystallized and hardened; it is in a soft, malleable state. High-pressure homogenization at this stage allows the powerful shear force to easily break up, crush, and reshape these soft, flocculent materials, enabling them to evenly coat the surface of the oyster shell calcium particles. Slowly lowering the temperature to -20°C allows the beeswax to solidify uniformly and stably on the surface of the oyster shell calcium during the cooling process. Vacuum filtration followed by vacuum drying yields a loose, free-flowing modified oyster shell calcium powder, preventing the beeswax particles from sticking together.

[0016] This invention significantly improves the dispersion stability and compatibility of oyster calcium in the oil phase by modifying the surface of oyster calcium, thus ensuring the filling accuracy and yield of soft capsules.

[0017] The researchers of this invention discovered that the composite soft capsules prepared by this method can improve the absorption and utilization rate of calcium. The surface modification of oyster calcium by beeswax delays the reaction of oyster calcium with large amounts of gastric acid, avoiding excessively high local calcium ion concentrations in the stomach, which can cause discomfort such as bloating and constipation. The beeswax layer acts as a slow-release agent, steadily releasing calcium ions, reducing the burden on the gastrointestinal tract, and achieving stable release and efficient absorption of calcium in the intestines. Stable and long-lasting blood calcium concentrations can inhibit osteoclast activity, reduce bone resorption, delay subarticular bone erosion and structural damage, indirectly alleviating swelling caused by bone erosion, and providing a continuous and stable calcium source for cartilage repair.

[0018] The oyster calcium sample to be tested was passed through an 80-mesh sieve. 0.40 g was accurately weighed and placed in a 13 mm inner diameter compression mold. The mold was pressurized at 12 MPa for 45 s to prepare a smooth, crack-free circular sample. The sample was placed on the sample stage of an optical contact angle measuring instrument. The testing environment was 25 ± 1℃ and 50 ± 5% RH. Ultrapure water (resistivity ≥ 18.2 MΩ·cm) was used as the probe liquid, with a droplet volume of 4 μL. Three points were randomly selected at different locations on the same sample (avoiding edges and defect areas) for testing. The contact angle value was recorded at each point 5 seconds after the droplet contacted the sample surface. The arithmetic mean of the three measurements was taken as the contact angle of the sample. The contact angle of modified oyster calcium was 112.53–116.15°. The contact angle of unmodified oyster calcium was 64.55–67.68°.

[0019] In step 2, 28 parts by weight of molten beeswax are added to 700 parts by weight of ethyl acetate preheated to 48°C, and stirred evenly under sealed conditions to obtain a beeswax solution.

[0020] The stirring speed in step 3 is 1500-3000 rpm, and the temperature is 40-50℃.

[0021] In step 4, 350 parts by weight of anhydrous ethanol are added at a dropping rate of 15-20 g / min under stirring conditions of 40-50℃ and 500-800 rpm.

[0022] Adding anhydrous ethanol slowly under stirring conditions avoids excessive local ethanol concentration, which could lead to a rapid and massive precipitation of beeswax. This prevents the beeswax from clumping together during precipitation and becoming ineffective on the surface of the oyster shell calcium powder. A dropping rate of 15-20 g / min is used to control the crystal growth rate of the beeswax. If the dropping rate is too fast, the beeswax will clump together (homogeneous nucleation); if the dropping rate is too slow, the coating layer will be too thin. Only this rate can induce beeswax to coat the surface of the pretreated calcium powder.

[0023] In step 4, the homogenization temperature is 45-50℃ and the homogenization pressure is 40 MPa.

[0024] After the beeswax antisolvent precipitates, it is homogenized again, causing the beeswax to deposit in flake / patch form and adhere to the surface of the pretreated powder. High-pressure shearing breaks up the wax agglomerates, forming a finer, more stable coating structure, which significantly improves the dispersibility and bioavailability of the modified powder. Under homogenization conditions of 45-50℃, the beeswax is in a semi-solid state, and the homogenization process forms a highly continuous and well-covered hydrophobic coating layer.

[0025] In step 4, the cooling rate is 5-10℃ / h, and the stirring is continuous during the cooling process at a stirring rate of 120r / min. After cooling to -20℃, the mixture is allowed to stand for 1-2 hours and then filtered to remove the solvent.

[0026] During the slow cooling process, beeswax slowly and evenly solidifies on the surface of the oyster calcium. Allowing it to stand at a low temperature for 1-2 hours allows the beeswax to fully solidify on the surface of the oyster calcium, preventing the particles from clumping together during filtration. The resulting modified oyster calcium has a loose structure and the powder has good flowability, facilitating subsequent processing.

[0027] The vacuum drying conditions in step 4 are 32-35℃ and -0.08MPa.

[0028] A drying temperature of 32-35℃ can prevent beeswax from softening and sticking together.

[0029] A compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health, prepared by the method described in any one of claims 1-8.

[0030] The beneficial effects of this invention are: 1. Solving production challenges and achieving stable industrialization. This invention fundamentally overcomes the industry's technical bottleneck of high-density, highly polar oyster calcium powder's tendency to agglomerate and settle in the oil phase. Through interface modification, this invention increases the water contact angle of oyster calcium powder from 64.55-67.68° (hydrophilic) to 112.53-116.15° (strongly hydrophobic), causing it to thermodynamically and spontaneously disperse in the oil phase. The modified oyster calcium powder can maintain a uniform and stable suspension in the oil phase for a long time, ensuring the filling accuracy and high yield of soft capsules in continuous filling processes, providing a key guarantee for large-scale production.

[0031] 2. Blocking the interfacial catalytic oxidation pathway and extending the shelf life of polyunsaturated fatty acids. The active calcium ions on the surface of unmodified oyster calcium readily adsorb trace amounts of moisture, forming a hydrolysis microenvironment. This invention utilizes a physical barrier constructed through a beeswax layer, which helps reduce / weaken the promoting effect of the interfacial micro-aqueous phase and surface catalytic sites on oxidation. Accelerated stability tests (40℃, 75% RH, 3 months) show (see Table 5) that the retention rates of DHA and EPA in the soft capsules of this invention remain above 90%, while the retention rate of Comparative Example 3 (without beeswax coating) is only about 70%, and the peroxide value is significantly increased.

[0032] 3. Regulating calcium ion release kinetics and optimizing bone metabolism homeostasis. Traditional calcium supplements release large amounts of calcium instantaneously in gastric juice, leading to excessively high local calcium ion concentrations, causing osmotic imbalance and gastrointestinal discomfort, with most of the calcium being excreted in feces. This invention utilizes the strong hydrophobicity and extremely low solubility / swelling in gastrointestinal juices of beeswax to form a lipid physical barrier on the surface of oyster calcium, delaying its instantaneous large-scale dissolution in the acidic gastric environment, allowing calcium ions to be transported to the intestines at a more stable rate, achieving a delayed gastric release effect. Animal experiments confirmed (see Table 7) that this sustained-release mode increased the calcium retention rate in rats from 49.58% in Comparative Example 3 to 64.53%, and simultaneously downregulated the level of the bone resorption marker CTX-I, indicating that it optimizes the osteoblast / osteoclast balance through stable calcium supply.

[0033] 4. The innovation of this invention lies not only in the simple superposition of components, but also in the pioneering two-step interface modification technology of 'dry heat primer coating + antisolvent wax coating'. The polar anchoring of mono- and diglyceride fatty acid esters and the physical barrier of beeswax are both indispensable (see Comparative Examples 1 and 3). This interface engineering not only solves the thermodynamic incompatibility problem of inorganic powders in the oil phase of polyunsaturated fatty acids (preventing oxidative rancidity), but also reshapes the pharmacokinetic release behavior of calcium ions (improving retention rate), which is the fundamental reason for the unexpected synergistic therapeutic effect. Attached Figure Description

[0034] Figure 1 This is a line graph showing the effect of different groups of gavage drugs on joint swelling in rats with osteoarthritis in Experiment Example 3 of this invention.

[0035] Figure 2 This is a bar chart showing the effect of different groups of gavage drugs on serum inflammatory factors in rats with osteoarthritis in Experiment Example 3 of this invention.

[0036] Figure 3 This is a bar chart showing the effect of different groups of gavage drugs on serum bone formation markers in rats with osteoarthritis in Experiment Example 3 of this invention.

[0037] Figure 4This is a bar chart showing the effect of different groups of gavage drugs on serum bone resorption markers in an osteoarthritis model rat in Experiment Example 3 of this invention.

[0038] Figure 5 This is a bar chart showing the effect of different groups of gavage drugs on the expression level of cartilage tissue proteins in rats with osteoarthritis in Experiment Example 3 of this invention. Specific Implementation Example 1

[0039] Step 1: Mix 320g of oyster calcium with 3g of mono- and diglyceride fatty acid esters evenly, then heat to 100-110℃ and maintain for 5 minutes to obtain pretreated powder; Step 2: Add 30g of molten beeswax to 700g of ethyl acetate preheated to 48°C, and stir evenly under sealed conditions to obtain a beeswax solution; Step 3: Add pretreated powder to beeswax solution at 1500 rpm, and stir at high speed to obtain a suspension. The stirring temperature is 48℃. Step 4: Slowly add 350g of anhydrous ethanol to the suspension under stirring at 600rpm, at a dropping rate of 20g / min. After the addition is complete, homogenize at a homogenization temperature of 48℃ and a homogenization pressure of 40MPa. Then cool down at a cooling rate of 8℃ / h, with a stirring rate of 120r / min during the cooling process. After cooling to -20℃, let stand for 2h, then filter to remove the solvent, and vacuum dry to obtain oyster calcium modified powder. The vacuum drying conditions are 32-35℃ and -0.08MPa. The contact angle of the oyster calcium modified powder is 115.27°. Step 5: Add oyster calcium modified powder and 10g colostrum basic protein to 260g perilla seed oil, mix and stir evenly, then add 80g krill oil, and homogenize under high pressure to obtain the contents. Step 6: Prepare capsule shells, compress into pellets, and obtain compound soft capsules that relieve osteoarthritis, joint swelling and maintain cartilage health.

[0040] The compound soft capsules are available in a specification of 0.7g / capsule. Example 2

[0041] Step 1: Mix 280g of oyster calcium with 4g of mono- and diglyceride fatty acid esters evenly, then heat to 100-110℃ and maintain for 5 minutes to obtain pretreated powder; Step 2: Add 25g of molten beeswax to 600g of ethyl acetate preheated to 50°C, and stir evenly under sealed conditions to obtain a beeswax solution; Step 3: Add pretreated powder to beeswax solution at 3000 rpm, and stir at high speed to obtain a suspension. The stirring temperature is 50℃. Step 4: Slowly add 300g of anhydrous ethanol to the suspension under stirring at 800rpm, at a dropping rate of 15-20g / min. After the addition is complete, homogenize at a homogenization temperature of 50℃ and a homogenization pressure of 40MPa. Then cool down at a cooling rate of 5℃ / h, with a stirring rate of 120r / min during the cooling process. After cooling to -20℃, let stand for 1h, then filter to remove the solvent. Vacuum dry to obtain oyster calcium modified powder. The vacuum drying conditions are 32-35℃ and -0.08MPa. The contact angle of the oyster calcium modified powder is 116.15°. Step 5: Add oyster calcium-modified powder and 10g colostrum basic protein to 295g perilla seed oil, mix and stir evenly, then add 90g krill oil, and homogenize under high pressure to obtain the contents. Step 6: Prepare capsule shells, compress into pellets, and obtain compound soft capsules that relieve osteoarthritis, joint swelling and maintain cartilage health.

[0042] The compound soft capsules are available in a specification of 0.7g / capsule. Example 3

[0043] Step 1: Mix 300g of oyster calcium with 6g of mono- and diglyceride fatty acid esters evenly, then heat to 100-110℃ and hold for 5 minutes to obtain pretreated powder. Step 2: Add 28g of molten beeswax to 800g of ethyl acetate preheated to 45°C, and stir evenly under sealed conditions to obtain a beeswax solution; Step 3: Add pretreated powder to the beeswax solution at 2500 rpm, and stir at high speed to obtain a suspension. The stirring temperature is 45℃. Step 4: Under stirring at 500 rpm, slowly add 400 g of anhydrous ethanol to the suspension at a rate of 15 g / min. After the addition is complete, homogenize at a temperature of 45℃ and a pressure of 40 MPa. Then cool down at a rate of 10℃ / h, stirring at 120 rpm. After cooling to -20℃, let stand for 2 hours, then filter to remove the solvent. Vacuum dry to obtain oyster calcium modified powder. The vacuum drying conditions are 32-35℃ and -0.08 MPa. The contact angle of the oyster calcium modified powder is 112.53°. Step 5: Add oyster calcium modified powder and 10g colostrum basic protein to 277g perilla seed oil, mix and stir evenly, then add 85g krill oil, and homogenize under high pressure to obtain the contents. Step 6: Prepare capsule shells, compress into pellets, and obtain compound soft capsules that relieve osteoarthritis, joint swelling and maintain cartilage health.

[0044] The compound soft capsules are available in a specification of 0.7g / capsule. Comparative Example 1

[0045] Step 1: Add 28g of molten beeswax to 700g of ethyl acetate preheated to 48°C, and stir evenly under sealed conditions to obtain a beeswax solution; Step 2: The beeswax solution is sheared and stirred at 2000 rpm. During the homogenization process, 300g of oyster calcium is slowly added to obtain a suspension. The stirring temperature is 48℃. Step 3: Under stirring at 600 rpm, 350 g of anhydrous ethanol was slowly added dropwise to the suspension at a rate of 15 g / min. The homogenization temperature was 48 ℃ and the homogenization pressure was 40 MPa. After high-pressure homogenization, the suspension was cooled at a rate of 8 ℃ / h. During the cooling process, the stirring rate was 120 r / min. After cooling to -20 ℃, the suspension was allowed to stand for 2 h. The solvent was then removed by filtration and vacuum drying to obtain modified oyster calcium. The vacuum drying conditions were 32-35 ℃ and -0.08 MPa. The contact angle of the modified oyster calcium was 102.36°. Step 4: Add oyster calcium modified powder and 10g colostrum basic protein to 277g perilla seed oil, mix and stir evenly, then add 85g krill oil, and homogenize under high pressure to obtain the contents. Step 5: Prepare capsule shells, compress into pellets, and obtain comparative example 1 soft capsules.

[0046] The compound soft capsules are available in a specification of 0.7g / capsule. Comparative Example 2

[0047] Step 1: Heat 277g of perilla seed oil and 28g of beeswax together to 70℃. After the beeswax melts, cool down to 35℃, add 85g of krill oil, 10g of colostrum basic protein, and 300g of oyster calcium, and mix well to obtain the contents. Step 2: Prepare capsule shells, compress into pellets, and obtain Comparative Example 2 soft capsules with a content of 0.7g / capsule. Comparative Example 3

[0048] Step 1: Mix 300g of oyster calcium with 3g of mono- and diglyceride fatty acid esters evenly, then heat to 100-110℃ and hold for 5 minutes to obtain pretreated powder. The contact angle of the pretreated powder is 95.51°. Step 2: Heat 277g of perilla seed oil and 28g of beeswax together to 70℃. After the beeswax melts, cool down to 35℃, add 85g of krill oil, 10g of colostrum basic protein, and 303g of pretreatment powder, mix evenly to obtain the contents, and homogenize under high pressure to obtain the contents. Step 3: Prepare capsule shells, compress into pellets, and obtain comparative example 3 soft capsules.

[0049] The compound soft capsules are available in a specification of 0.7g / capsule.

[0050] Experimental Example 1: Content Uniformity Detection Referring to the fourth part of the Chinese Pharmacopoeia 2020 Edition, the uniformity of dosage test method 0941, take 10 soft capsules prepared in Examples 1-3 and Comparative Examples 1-3 of the present invention respectively, and determine the calcium carbonate content in the content of each soft capsule. The relative content x with the labeled amount as 100 i , calculate its mean value X and standard deviation S, and the absolute value A of the difference between the labeled amount and the mean value. Among them, ; .

[0051] Table 1 Uniformity of calcium carbonate content in soft capsules.

[0052] Example 1 99.53 0.47 3.91 9.07 Example 2 99.51 0.49 3.64 8.50 Example 3 99.77 0.23 3.41 7.73 Comparative Example 1 99.46 0.54 11.24 25.27 Comparative Example 2 99.45 0.55 11.73 26.36 Comparative Example 3 99.56 0.44 10.97 24.57 For the soft capsules prepared in Examples 1-3 of the present invention, A + 2.2S < L (L = .0), which indicates that through the synergistic process of "heating treatment after dry mixing of mono- and diglycerol fatty acids + embedding and modification with beeswax", the stable dispersion and surface functionalization of oyster calcium particles have been successfully achieved. This process significantly improves the compatibility of the calcium source in the oil matrix, effectively inhibits agglomeration and precipitation, and ensures the precise consistency of the active ingredients in each capsule. For the soft capsules prepared in Comparative Examples 1-3, A + 2.2S > L, indicating that the uniformity of the calcium carbonate content in the content of the soft capsules prepared in Comparative Examples 1-3 does not meet the requirements, and part of the oyster calcium agglomerates and precipitates, with poor homogeneity. In Comparative Example 1, beeswax was directly used to modify the surface of oyster calcium. Although beeswax has hydrophobicity, there is no chemical bonding or strong physical adsorption between it and oyster calcium particles, resulting in a loose and easily detachable coating layer, low modification efficiency, and easy aggregation of calcium particles. Therefore, the mixed pretreatment of oyster calcium and mono- and diglycerol fatty acids is the basis for firm coating. Mono- and diglycerol fatty acids may interact with the calcium surface under heating, providing a hydrophobic interface for beeswax to tightly anchor, which is a prerequisite for achieving durable and stable coating. In Comparative Example 2, the oyster calcium was not surface-treated and was directly mixed with other components as the content. Due to the strong hydrophilicity of oyster calcium itself, it is extremely easy to settle and agglomerate in the oily soft capsule content, resulting in the worst uniformity. In Comparative Example 3, the oyster calcium was not embedded and modified with beeswax. Although the dry mixing and heating treatment of mono- and diglycerol fatty acids were used to improve the hydrophobicity of the particle surface, the beeswax embedding layer was missing. As a natural polymer, beeswax can not only form a physical barrier to block particle contact, but also further stabilize the dispersion system through its compatibility with oil. Without this "molecular-level buffer", only the surface modification of mono- and diglycerol fatty acids is not enough to maintain homogeneity for a long time. Experimental Example 2 Stability Accelerated Experiment

[0053] One hundred capsules each of the composite soft capsules obtained in Examples 1-3 and Comparative Examples 1-3 of this invention were randomly selected and subjected to an accelerated stability test under constant temperature and humidity. Experimental method: The soft capsules of Examples 1-3 and Comparative Examples 1-3 of this invention were placed in a constant temperature and humidity incubator. The temperature inside the incubator was maintained at 40±2℃, the relative humidity at 75±5%, and direct sunlight was avoided for storage for 3 months. Samples stored for 0, 1, 2, and 3 months were examined.

[0054] Table 2. Test results for month 0 Ash ≤50 % 39.2 39.5 39.4 39.4 39.5 38.8 Filling volume difference Compliant \ Compliant Compliant Compliant Non-compliant Non-compliant Non-compliant Benzo(a)pyrene ≤10 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected coliform bacteria ≤0.92 MPN / g <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 Staphylococcus aureus ≤0 / 25g / 25g Not detected Not detected Not detected Not detected Not detected Not detected Total bacterial count ≤30000 CFU / g <10 <10 <10 <10 <10 <10 Mold and yeast count ≤50 CFU / g <10 <10 <10 <10 <10 <10 salmonella ≤0 / 25g / 25g Not detected Not detected Not detected Not detected Not detected Not detected Aflatoxin B1 ≤10 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected Eicosapentaenoic acid (EPA) ≥0.58 g / 100g 1.4 1.6 1.5 1.5 1.5 1.5 Docosahexaenoic acid (DHA) ≥0.29 g / 100g 0.77 0.83 0.81 0.79 0.78 0.80 Disintegration time limit ≤60 min 5 5 5 5 5 5 cadmium ≤0.1 mg / kg Not detected Not detected Not detected Not detected Not detected Not detected lead ≤2.0 mg / kg 0.066 0.061 0.079 0.072 0.067 0.073 calcium 14.5-17.7 g / 100g 17.6 16.2 17.1 17.2 17.0 16.9 Total Mercury ≤0.3 mg / kg Not detected Not detected Not detected Not detected Not detected Not detected Total arsenic ≤1.0 mg / kg 0.578 0.549 0.486 0.550 0.574 0.545 Peroxide value (based on fat content) ≤0.25 g / 100g 0.021 0.016 0.019 0.017 0.020 0.018 Acid value (based on fat content) (KOH) ≤7.5 mg / g 2.3 2.3 2.4 2.3 2.4 2.3 PCBs ≤200 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected Table 3: Test Results for Month 1 Ash ≤50 % 38.5 38.5 38.6 38.5 38.3 38.5 Filling volume difference Compliant \ Compliant Compliant Compliant Non-compliant Non-compliant Non-compliant Benzo(a)pyrene ≤10 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected coliform bacteria ≤0.92 MPN / g <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 Staphylococcus aureus ≤0 / 25g / 25g Not detected Not detected Not detected Not detected Not detected Not detected Total bacterial count ≤30000 CFU / g <10 <10 <10 <10 <10 <10 Mold and yeast count ≤50 CFU / g <10 <10 <10 <10 <10 <10 salmonella ≤0 / 25g / 25g Not detected Not detected Not detected Not detected Not detected Not detected Aflatoxin B1 ≤10 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected Eicosapentaenoic acid (EPA) ≥0.58 g / 100g 1.4 1.5 1.4 1.4 1.4 1.4 Docosahexaenoic acid (DHA) ≥0.29 g / 100g 0.70 0.74 0.71 0.70 0.68 0.72 Disintegration time limit ≤60 min 6 6 6 6 6 6 cadmium ≤0.1 mg / kg Not detected Not detected Not detected Not detected Not detected Not detected lead ≤2.0 mg / kg 0.067 0.065 0.071 0.072 0.067 0.068 calcium 14.5-17.7 g / 100g 17.5 16.1 17.2 17.3 17.1 17.1 Total Mercury ≤0.3 mg / kg Not detected Not detected Not detected Not detected Not detected Not detected Total arsenic ≤1.0 mg / kg 0.552 0.536 0.535 0.541 0.534 0.537 Peroxide value (based on fat content) ≤0.25 g / 100g 0.037 0.039 0.041 0.052 0.061 0.063 Acid value (based on fat content) (KOH) ≤7.5 mg / g 2.3 2.3 2.3 3.8 4.4 3.6 PCBs ≤200 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected Table 4: Test Results for the Second Month Ash ≤50 % 38.4 38.5 38.8 38.4 38.2 38.1 Filling volume difference Compliant \ Compliant Compliant Compliant Non-compliant Non-compliant Non-compliant Benzo(a)pyrene ≤10 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected coliform bacteria ≤0.92 MPN / g <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 Staphylococcus aureus ≤0 / 25g / 25g Not detected Not detected Not detected Not detected Not detected Not detected Total bacterial count ≤30000 CFU / g <10 <10 <10 <10 <10 <10 Mold and yeast count ≤50 CFU / g <10 <10 <10 <10 <10 <10 salmonella ≤0 / 25g / 25g Not detected Not detected Not detected Not detected Not detected Not detected Aflatoxin B1 ≤10 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected Eicosapentaenoic acid (EPA) ≥0.58 g / 100g 1.4 1.4 1.4 1.2 1.1 1.2 Docosahexaenoic acid (DHA) ≥0.29 g / 100g 0.70 0.74 0.71 0.60 0.62 0.62 Disintegration time limit ≤60 min 6 6 6 6 6 6 cadmium ≤0.1 mg / kg Not detected Not detected Not detected Not detected Not detected Not detected lead ≤2.0 mg / kg 0.067 0.065 0.071 0.072 0.067 0.068 calcium 14.5-17.7 g / 100g 17.5 16.1 17.2 17.3 17.1 17.1 Total Mercury ≤0.3 mg / kg Not detected Not detected Not detected Not detected Not detected Not detected Total arsenic ≤1.0 mg / kg 0.552 0.536 0.535 0.541 0.534 0.537 Peroxide value (based on fat content) ≤0.25 g / 100g 0.043 0.039 0.041 0.157 0.166 0.158 Acid value (based on fat content) (KOH) ≤7.5 mg / g 2.3 2.3 2.3 6.7 6.7 6.8 PCBs ≤200 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected Table 5. Test results for the third month. Ash ≤50 % 39.3 39.2 38.9 39.1 39.3 39.2 Filling volume difference Compliant \ Compliant Compliant Compliant Non-compliant Non-compliant Non-compliant Benzo(a)pyrene ≤10 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected coliform bacteria ≤0.92 MPN / g <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 Staphylococcus aureus ≤0 / 25g / 25g Not detected Not detected Not detected Not detected Not detected Not detected Total bacterial count ≤30000 CFU / g <10 <10 <10 <10 <10 <10 Mold and yeast count ≤50 CFU / g <10 <10 <10 <10 <10 <10 salmonella ≤0 / 25g / 25g Not detected Not detected Not detected Not detected Not detected Not detected Aflatoxin B1 ≤10 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected Eicosapentaenoic acid (EPA) ≥0.58 g / 100g 1.4 1.4 1.4 1.0 0.9 1.0 Docosahexaenoic acid (DHA) ≥0.29 g / 100g 0.70 0.74 0.75 0.54 0.52 0.53 Disintegration time limit ≤60 min 6 6 6 7 8 8 cadmium ≤0.1 mg / kg Not detected Not detected Not detected Not detected Not detected Not detected lead ≤2.0 mg / kg 0.062 0.062 0.063 0.062 0.065 0.063 calcium 14.5-17.7 g / 100g 17.5 16.1 17.1 16.3 16.2 16.2 Total Mercury ≤0.3 mg / kg Not detected Not detected Not detected Not detected Not detected Not detected Total arsenic ≤1.0 mg / kg 0.523 0.540 0.534 0.540 0.525 0.535 Peroxide value (based on fat content) ≤0.25 g / 100g 0.053 0.050 0.051 0.261 0.285 0.262 Acid value (based on fat content) (KOH) ≤7.5 mg / g 2.5 2.5 2.5 7.8 8.5 7.6 PCBs ≤200 μg / kg Not detected Not detected Not detected Not detected Not detected Not detected Throughout the entire 3-month accelerated storage period, the test results for all items in Examples 1-3 met the preset technical requirements. Ash content, calcium content, total arsenic, lead, and other heavy metal indicators fluctuated slightly, remaining within the normal testing error range and generally stable. Benzo(a)pyrene, aflatoxin B1, and polychlorinated biphenyls were not detected. Total bacterial count, mold and yeast, coliform bacteria, Staphylococcus aureus, Salmonella, and other microbial indicators were consistently well below the limits, indicating stable and controllable product hygiene and safety.

[0055] The disintegration time limit increased slightly and slowly, but remained well below the upper limit of 60 min, indicating stable dissolution performance of the capsule shell. The content of characteristic active components EPA and DHA fluctuated very little, and the degradation was minimal. The peroxide value and acid value, which characterize the degree of lipid oxidation, increased only slightly and gradually with storage time, and remained well below the limit threshold until the end of storage, indicating that the oxidation of polyunsaturated fatty acids was effectively inhibited.

[0056] In terms of appearance, the soft capsules in Examples 1-3 were uniformly white throughout, without any deflated capsules, deformation, leakage, or stickiness; the internal contents were a uniform yellow to brownish-yellow oily substance, without layering, sedimentation, or visible foreign impurities, with a normal odor and no rancid smell, and the system had good homogeneity.

[0057] At the initial storage stage (month 0), the physicochemical indicators of Comparative Examples 1-3, such as microorganisms, toxins, heavy metals, ash, EPA, DHA, peroxide value, and acid value, were basically equivalent to those of the Examples. There were no significant differences in the appearance, color, and odor of the soft capsules and contents. However, due to the strong hydrophilicity of unmodified oyster calcium and its poor dispersibility and compatibility in the oil system, the density of the contents system was uneven, the flowability fluctuated greatly, the filling uniformity was difficult to control, and the filling volume difference did not meet the requirements from the beginning.

[0058] Starting from the first month of storage, the contents of the comparative soft capsules showed obvious oil phase separation with dark banded phase separation; EPA and DHA showed slight degradation, and the peroxide value and acid value increased at a significantly higher rate than in the example, indicating a significantly accelerated oil oxidation process.

[0059] By the second month of storage, the stratification problem in the comparative system had worsened further, the degradation of EPA and DHA had increased, and the peroxide value and acid value showed an accelerated upward trend, with the oxidative deterioration of the oils continuing to intensify.

[0060] After three months of storage, the peroxide values ​​of Comparative Examples 1-3 were 0.261 g / 100g, 0.285 g / 100g, and 0.262 g / 100g, respectively, exceeding the limit of ≤0.25 g / 100g; the acid values ​​were 7.8 mg / g, 8.5 mg / g, and 7.6 mg / g, respectively, exceeding the limit of ≤7.5 mg / g. Compared with the initial values, the overall decrease in EPA and DHA content in the comparative examples was approximately 30-35%. Although the content values ​​were still higher than the minimum content limit, considering the excessive levels of lipid oxidation indicators, continuous stratification of contents, persistent non-compliance with fill volume differences, and disruption of system homogeneity, the product could no longer meet the requirements for long-term storage stability. Furthermore, the disintegration time of the later comparative examples was slightly prolonged, presumably related to the viscous stratification of contents and the oxidative deterioration of the capsule shell due to prolonged contact, but the absolute values ​​were still far below the upper limit and did not pose a quality risk.

[0061] Unmodified oyster shell calcium exhibits strong hydrophilicity and poor compatibility with oil matrices. In oil systems, it is prone to particle aggregation and sedimentation, directly disrupting the homogeneous emulsion of the contents and causing phase separation and stratification. The micro-aqueous environment at the calcium carbonate-oil interface, coupled with trace metal ions in oyster shell calcium acting as catalysts, accelerates the oxidative decomposition of polyunsaturated fatty acids such as EPA and DHA, resulting in a continuous increase in acid value and peroxide value. Uneven system density further leads to non-compliance in filling volume, resulting in a continuous deterioration in overall product stability.

[0062] By modifying the surface of oyster calcium with hydrophobicity, the interfacial compatibility between inorganic calcium powder and oil matrix is ​​improved, ensuring long-term uniformity of contents without stratification, effectively delaying the oxidative degradation of unsaturated fatty acids, stabilizing the uniformity of the filling amount, and significantly improving the long-term storage stability of the composite soft capsules. This demonstrates that the surface hydrophobic modification effect of oyster calcium is the core key factor determining the compatibility and storage stability of this composite soft capsule system. Experiment Example 3: Animal Experiment

[0063] 1.1 Experimental animals: 48 SPF-grade female SD rats, weighing 250-270g, were housed in an environment with a temperature of 20-26 ℃, relative humidity of 30-70%, and alternating light and dark conditions for 12 hours. All rats had free access to food and water.

[0064] 1.2 Animal grouping: After the rats were adapted to feeding, they were randomly divided into 6 groups according to their body weight, with 8 rats in each group: osteoarthritis (OA) model group, OA + low-dose group, OA + medium-dose group, OA + high-dose group, OA + control group 3, and sham operation group.

[0065] 1.3 Establishment of a rat osteoarthritis model: A mixture of 4% (w / v) papain solution and 0.03 mol / L cysteine ​​solution was prepared by mixing at a 1:1 ratio and allowing to stand for 0.5 hours. After anesthetizing the rats, 0.2 mL of the mixture was injected into the joint cavities of both knees of rats in the osteoarthritis (OA) model group, OA + low-dose group, OA + medium-dose group, OA + high-dose group, and OA + control group. Injections were given every 2 days for a total of 2 weeks. Successful model establishment was indicated by knee swelling and impaired function. The sham-operated group received saline injection into the joint cavity.

[0066] 1.4 Drug Treatment: Gavage treatment began one week post-surgery. Before gavage, the capsules were ruptured and the contents were precisely administered by weight. Rats in the OA+ low, medium, and high dose groups were administered the composite soft capsules prepared in Example 3 of this invention by gavage at doses of 0.35 g / kg·BW, 0.70 g / kg·BW, and 2.10 g / kg·BW, respectively. The OA+ comparative group 3 received 2.10 g / kg·BW, which was equivalent to the high dose group. The OA model group and sham-operated group received only deionized water by gavage. Gavage was administered continuously for 8 weeks, with weekly weighing and adjustments to the gavage volume. All animals had free access to low-calcium feed and deionized water.

[0067] 2 Experimental Testing 2.1 Knee Joint Swelling Assessment: Before modeling, the diameter of the right knee joint of each group of rats was measured with calipers as the baseline diameter (mm) before modeling. The diameter of the right knee joint of each group of rats was measured before drug administration and at 4 and 8 weeks after drug administration, and the swelling degree of the right knee joint was calculated. The calculation formula was: Right knee joint swelling degree (%) = (Right knee joint diameter (mm) - Baseline diameter before modeling (mm)) / Baseline diameter before modeling (mm). The results are shown in Table 6.

[0068] Table 6. Effects of different gavage drugs on joint swelling in rats with osteoarthritis model. Model group 13.14±0.74 6.6±1.15 4.88±1.63 low-dose group 13.22±1.65 5.19±1.35 1.99±0.61 medium dose group 13.33±1.11 3.35±1.27 1.14±0.76 High-dose group 13.53±1.53 2.81±0.65 0.43±0.42 Comparative Example 3 Groups 13.75±1.28 3.58±0.26 2.26±0.35 Sham surgery group 5.87±2.18 0.35±0.29 0.38±0.26 As shown in Table 6, the natural remission of osteoarthritis in the model group of this invention was slow, and joint swelling persisted until week 8, indicating successful modeling. The sham-operated group showed no modeling damage; the acute joint swelling was caused by the sham surgery procedure and subsided and returned to normal after 4 weeks of administration. The high / medium dose group of this invention showed a continuous decrease in joint swelling after 8 weeks of administration, exhibiting a rapid and sustained anti-swelling effect. The joint swelling in the high-dose group after 8 weeks of administration was close to the normal level of the sham-operated group. The low-dose group was effective in relieving joint swelling, but the effect was lower than that in the medium / high dose groups, indicating that this invention is dose-dependent. The anti-swelling efficacy of the three comparative groups weakened from week 4 to week 8 of administration, presumably due to poor dispersibility of oyster calcium during storage. The unstable solid-liquid dispersion system led to the formation of catalytic sites for lipid oxidation at the solid-liquid interface, and the accumulation of micro-regions of water around the aggregated calcium particles easily accelerated the oxidative rancidity of the lipids, thereby destroying the stability of the core anti-inflammatory component, ω-3 fatty acids, and indirectly weakening the therapeutic effect.

[0069] In week 4, rats from each group were transferred to metabolic cages for calcium metabolism assays. The feed intake of the rats over 3 days was accurately measured to calculate calcium intake. Fecal samples were collected daily for 3 consecutive days to determine calcium content and calculate the apparent calcium absorption rate.

[0070] Table 7. Effects of different groups of intragastric administration drugs on calcium balance test in rats. low-dose group 290.63±5.29 43.59±0.79 13.25±0.65 1.59±0.3 65.95±0.9 medium dose group 296.66±6.17 89±1.85 29.54±0.74 1.99±0.21 64.58±0.31 High-dose group 303.47±8.62 273.12±7.76 93.99±3.21 2.89±0.09 64.53±0.64 Comparative Example 3 Groups 303.22±8.68 272.9±7.81 133.43±13.94 3.29±1.2 49.92±4.85 As shown in Table 7, the retention rate of pretreated powder calcium obtained by heat treatment after dry mixing of oyster calcium with mono- and diglycerides in Comparative Example 3 was lower than that of the low, medium, and high dosage groups. This indicates that hydrophobic modification with mono- and diglycerides alone cannot improve calcium retention. The improvement in calcium retention depends on the beeswax coating after pretreatment. Mono- and diglycerides pretreatment forms a hydrophobic film on the surface of oyster calcium, providing a basis for uniform and stable beeswax coating. However, it does not provide an effective sustained-release barrier. The beeswax layer forms a physical diffusion barrier in gastric juice, slowing the dissolution and diffusion rate of calcium ions, thus achieving sustained release. This avoids a sudden, large release of calcium in the stomach, allowing it to enter the intestines more smoothly for absorption, thereby significantly improving retention and reducing gastrointestinal irritation.

[0071] 2.3 Serum Indicator Detection: After the experiment, rats were anesthetized and blood was collected from the abdominal vein. Serum was separated by centrifugation at 3000 r / min for 10 min, and the levels of alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), osteocalcin (OC), type I procollagen amino acid propeptide (PINP), type I collagen C-terminal peptide (CTX-I), tartrate-resistant acid phosphatase (TRAP), osteoprotegerin (OPG), interleukins (IL-1β, IL-6, IL-8), and tumor necrosis factor-α (TNF-α) were measured. Experimental results are expressed as mean ± standard deviation, and the data were statistically analyzed using SPSS 20.0 software.

[0072] Table 8. Effects of different gavage drugs on serum inflammatory factors in rats with osteoarthritis. Model group 23.98±2.62 121.48±12.85 35.41±2.63 91.71±7.8 low-dose group <![CDATA[11.87±2.44 ** ]]> <![CDATA[102.73±11.49 *# ]]> <![CDATA[32.68±4.31 * ]]> <![CDATA[78.44±6.77 **## ]]> medium dose group <![CDATA[11.34±2.01 ** ]]> <![CDATA[89.09±20.76 ** ]]> <![CDATA[31.03±3.2 * ]]> <![CDATA[46.67±8.43 **## ]]> High-dose group <![CDATA[10.99±1.27 ** ]]> <![CDATA[88.9±16.19 ** ]]> <![CDATA[30.83±2.64 ** ]]> <![CDATA[41.35±8.09 **# ]]> Comparative Example 3 Groups <![CDATA[11.06±0.73 ** ]]> <![CDATA[90.72±16.36 ** ]]> <![CDATA[33.06±2.25 * ]]> <![CDATA[46.8±9.02 **## ]]> Sham surgery group <![CDATA[12.17±0.99 **# ]]> <![CDATA[81.44±19.97 * ]]> 31.47±2.95 <![CDATA[33.22±5.7 ** ]]> Note: * This indicates that P < 0.05 compared to the model group; ** This indicates that P < 0.01 compared to the model group; # This indicates a P<0.05 difference compared to the sham surgery group; ## This indicates that the P value was less than 0.01 compared to the sham surgery group.

[0073] All dosage groups of this invention significantly inhibited the levels of core inflammatory factors in osteoarthritis model rats. As shown in Table 8, the high, medium, and low dose groups of this invention significantly (P<0.01) reduced serum IL-1β and IL-8 levels, and the effect was not statistically different from that of the sham-operated group. This indicates that this invention can effectively inhibit the core inflammatory pathways closely related to the progression of osteoarthritis. The high and medium dose groups significantly reduced serum TNF-α levels, showing good anti-inflammatory effects. The low dose group also showed improvement, but the effect was weaker than that of the medium and high dose groups, indicating that the efficacy is dose-dependent. The three comparative groups were similar to the high dose group of this invention in controlling early inflammatory factors (IL-1β, TNF-α), but were weaker than the high dose group in maintaining long-term immune balance (such as the continuous control of IL-8) and deep bone mineralization repair (OC, SOX9 indicators). This is because the lack of beeswax-coated sustained-release effect cannot provide continuous and stable calcium signal support for cartilage repair. Calcium, as a second messenger, plays a key role in regulating immune processes such as T cell activation and macrophage polarization. Transient signals can easily lead to regulatory disruptions and affect the control of IL-8.

[0074] The sustained-release design of this invention enables continuous calcium release, which, combined with stabilized ω-3 fatty acids (EPA / DHA), works synergistically on common inflammatory pathways such as NF-κB and MAPK. This not only inhibits the excessive production of pro-inflammatory factors (such as TNF-α and IL-1β) but also suppresses the levels of other inflammatory mediators such as IL-8, achieving broader immune balance regulation. This dual optimization of the intervention effect on chronic inflammation of osteoarthritis in terms of both time dimension and pathway breadth.

[0075] Table 9. Effects of different gavage drugs on serum bone formation markers in rats with osteoarthritis. Model group 38.59±10.07 13.11±2.56 16.61±3.65 4.84±0.57 low-dose group 45.29±16.32 15.53±2.17 <![CDATA[20.51±4.21 ## ]]> <![CDATA[2.89±0.58 ** ]]> medium dose group 46.77±11.7 16.18±3.73 <![CDATA[21.23±5.68 ## ]]> <![CDATA[2.56±0.65 ** ]]> High-dose group <![CDATA[52.75±7.42 * ]]> <![CDATA[17.76±3.85 * ]]> <![CDATA[22.18±2.94 *## ]]> <![CDATA[2.52±0.63 ** <!-- 12 -->]]> Comparative Example 3 Groups <![CDATA[52.57±13.45 * ]]> <![CDATA[16.77±1.77 ** ]]> <![CDATA[21.85±4.58 **## ]]> <![CDATA[2.65±0.63 ** ]]> Sham surgery group <![CDATA[51.87±11.28 * ]]> <![CDATA[27.82±5.23 ** ]]> <![CDATA[22.12±4.66 ** ]]> <![CDATA[2.44±0.38 ** ]]> Note: * This indicates that P < 0.05 compared to the model group; ** This indicates that P < 0.01 compared to the model group; # This indicates a P<0.05 difference compared to the sham surgery group; ## This indicates that the P value was less than 0.01 compared to the sham surgery group.

[0076] Type I procollagen N-terminal propeptide (PINP) is one of the most specific markers of bone collagen synthesis, directly reflecting osteoblast activity and bone formation rate. Elevated levels indicate active bone matrix synthesis. Osteocalcin (OC), secreted by osteoblasts, is a key protein in bone mineralization and directly participates in calcium deposition. Its level reflects the late-stage function of osteoblasts and the activity of bone mineralization. Alkaline phosphatase (ALP) is secreted by osteoblasts in bone tissue and participates in bone mineralization. Elevated total ALP usually indicates enhanced overall osteoblast activity. Bone alkaline phosphatase (BALP), the bone-derived portion of ALP, is more specific and is a classic early marker of bone formation.

[0077] In the model group, serum levels of PINP, OC, and ALP were the lowest among all groups, confirming that osteoblast activity and bone formation were systematically inhibited under the pathological state of osteoarthritis. The highest level of bone alkaline phosphatase indicated an imbalance in bone metabolism and active compensatory osteoblast activity in osteoarthritis rats. In the low-dose group, serum levels of PINP, OC, and ALP were higher than in the model group (P<0.05), indicating a preliminary recovery of bone formation activity. BALP was significantly lower than in the model group (P<0.01), suggesting partial inhibition of abnormal bone turnover. However, ALP remained significantly lower than in the sham-operated group (P<0.01), indicating that bone metabolism had not fully recovered to normal. In the medium-dose group, serum indicators further improved, with PINP, OC, and ALP continuously increasing, while BALP continued to decrease (P<0.01 vs. model group). There was no significant difference in ALP between the two groups, indicating that bone mineralization activity was approaching normal. Compared with the model group, the serum levels of PINP, OC, and BALP in the high-dose group of rats were statistically significantly increased (P<0.05), demonstrating that it can effectively activate osteoblast function, promote bone collagen synthesis and mineralization, and effectively restore bone metabolic homeostasis. The increase in PINP and ALP in Comparative Example 3 was not substantially different from that in the high-dose group of this invention, while the regulatory effect on BALP was slightly lower than that in the medium and high-dose groups of this invention, indicating that bone metabolism tends to stabilize after gavage treatment. Although the increase in OC in Comparative Example 3 (16.77) was better than that in the model group, it was significantly lower than that in the sham-operated group (27.82), and slightly lower than that in the high-dose group of this invention (17.76). This indicates that due to the lack of a long-term stable dispersion system embedded in beeswax and the sustained-release effect of beeswax coating, the bioavailability of calcium in Comparative Example 3 could not be maintained during long-term treatment, resulting in an inability to support deep and continuous bone mineralization repair.

[0078] Table 10 Effects of different gavage drugs on serum bone resorption markers in osteoarthritis model rats Model group 35.9±2.8 812.52±95.03 334.41±45.54 low-dose group <![CDATA[32.9±3.17 ## ]]> <![CDATA[700.08±83.03 * ]]> <![CDATA[268.62±40.38 * ]]> medium dose group <![CDATA[33.25±3.19 ## ]]> <![CDATA[655.88±75.75 ** ]]> <![CDATA[267.78±13.91 ** ]]> High-dose group <![CDATA[34.76±1.57 ## ]]> <![CDATA[651.34±91.41 ** ]]> <![CDATA[258.41±31.98 ** ]]> Comparative Example 3 Groups <![CDATA[33.82±4.8 ## ]]> <![CDATA[663.31±85.14 * ]]> <![CDATA[259.69±35.24 ** ]]> Sham surgery group <![CDATA[27.04±1.91 ** ]]> <![CDATA[641.69±52.12 ** ]]> 233.5±45.51 Note: * indicates P<0.05 compared with the model group; ** indicates P<0.01 compared with the model group; # indicates P<0.05 compared with the sham surgery group; ## indicates P<0.01 compared with the sham surgery group.

[0079] Tartrate-resistant acid phosphatase (TRAP), primarily secreted by activated osteoclasts, is a direct marker of osteoclast activity and quantity. Elevated TRAP levels directly indicate enhanced bone resorption. Type I collagen C-terminal peptide (CTX-I) is a specific product of type I collagen degradation and one of the most sensitive and specific serum markers reflecting bone resorption rate. Elevated CTX-I levels indicate accelerated bone matrix degradation and vigorous bone resorption. Osteoporosis inhibitor (OPG), secreted by osteoblasts, is a negative regulator (decoy receptor) of osteoclast production. It binds to and neutralizes RANKL, thereby inhibiting osteoclast differentiation and activity. Elevated OPG levels are usually a compensatory protective response against excessive bone resorption.

[0080] The model group rats had the highest serum TRAP and CTX-I levels, indicating extremely active bone resorption. Simultaneously, OPG was also highest, representing a compensatory increase in the body's attempt to inhibit excessive bone resorption, but insufficient to offset the damage. The sham-operated group rats had the lowest serum TRAP, CTX-I, and OPG levels, indicating a balance between bone resorption and bone formation metabolism. The low-dose group rats had significantly lower serum CTX-I and OPG levels than the model group, indicating that bone resorption was beginning to be inhibited, but TRAP remained significantly higher than normal, indicating incomplete inhibition of osteoclast activity. The medium- and high-dose groups rats had reduced serum CTX-I to levels not statistically different from the sham-operated group, indicating that the bone resorption rate had essentially returned to normal. OPG decreased synchronously, indicating reduced compensatory pressure and enhanced inhibitory effect. The high-dose group rats effectively inhibited abnormally high bone resorption to near-normal levels. The three comparative groups showed effects on inhibiting bone resorption that were essentially equivalent to the medium- and high-dose groups of this invention. CTX-I and OPG both returned to normal ranges.

[0081] 2.4 Western blot detection: Rat cartilage tissue samples were ground with liquid nitrogen and proteins were extracted. The relative expression levels of matrix metalloproteinase-13 (MMP-13), platelet-reactive protein motif-5 (ADAMTS-5), and SRY-associated high-mobility group 9 (SOX9) proteins in the cartilage tissue of each group of rats were detected.

[0082] Table 11 Effects of different gavage drugs on protein expression in cartilage tissue of osteoarthritis model rats Model group 0.72±0.03 6.62±0.9 0.38±0.06 low-dose group 0.62±0.03 4.45±0.26 0.59±0.17 medium dose group 0.57±0.04 2.84±0.41 0.81±0.05 High-dose group 0.49±0.02 2.55±0.28 0.83±0.03 Comparative Example 3 Groups 0.53±0.04 2.94±0.35 0.73±0.05 Sham surgery group 0.32±0.04 1.18±0.28 0.99±0.08 Matrix metalloproteinase-13 (MMP-13) is the most critical enzyme for degrading type II collagen (the main structural protein of the cartilage matrix). Increased MMP expression directly leads to the disruption of the cartilage matrix's structural network and is a core marker of cartilage degeneration and defects. Platelet-reactive protein motif-5 (ADAMTS-5) is the most important proteolytic enzyme for degrading proteoglycans (responsible for maintaining cartilage moisture and elasticity). Increased ADAMTS activity leads to significant proteoglycan loss, resulting in loss of cartilage elasticity and compressive strength. SRY-associated high-mobility group box 9 (SOX9) is a core transcription factor regulating chondrocyte differentiation, survival, and synthesis of matrix components such as type II collagen and proteoglycans; its expression level directly reflects the chondrocyte's synthetic repair capacity.

[0083] In the model group rats, the relative expression levels of MMP-13 and ADAMTS-5 proteins were the highest, while the relative expression level of SOX9 protein was the lowest. This indicates that the model group rats exhibit a typical osteoarthritis-related cartilage pathological state: extremely high activity of destructive enzymes and severely impaired self-repair capacity of chondrocytes. Compared with the model group, the relative expression levels of MMP-13 and ADAMTS-5 proteins in the low, medium, and high dose groups and the three control groups were significantly lower in the cartilage tissue samples, while the relative expression level of SOX9 protein was significantly higher. The data from the high-dose group were similar to those of the sham-operated group, indicating that the high-dose group can effectively block the cartilage degradation pathway and almost completely restore the synthetic function of chondrocytes, thus improving cartilage damage caused by osteoarthritis and maintaining the structural stability of diseased cartilage. The three control groups showed comparable effects to the high-dose group in inhibiting destructive factors (MMP-13 and ADAMTS-5), but were lower than the high-dose group and the sham-operated group in increasing the repair factor SOX9.

[0084] This invention's sustained-release formulation achieves superior regulation of the "destruction-repair" dual process of articular cartilage by providing a continuously stable bioactive environment. This invention not only effectively inhibits key enzymes leading to cartilage degradation but also deeply activates the chondrocytes' own synthetic repair capabilities (high expression of SOX9), thus achieving a leap from "preventing destruction" to "active repair." The shortcomings of Comparative Example 3 in promoting deep repair further confirm that maintaining a long-term stable local microenvironment is crucial for achieving structural joint repair.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling, and maintaining cartilage health, characterized in that: Step 1: Mix 280-320 parts by weight of oyster calcium with 3-6 parts by weight of mono- and diglycerides of fatty acids, then heat to 100-110℃ and maintain for 5 minutes to obtain a pretreated powder. Step 2: Add 25-30 parts by weight of molten beeswax to 600-800 parts by weight of ethyl acetate preheated to 40-50℃, and stir evenly under sealed conditions to obtain a beeswax solution. Step 3: Add the pretreated powder to the beeswax solution and stir at high speed until homogeneous to obtain a suspension. Step 4: Slowly add [the following to be added] dropwise to the suspension under stirring. 300-400 parts by weight of anhydrous ethanol, homogenized under high pressure, then cooled to -20℃ until completely solidified, with continuous stirring during cooling to prevent caking, solvent removed by filtration, and vacuum dried to obtain oyster calcium modified powder; Step 5, add oyster calcium modified powder and 10 parts by weight of colostrum basic protein to 260-295 parts by weight of perilla seed oil, mix and stir evenly, then add 80-90 parts by weight of krill oil, homogenize under high pressure to obtain the contents; Step 6, prepare capsule shell, compress into pellets, to obtain a composite soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health.

2. The method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health according to claim 1, characterized in that: Step 1: Mix 300 parts by weight of oyster calcium with 3-6 parts by weight of mono- and diglycerides of fatty acids, then heat to 100-110℃ and maintain for 5 minutes to obtain a pretreated powder. Step 2: Add 28 parts by weight of molten beeswax to 600-800 parts by weight of ethyl acetate preheated to 40-50℃, and stir evenly under sealed conditions to obtain a beeswax solution. Step 3: Add the pretreated powder to the beeswax solution and stir at high speed until homogeneous to obtain a suspension. Step 4: Slowly add [the pretreated powder] dropwise to the suspension under stirring. 300-400 parts by weight of anhydrous ethanol were homogenized under high pressure and then cooled to -20°C until completely solidified. During the cooling process, the mixture was continuously stirred to prevent caking. The solvent was removed by filtration and vacuum drying to obtain oyster calcium modified powder. Step 5: The oyster calcium modified powder and 10 parts by weight of colostrum basic protein were added to 277 parts by weight of perilla seed oil and mixed evenly. Then, 85 parts by weight of krill oil were added and homogenized under high pressure to obtain the contents. Step 6: The capsule shell was prepared, and the capsules were pressed to obtain a composite soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health.

3. The method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health according to claim 2, characterized in that: In step 2, 28 parts by weight of molten beeswax are added to 700 parts by weight of ethyl acetate preheated to 48°C, and stirred evenly under sealed conditions to obtain a beeswax solution.

4. A method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling, and maintaining cartilage health according to claim 2 or 3, characterized in that: The stirring speed in step 3 is 1500-3000 rpm, and the temperature is 40-50℃.

5. The method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health according to claim 4, characterized in that: In step 4, 350 parts by weight of anhydrous ethanol are added at a dropping rate of 15-20 g / min under stirring conditions of 40-50℃ and 500-800 rpm.

6. The method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health according to claim 5, characterized in that: In step 4, the homogenization temperature is 45-50℃ and the homogenization pressure is 40 MPa.

7. The method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health according to claim 6, characterized in that: In step 4, the cooling rate is 5-10℃ / h, and the stirring is continuous during the cooling process at a stirring rate of 120r / min. After cooling to -20℃, the mixture is allowed to stand for 1-2 hours and then filtered to remove the solvent.

8. The method for preparing a compound soft capsule for relieving osteoarthritis, joint swelling and maintaining cartilage health according to claim 5, characterized in that: The vacuum drying conditions in step 4 are 32-35℃ and -0.08MPa.

9. A compound soft capsule for relieving osteoarthritis, joint swelling, and maintaining cartilage health, characterized in that: Prepared by the method described in any one of claims 1-8.

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