Stable Pickering emulsion based on D-glucosamine-magnesium compound as well as preparation method and application of stable Pickering emulsion

Abstract

The invention provides a stable Pickering emulsion based on a D-glucosamine-magnesium compound, a preparation method and application, and relates to the technical field of emulsion preparation. An emulsion system is stabilized through a D-glucosamine-magnesium compound, collaborative delivery of D-glucosamine and magnesium ions is completed, Mg < 2 + > and amino groups and hydroxyl groups of the D-glucosamine form weak coordinate bonds, Ostwald ripening and gravity settling in long-term storage are resisted, it is ensured that oil drops are uniform in size, and the shelf life is prolonged. Through weak coordinate bonds and response of pH response synthase, efficient release of targeted intestinal tracts is realized, and bioavailability is improved. Meanwhile, due to the large hydration radius of Mg < 2 + >, a thick hydration layer is formed on the surface of Mg < 2 + >, mechanical damage of ice crystals to an interfacial film is inhibited at low temperature, and film breakage caused by particle dehydration is slowed down at high temperature; coordination bonds in the D-glucosamine-magnesium compound are stably adsorbed at low temperature and moderately dissociated at high temperature without damaging the overall structure, so that the stable temperature range of the emulsion is further widened.

Description

Technical Field

[0001] The present invention relates to the technical field of emulsion preparation, and in particular to a Pickering emulsion stabilized by a D-glucosamine-magnesium complex, a preparation method and an application thereof. Background Art

[0002] In the field of drug delivery, traditional emulsions, as commonly used drug carriers, are relatively simple to prepare and can encapsulate a wide range of drugs, meeting some of the drug delivery needs to a certain extent. However, traditional emulsions rely on surfactants to maintain stability. Surfactants can not only cause toxicity and irritation issues, but their stability is also easily affected by external environmental factors such as temperature and pH. During storage and use, emulsion stratification and demulsification are prone to occur, which greatly limits their further application in drug delivery. In addition, the release rate of existing emulsions in simulated gastrointestinal digestion is uncontrollable, making it difficult to meet the needs of nutrient delivery.

[0003] Compared to traditional emulsions, Pickering emulsions offer advantages such as low toxicity, environmental friendliness, relatively high stability, and tunable particle surface properties, showing broad application prospects in the field of drug delivery. As a solid particle-stabilized emulsion system, Pickering emulsions can achieve targeted drug delivery, control drug release rate, and increase drug loading. These solid particles can adsorb at the oil-water interface, forming a stable interfacial film and thus stabilizing the emulsion. Common solid stabilizing particles include silica, clay, proteins (such as gelatin and whey protein), and polysaccharides (such as chitosan and cellulose). However, proteins are susceptible to pH and temperature, leading to emulsion instability. Polysaccharide particles have low emulsification efficiency and require high concentrations to stabilize the emulsion.

[0004] Glucosamine (GlCN) is a commonly used ingredient in cartilage repair. When joint cartilage is damaged, exogenous glucosamine supplementation can serve as a raw material for cartilage matrix synthesis. However, after oral administration, glucosamine is subject to the action of various digestive enzymes in the gastrointestinal tract, partially destroying its structure. Furthermore, its absorption efficiency by the gastrointestinal mucosa is limited, and it undergoes first-pass metabolism in the liver before entering the bloodstream, causing a large amount of the drug to be metabolized and inactivated. This results in extremely low bioavailability of oral glucosamine, typically less than 20%. 2+ It can reduce local inflammatory response and relieve pain symptoms. 2+ Precise delivery to the joint cavity is difficult. With conventional routes of administration, such as oral or intravenous injection, only a very small amount of the drug can passively diffuse into the joint cavity after being distributed throughout the body, making it difficult to achieve an effective therapeutic concentration within the joint cavity. Furthermore, systemic administration can cause numerous adverse reactions, limiting its clinical application.

[0005] Therefore, developing a Pickering emulsion with high safety, good stability, strong environmental adaptability and targeted delivery is of great significance for promoting its practical application in the field of drug delivery. Summary of the Invention

[0006] In view of the technical problems existing in the prior art, the present invention aims to provide a Pickering emulsion stabilized by a D-glucosamine-magnesium complex, a preparation method and an application thereof.

[0007] One of the objects of the present invention is to provide a method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex, comprising the following steps: Preparation of D-glucosamine-magnesium complex; uniformly dispersing the D-glucosamine-magnesium complex in water to form a complex dispersion; The oil component is added to the complex dispersion and homogenized to obtain a Pickering emulsion stabilized by the D-glucosamine-magnesium complex.

[0008] Preferably, the molar ratio of D-glucosamine to magnesium in the D-glucosamine-magnesium complex is 1:1.

[0009] Preferably, the mass volume fraction of the D-glucosamine-magnesium complex in the complex dispersion is 0.3-1.3%.

[0010] Preferably, the oil component comprises vegetable oil and / or biological oil; the vegetable oil comprises one or more of soybean oil, olive oil or almond oil; the biological oil comprises one or more of fish oil, beef tallow or sheep oil.

[0011] Preferably, the volume ratio of the oil component to the complex dispersion is 3-8:2-7.

[0012] Preferably, the speed of the homogenization process is 5000~25000 rpm.

[0013] Preferably, the homogenization time is 2 to 4 minutes.

[0014] The second object of the present invention is to provide a Pickering emulsion prepared by the preparation method of the Pickering emulsion stabilized by the D-glucosamine-magnesium complex as described above.

[0015] Preferably, the raw materials of Pickering emulsion further include auxiliary functional ingredients; the auxiliary functional ingredients include one or more of hyaluronic acid, hyaluronic acid, vitamin C, chondroitin sulfate, curcumin, resveratrol, chlorogenic acid or asiaticoside.

[0016] A third object of the present invention is to provide a use of the Pickering emulsion described above in the preparation of medicines for treating osteoarthritis.

[0017] Beneficial effects of the present invention: The present invention stabilizes the emulsion system by using a D-glucosamine-magnesium complex to achieve the coordinated delivery of D-glucosamine and magnesium ions without using surfactants, thereby reducing potential toxic side effects and being safer for the human body. 2+ It forms a weak coordination bond with the amino and hydroxyl groups of D-glucosamine (GLCN), thus avoiding the 2+ 、Cu 2+ Strong coordinating ions can easily lead to the problem of particle rigidity agglomeration. After entering the weak alkaline environment of the intestine (pH 7.0-8.5), the protonation degree of the amino group of GLCN decreases, the coordination bond dissociates, and the Mg is released. 2+ At the same time, digestive enzymes in the intestine can degrade GLCN molecules and accelerate the disintegration of the complex. The D-glucosamine-magnesium complex achieves efficient release in the targeted intestine through weak coordination bonds and pH-responsive synthase response, thereby improving bioavailability. The elastic network interface membrane formed by the dynamic coordination bonds in the D-glucosamine-magnesium complex can resist Ostwald ripening and gravity sedimentation during long-term storage, ensuring the uniformity of oil droplet size and significantly extending the shelf life.

[0018] At the same time, Mg 2+ The large hydration radius forms a thick hydration layer on its surface, which inhibits the mechanical damage of ice crystals to the interfacial film at low temperatures and slows down the membrane rupture caused by particle dehydration at high temperatures; the coordination bonds in the D-glucosamine-magnesium complex are stably adsorbed at low temperatures and moderately dissociated at high temperatures without destroying the overall structure, further broadening the stable temperature range of the emulsion and solving the problem of easy demulsification of traditional strong coordination systems in cold chain or high-temperature processing. It is suitable for cold chain food or high-temperature processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The photographs show that the GLCN-Mg Pickering emulsion was dropped into a sample bottle containing an equal volume of ultrapure water or soybean oil, and the photographs show that the GLCN-Mg Pickering emulsion was dropped into a sample bottle containing ultrapure water and soybean oil, respectively; Figure 2 The appearance and droplet morphology of GLCN-Mg PE at different homogenizer speeds; Figure 3 is the appearance and droplet morphology of GLCN-Mg PE with different GLCN-Mg mass volume fractions; Figure 4 is the appearance and droplet morphology of GLCN-Mg PE with different oil-water ratios; Figure 5 is the centrifugal stability and average particle size of GLCN-Mg PE at different homogenizer speeds; Figure 6is the centrifugal stability and average particle size of GLCN-Mg PE with different GLCN-Mg mass volume fractions; Figure 7 is the centrifugal stability and average particle size of GLCN-Mg PE with different oil-water ratios; Figure 8 is the temperature stability of GLCN-Mg PE at different homogenizer speeds; Figure 9 is the temperature stability of GLCN-Mg PE with different GLCN-Mg mass volume fractions; Figure 10 is the temperature stability of GLCN-Mg PE with different oil-water ratios; Figure 11 This is the appearance of GLCN-Mg PE at different homogenizer speeds after storage at room temperature for 30 days; Figure 12 This is the appearance of GLCN-Mg PE with different GLCN-Mg mass volume fractions after storage at room temperature for 30 days; Figure 13 This is the appearance of GLCN-Mg PE with different oil-water ratios after storage at room temperature for 30 days; Figure 14 is the gastrointestinal digestibility of GLCN-Mg PE at different homogenizer speeds; Figure 15 is the gastrointestinal digestibility of GLCN-Mg PE with different GLCN-Mg mass volume fractions; Figure 16 is the gastrointestinal digestibility of GLCN-Mg PE with different oil-water ratios. DETAILED DESCRIPTION

[0020] According to a first aspect of the present invention, there is provided a method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex, comprising the following steps: Preparation of D-glucosamine-magnesium complex; uniformly dispersing the D-glucosamine-magnesium complex in water to form a complex dispersion; The oil component is added to the complex dispersion and homogenized to obtain a Pickering emulsion stabilized by the D-glucosamine-magnesium complex.

[0021] In the present invention, the amino group, hydroxyl group and Mg 2+ Form coordination bonds to form complex particles with specific spatial structures. 2+ As a divalent cation, it has a moderate charge density and a moderate coordination bond strength with GLCN, which is weaker than that of Zn 2+ 、Cu 2+The strong coordination bond of Mg makes the adsorption-desorption equilibrium of the composite particles at the oil-water interface easier to control, and can form a dense adsorption layer at the interface at a lower concentration, effectively preventing the aggregation and fusion of oil droplets. 2+ The coordination effect of GLCN makes the hydrophobic groups of GLCN molecules oriented on the particle surface, and the hydrophilic groups face the water phase, forming a "hydrophilic-hydrophobic" dual-functional interface layer, reducing the interfacial tension while enhancing the steric effect. Compared with other metal ions that may cause excessive hydrophobicity or hydrophilic aggregation, GLCN has better stability. 2+ 、Cu 2+ The coordination system is prone to form rigid aggregates due to the strong coordination bond of metal ions, and a higher particle concentration is required to stabilize the emulsion. 2+ The weak bonding characteristics of coordination achieve "low concentration, high efficiency and stability" and reduce costs.

[0022] In the present invention, Mg 2+ The coordination bond with GLCN is reversible when the temperature changes. At low temperatures, the coordination bond remains stable to avoid particle desorption. At high temperatures, the Mg 2+ The hydration radius is large, the coordination bond length is long, the thermal motion tolerance is stronger, and it slightly dissociates at high temperature without destroying the overall interface film structure; Mg 2+ The strong hydration of ZnO forms a thicker hydration layer on the surface of the composite particles. At high temperatures, the hydration layer can slow down the dehydration and aggregation of the particles. At low temperatures, the thicker hydration layer inhibits the mechanical damage of ice crystals to the interfacial film, broadening the temperature range of the emulsion's stability and enabling the emulsion to maintain the integrity of the interfacial film during cold chain or high-temperature processing. 2+ 、Cu 2+ The coordination system has strong rigidity of coordination bond, and the interface film is easily broken due to particle dehydration at high temperature. At low temperature, the hydration layer is thin and easily impacted by ice crystals, and the temperature range stability is poor. 2+ The system achieves stability in a wide temperature range through "flexible coordination + hydration protection".

[0023] In the present invention, the adsorption layer formed by the D-glucosamine-magnesium complex at the interface has an "elastic network structure" that can both combine and slightly dissociate, similar to the adsorption force of a magnet, which can attract when close together and separate when slightly forced but still maintain contact. This characteristic makes the connection between the complex particles like a "spring net", which has a certain strength and can be flexibly deformed. 2+The dynamic reversibility of coordination bonds allows for weak cross-linking between particles. When a small oil droplet approaches a larger one, the "spring net" of the composite particles at the interface adaptively adjusts due to the dynamic coordination bonds. The connections between the particles can be slightly stretched or broken, and quickly rearranged at the interface of the newly contacted oil droplet, forming a new protective layer that prevents the oil droplets from merging and resists Ostwald ripening during long-term storage. When the oil droplet floats slightly upward due to gravity, the network can deform slightly without breaking, like an elastic net holding the oil droplet. The connections between the particles can adjust with the slight movement of the oil droplet, maintaining the overall structure while allowing for slight position changes, avoiding large-area aggregation caused by the fracture of the rigid structure, ensuring the uniformity of the oil droplet size, and extending the shelf life.

[0024] In the present invention, Mg 2+ The coordination dissociation energy with GLCN is low, and it is easier to trigger dissociation under mild intestinal conditions. The D-glucosamine-magnesium complex is stable under neutral conditions, and the coordination bond is not dissociated at this time. However, after entering the weak alkaline environment of the intestine (pH 7.0-8.5), the protonation degree of the amino group of GLCN decreases, the coordination bond dissociates, and Mg is released. 2+ At the same time, digestive enzymes in the intestine can degrade GLCN molecules and accelerate the disintegration of the complex. The triple effect makes Mg 2+ The release rate is >80%. Compared with other systems that rely on the strong acid environment in the stomach for release, the D-glucosamine-magnesium complex achieves efficient intestinal targeted release through weak coordination bonds and pH-responsive synthase response, thereby improving bioavailability.

[0025] In the present invention, Pickering emulsion relies on solid particle D-glucosamine-magnesium complex to stabilize the interface, without adding traditional surfactants, thus avoiding the cytotoxicity and mucosal irritation of surfactants. GLCN is an amino sugar naturally present in synovial fluid, Mg 2+ It is an essential cation in the human body. The complex has no chemically synthesized components, and its degradation products are all endogenous substances with no obvious stimulation to human tissues.

[0026] In inflammatory response, Mg 2+It can reduce the local inflammatory response and relieve pain symptoms by inhibiting the activation and aggregation of inflammatory cells and reducing the synthesis and release of inflammatory mediators such as prostaglandins and leukotrienes. Glucosamine participates in the metabolic activities of chondrocytes, promotes the synthesis of cartilage matrix, and inhibits the degradation of cartilage matrix by matrix metalloproteinases, thereby maintaining the structural and functional integrity of cartilage. When articular cartilage is damaged due to wear, trauma or disease, exogenous glucosamine supplementation can be used as a raw material for synthesizing cartilage matrix, assisting in the repair and regeneration of cartilage. Pickering emulsion stabilized by D-glucosamine-magnesium complex improves stability and environmental adaptability through D-glucosamine-magnesium complex, while also achieving the synergistic delivery of D-glucosamine and magnesium ions, thereby improving the therapeutic effect of arthritis drugs.

[0027] In a preferred embodiment of the present invention, the molar ratio of D-glucosamine to magnesium in the D-glucosamine-magnesium complex is 1:1.

[0028] In the present invention, the 1:1 molar ratio directly determines the surface hydrophilicity and hydrophobicity balance of the composite particles, which is the key to the stability of the Pickering emulsion. Each GLCN molecule fixes a Mg 2+ After that, Mg 2+ The hydration of Mg makes the coordinated ionized groups, i.e., hydrophilic groups, face the water phase, while the hydrophobic structure of the sugar ring of GLCN is oriented on the particle surface, forming a stable "hydrophilic-hydrophobic" dual-functional interface layer. 2+ If the amount is excessive, the particle surface may become too hydrophilic or the coordination bonds may be excessively cross-linked, forming rigid aggregates. If the amount of GLCN is excessive, the hydrophobic groups may be insufficiently exposed, thereby reducing the interfacial adsorption capacity.

[0029] Mg 2+ The coordination bond with a single GLCN molecule is neither too strong nor too weak, forming a controllable adsorption-desorption equilibrium at the oil-water interface. The coordination bond is stable at low temperatures and slightly dissociates at high temperatures without destroying the overall structure. 2+ Only one GLCN is coordinated, and the dissociation energy is uniform, avoiding rigid aggregation or excessive dissociation caused by multi-molecule cross-linking. In the weak alkaline environment of the intestine, a single GLCN molecule and Mg 2+ The coordination bond is easier to dissociate, combined with enzyme degradation, to ensure that Mg 2+ Release rate>80%. If Mg 2+ Coordination with multiple GLCNs may form multinuclear complexes, which increases the difficulty of dissociation and reduces the release efficiency.

[0030] In the present invention, the preparation method of the D-glucosamine-magnesium complex specifically includes: weighing 1.0 g of GLCN-HCl and 0.5582 g of anhydrous MgSO4 in 20 mL of distilled water, stirring at room temperature for 1 hour, adjusting the pH value to 6.5 with a NaOH solution, and then allowing to react for 4 hours, adding 3 times the volume of acetone, allowing to stand, discarding the supernatant, adding anhydrous methanol, stirring, filtering, and repeatedly washing the filter residue with anhydrous ethanol until it is no longer viscous, and drying through ventilation to obtain a white powder, which is recrystallized in ethanol to obtain the D-glucosamine-magnesium complex.

[0031] In a preferred embodiment of the present invention, the mass volume fraction of the D-glucosamine-magnesium complex in the complex dispersion is 0.3-1.3%.

[0032] In the present invention, the Pickering emulsion relies on solid particles to form a tightly adsorbed interfacial film at the oil-water interface to prevent oil droplets from aggregating. If the concentration of the complex is less than 0.3%, the number of particles is insufficient to completely cover the surface of the oil droplets, resulting in an incomplete interfacial film. The oil droplets are easily demulsified due to collision, merging, or gravity sedimentation. The D-glucosamine-magnesium complex stabilizes the interface through coordination bonds and hydration layers. A sufficient particle concentration is required to form a continuous interfacial adsorption layer to ensure that there are enough complex particles adsorbed on the surface of each oil droplet to inhibit Ostwald ripening and aggregation. When the concentration is higher than 1.3%, the interaction between particles in the solution is enhanced, and the particles may agglomerate due to coordination bonds or hydrogen bonds to form irreversible aggregates, which in turn reduces the number of effectively dispersed particles and reduces the interfacial adsorption efficiency. Excessively high particle concentrations will increase the viscosity of the water phase, affecting the dispersion effect of the oil droplets during the homogenization process. The oil droplets are difficult to be sheared and refined, and at the same time, the interfacial film is too thick or too rigid, losing the dynamic adjustment ability, and destroying the stability of the emulsion.

[0033] In a preferred embodiment of the present invention, the oil component comprises vegetable oil and / or biological oil; The vegetable oil includes one or more of soybean oil, olive oil or almond oil; The biological oil includes one or more of fish oil, beef tallow or sheep oil.

[0034] In a preferred embodiment of the present invention, the volume ratio of the oil component to the complex dispersion is 3-8:2-7.

[0035] In the present invention, the stability of the Pickering emulsion depends on the adsorption of the complex particles at the interface of the oil droplets, and the total interface area is positively correlated with the volume of the oil phase. If the volume ratio of the oil component and the complex dispersion is less than 3:7, the oil component is too little, the total interface area is insufficient, and the complex particles are excessive, resulting in agglomeration or liberation of the particles in the water phase, which cannot be effectively utilized; too much water phase will dilute the particle concentration, below the critical stability concentration, and the interface coverage will be insufficient. If the volume ratio of the oil component and the complex dispersion is greater than 8:2, the oil phase volume is too large, the total interface area increases dramatically, and even if the complex concentration reaches the upper limit, the number of particles may still not be able to completely cover the surface of the oil droplets, resulting in an incomplete interface film and easy aggregation of the oil droplets; too little water phase will make the complex concentration too high, causing particle agglomeration or a sudden increase in the viscosity of the water phase, hindering homogeneous dispersion.

[0036] The experimentally determined mass volume fraction of the complex (0.3-1.3%) corresponds to its effective concentration in the aqueous phase and must be matched to the oil phase volume to achieve a balance between particle concentration and interfacial area. When the oil:water ratio is 3:7, the aqueous phase volume is large, and the complex concentration needs to be close to the lower limit (0.3%) to cover the relatively small interface. When the oil:water ratio is 8:2, the aqueous phase volume is small, and the complex concentration needs to be close to the upper limit (1.3%) to accommodate the larger interfacial area and ensure sufficient particle adsorption per unit interface. Increasing the oil phase ratio increases the probability of collisions between oil droplets, requiring the interfacial film to possess higher extrusion resistance. Within the oil phase ratio range of 3-8, the elastic network membrane formed by dynamic coordination bonds maintains membrane integrity through reversible dissociation and recombination of coordination bonds, preventing fracture and coalescence. Outside this range, the interfacial film either lacks crosslinking due to insufficient stress or lacks density due to excessive stress, resulting in decreased stability.

[0037] The volume ratio of 3~8:2~7 is the optimal balance range among the oil phase interface area requirement, the concentration of the complex particles, the mechanical properties of the interfacial film, and the process feasibility. The lower limit of 3:7 ensures that the oil phase has sufficient volume to form an effective emulsion while avoiding excessive agglomeration of the complex. The upper limit of 8:2 relies on the high concentration of the complex (1.3%) and the strength of the dynamic coordination bond to maintain interface stability under high oil phase loading and prevent oil droplet aggregation.

[0038] In a preferred embodiment of the present invention, the rotation speed of the homogenization process is 5000-25000 rpm.

[0039] In the present invention, if the rotation speed is lower than 5000rpm, the shear force is not enough to overcome the oil-water interfacial tension, resulting in the inability to effectively break the oil phase into small droplets. When it is lower than 5000rpm, the average particle size of the oil droplets is greater than 10μm, the total interface area is insufficient, and the composite particles with a mass volume fraction of 0.3~1.3% cannot completely cover the surface of the oil droplets. After the emulsion has been allowed to stand for 24 hours, obvious stratification occurs. Sufficient shear force can quickly update the oil-water interface, prompting the composite particles to be adsorbed to the newly generated interface in time to form a stable particle film. At low rotation speeds, the interface is updated slowly, and the particles are prone to agglomeration in the aqueous phase, reducing the interface coverage. A rotation speed greater than 25000rpm will generate extremely strong shear force, which will destroy the composite particle structure, and the glycosidic bond or coordination bond of D-glucosamine may break, Mg 2+ The binding with the ligand is weakened and the interfacial adsorption capacity decreases; the mechanical stress generated by high-speed shear causes uneven charge distribution on the particle surface, triggering secondary agglomeration and forming particle clusters instead of monodisperse adsorption, which increases the risk of bridging agglomeration between oil droplets.

[0040] In the present invention, the rotation speed of the homogenization process is 20000 rpm.

[0041] In a preferred embodiment of the present invention, the homogenization time is 2 to 4 minutes.

[0042] In the present invention, the homogenization time is 3 minutes.

[0043] According to a second aspect of the present invention, a Pickering emulsion prepared by the method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex as described above is provided.

[0044] In a preferred embodiment of the present invention, the raw materials of Pickering emulsion further include auxiliary functional ingredients; the auxiliary functional ingredients include one or more of hyaluronic acid, hyaluronic acid, vitamin C, chondroitin sulfate, curcumin, resveratrol, chlorogenic acid or asiaticoside.

[0045] A third object of the present invention is to provide a use of the Pickering emulsion described above in the preparation of medicines for treating osteoarthritis.

[0046] Example The present invention will be further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the scope of protection of the present invention is not limited thereby. Unless otherwise specified, the materials and instruments used in the following examples are commercially available.

[0047] The preparation method of Pickering emulsion stabilized by D-glucosamine-magnesium complex comprises the following steps: 1. Preparation of GLCN-Mg 1.0 g of GLCN-HCl and 0.5582 g of anhydrous MgSO4 were weighed separately in 20 mL of distilled water, stirred at room temperature for 1 h, and then the pH value was adjusted to 6.5 with NaOH solution. The reaction was allowed to react for 4 h, and 3 times the volume of acetone was added. The mixture was allowed to stand, and the supernatant was discarded. Anhydrous methanol was added and stirred, filtered, and the filter residue was repeatedly washed with anhydrous ethanol until it was no longer viscous. The powder was then ventilated and dried to obtain a white powder, which was recrystallized from ethanol to obtain D-glucosamine-magnesium complex (GLCN-Mg).

[0048] 2. Preparation of Emulsion A D-glucosamine-magnesium complex with a mass volume fraction of 0.3-1.3% was weighed and uniformly dispersed in water to form a complex dispersion; soybean oil was added to the complex dispersion with a volume ratio of the oil component to the complex dispersion of 3-8:2-7, and homogenized using a homogenizer at a speed of 5000-25000 rpm for 3 minutes to obtain a Pickering emulsion stabilized by the D-glucosamine-magnesium complex.

[0049] Preparation of GLCN-Mg PE at different homogenizer speeds A D-glucosamine-magnesium complex with a mass volume fraction of 0.7% (w / v) was weighed and uniformly dispersed in water to form a complex dispersion; soybean oil was added to the complex dispersion, and the volume ratio of the oil component to the complex dispersion was 7:3; a homogenizer was used to homogenize at speeds of 5000, 10000, 15000, 20000 and 25000 rpm for 3 minutes to obtain GLCN-Mg PE prepared at five different homogenizer speeds.

[0050] Preparation of GLCN-Mg PE with different GLCN-Mg mass volume fractions D-glucosamine-magnesium complex with a mass volume fraction of 0.3%, 0.5%, 0.7%, 0.9%, 1.1% or 1.3% (w / v) was weighed respectively and then uniformly dispersed in water to form a complex dispersion; soybean oil was added to the complex dispersion, and the volume ratio of the oil component to the complex dispersion was 7:3; a homogenizer was used to homogenize at a speed of 20,000 rpm for 3 minutes to obtain six GLCN-Mg PEs with different GLCN-Mg concentrations.

[0051] Preparation of GLCN-Mg PE with different oil-water ratios A 0.7% (w / v) D-glucosamine-magnesium complex was weighed and uniformly dispersed in water to form a complex dispersion. Soybean oil was then added to the complex dispersion at oil-to-complex ratios of 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2. Six different GLCN-Mg PEs with different oil-to-water ratios were prepared by homogenizing the mixture at 20,000 rpm for 3 minutes using a high-speed shear homogenizer.

[0052] Performance Testing Determination of GLCN-Mg PE type 20 ml of ultrapure water and 20 ml of soybean oil were added to a transparent sample bottle, and 1 ml of freshly prepared GLCN-Mg PE was added. After 30 seconds, the morphology of the droplets in the water phase and the oil phase was observed. If the emulsion droplets were dispersed in the water phase and aggregated into droplets in the oil phase, the emulsion was defined as an oil-in-water (O / W) type; if the emulsion droplets were dispersed in the oil phase and aggregated into droplets in the water phase, the emulsion was defined as a water-in-oil (W / O) type. The results are shown in Figure 2. Figure 1 As shown, the emulsion droplets are dispersed in the oil phase and aggregated into droplets in the water phase. The emulsion is a water-in-oil (W / O) type.

[0053] Microstructure of emulsion Observe the appearance and droplet morphology of GLCN-Mg PE by optical microscopy. Take 0.5 ml of the emulsion sample and drop it on a microscope slide, cover the drop with a cover glass, and observe the droplet morphology under a 100x objective lens. Figures 2-4 Shown are the appearance and droplet morphology of GLCN-Mg PE at different homogenizer speeds, the appearance and droplet morphology of GLCN-Mg PE with different GLCN-Mg mass volume fractions, and the appearance and droplet morphology of GLCN-Mg PE with different oil-water ratios.

[0054] The size of emulsion droplets and their uniformity have a decisive influence on the properties of the emulsion. Figure 2 The optical microscopic images and particle size distribution characteristics of GLCN-Mg PE at different homogenization speeds are shown. When the homogenization speed is in the lower range of 5000-10000 rpm, the insufficient coverage of GLCN-Mg at the interface leads to the formation of irregular droplets of uneven size. After increasing the homogenization speed to 15000 rpm, the directional migration of GLCN-Mg to the interface is significantly enhanced, and regular droplets with uniform particle size and optimized size are obtained. However, when the rate exceeds 20000 rpm, excessive shear force causes the droplets to break. This phenomenon reveals the dual effect of the homogenization process: moderate homogenization enhances the stability of the emulsion system by improving the integrity of the droplets, while excessive shearing causes the droplet structure to be destroyed. Figure 3The effects of different GLCN-Mg concentrations on the droplet morphology are presented. At a concentration of 0.3%, incomplete coverage of GLCN-Mg at the oil-water interface results in the formation of small, irregularly shaped droplets. When the concentration is increased to 0.9%, the packing density of GLCN-Mg at the interface reaches its maximum, forming uniformly sized, larger droplets. When the concentration exceeds 0.9%, the adsorption of GLCN-Mg at the interface approaches saturation, and the increase in droplet size enters a plateau phase. Excessive GLCN-Mg particles thicken the interface layer of the droplets but do not change the droplet size. Figure 4 The results reveal the influence of the oil-water ratio on droplet morphology. At an oil-water ratio of 3:7, the system formed small, unevenly dispersed droplets. Increasing the oil-phase ratio to 6:4 resulted in enhanced interfacial adhesion of GLCN-Mg, leading to a maximum droplet size and uniform distribution. However, when the oil-water ratio increased to 8:2, phase transition and insufficient emulsifier coverage led to a significant decrease in droplet size. This indicates that droplet size initially increases and then decreases with increasing oil-phase proportion.

[0055] Determination of centrifugal stability of GLCN-Mg PE Measure 50 ml of the prepared GLCN-Mg PE into a centrifuge tube and centrifuge at 3000 rpm for 5 minutes at 4°C in a high-speed refrigerated centrifuge. Observe the state of the emulsion after centrifugation. Determine the stability index (SI) by measuring the observed emulsion volume and the volume of all phases in the centrifuge tube. The formula for calculating the stability index is as follows: SI=(Ve / Vs)×100% Where: Vs—the total volume of the sample (mL); Ve—volume of the emulsion phase (mL).

[0056] like Figures 5 to 7 As shown, the centrifugal stability and average particle size of GLCN-Mg PE at different homogenizer speeds, the centrifugal stability and average particle size of GLCN-Mg PE with different GLCN-Mg mass volume fractions, and the centrifugal stability and average particle size of GLCN-Mg PE with different oil-water ratios.

[0057] Centrifugation allows for rapid assessment of the stability and uniformity of emulsion systems. Therefore, the stability index (SI) of GLCN-Mg PE was investigated under varying process parameters. As shown in the dual-bar chart, the SI exhibits a significant synergistic relationship with droplet size, stemming from the dual functions of GLCN-Mg as both a stabilizer and a structure modulator. A moderate amount of GLCN-Mg increases droplet volume by enhancing interfacial coverage until a critical threshold is reached. Once this threshold is exceeded, interfacial overload or shear failure triggers a simultaneous deterioration in centrifugal stability and droplet structural integrity. This correlation strongly supports the precise control of droplet morphology by GLCN-Mg. Figure 5 The effect of homogenization speed on the centrifugal stability of GLCN-Mg PE was revealed. When the speed was below a critical threshold (≤15,000 rpm), the SI increased with increasing homogenization speed, reaching a peak of 46.10±0.07% at 15,000 rpm. This phenomenon was attributed to the enhanced colloidal anchoring effect, which significantly improved the anti-agglomeration ability of the emulsion droplets. However, when the speed exceeded 20,000 rpm, excessive shearing triggered the desorption of GLCN-Mg at the oil-water interface, and the SI dropped to 36.03±0.11%. Further increasing the speed to 25,000 rpm, the intense shearing caused the emulsion droplets to dissociate, resulting in complete instability of the emulsion system, with the SI remaining at only 2.00±0.07%. Figure 6 The effect of GLCN-Mg concentration on the centrifugal stability of the system was demonstrated. In the 0.3%-0.9% concentration range, the SI increased from 10.17±0.11% to 40.10±0.07%, attributed to an increase in the interfacial packing density. However, above the critical 0.9% concentration, the SI remained essentially stable at 34.07±0.09% (1.1% GLCN-Mg), despite continued increases in the GLCN-Mg dosage. This phenomenon stems from interfacial structural defects caused by interparticle repulsion. Figure 7 The effects of different oil-water ratios on the centrifugal stability of GLCN-Mg PE were demonstrated. As the oil-water ratio increased from 3:7 to 6:4, the SI increased from 2.01±0.53% to 49.47±0.98%, corresponding to the optimal emulsion droplet packing efficiency. However, when the oil-water ratio increased to 8:2, the SI dropped back to 36.73±1.51%. This is attributed to the system's instability caused by a phase transition mechanism initiated by excess oil. The incomplete phase transition and encapsulation defects, combined with the disruption of the directional distribution of the emulsifier, disrupted the emulsifier's distribution.

[0058] Determination of rheological properties of GLCN-Mg PE 300 ml of the prepared GLCN-Mg PE was placed in a beaker. The viscosity of the emulsion was continuously measured for 5 minutes at 23°C and 60 rpm using an NDJ-1B rotational viscometer (spindle 1). Viscosity values ​​were recorded every 30 seconds and averaged to estimate the viscosity of the emulsion. The test results are shown in Tables 1-3.

[0059] Table 1 Viscosity of GLCN-Mg PE at different homogenizer speeds

[0060] Table 2 Viscosity of GLCN-Mg PE with different GLCN-Mg mass volume fractions

[0061] Table 3 Viscosity of GLCN-Mg PE at different oil-water ratios

[0062] The effect of homogenization speed on the rheological properties of GLCN-Mg PE is shown in Table 1. The viscosity of the system initially increased and then decreased with increasing homogenization speed: from 26.47±0.46 mPa·s at 5000 rpm to 176.04±0.64 mPa·s at 20000 rpm, and then decreased to 156.84±0.44 mPa·s at 25000 rpm. Moderate homogenization (≤20000 rpm) promoted the formation of a three-dimensional network structure at the oil-water interface by enhancing the anchoring effect of GLCN-Mg at the interface and causing moderate expansion of the emulsion droplets. Conversely, excessively high speeds (>20000 rpm) disrupted this network through shear-induced structural degradation. Statistical analysis confirmed significant differences in viscosity between treatments (p<0.05). Furthermore, the system exhibited a weak time-dependent shear-thinning characteristic, consistent with the viscoelastic behavior of Pickering emulsions. Table 2 reveals the effect of GLCN-Mg concentration on the rheological properties of the system. The viscosity increased from 21.10 ± 0.24 mPa·s at a 0.3% concentration to a peak of 112.98 ± 0.38 mPa·s at 1.1% before decreasing to 101.50 ± 0.28 mPa·s at 1.3%. The optimal concentration range (0.9%-1.1%) optimizes the oil-water interface structure to achieve a three-dimensional network. However, at concentrations exceeding 1.1%, steric hindrance and synergistic binding induced by GLCN-Mg overloading compromise the integrity of the interfacial network, manifesting as a decrease in viscosity. Table 3 shows the effect of the oil-water ratio on the rheological behavior of the system. The viscosity gradually increased from 13.37 ± 0.35 mPa·s at a 3:7 ratio to a peak of 123.91 ± 0.46 mPa·s at a 7:3 ratio before decreasing to 95.69 ± 0.57 mPa·s at an 8:2 ratio. Increasing the oil-to-water ratio (3:7-7:3) significantly improves the rheological properties of the emulsion by dense droplet packing and enhanced interactions between droplets, manifesting as a step-like increase in viscosity. However, when the oil-to-water ratio exceeds 7:3, interfacial instability driven by phase transition disrupts the continuity of the emulsion architecture, weakening the connectivity between droplets and causing a drop in emulsion viscosity.

[0063] Determination of temperature stability of GLCN-Mg PE 10 ml of the prepared GLCN-Mg PE was placed in a centrifuge tube and stored at -20°C for 24 hours, then at 25°C for 4 hours. The emulsion was then observed. Equal amounts of the emulsion were then placed in centrifuge tubes and stored at 4, 25, 50, or 80°C for 1 hour. The emulsion was then observed to assess its temperature stability. The temperature stability of the emulsion is expressed as the emulsification index (EI), which is calculated as follows: EI=(Ve / Vs)×100% Where: Vs—the total volume of the sample (mL); Ve—volume of the emulsion phase (mL).

[0064] like Figures 8-10 As shown, the temperature stability of GLCN-Mg PE at different homogenizer speeds, the temperature stability of GLCN-Mg PE with different GLCN-Mg mass volume fractions, and the temperature stability of GLCN-Mg PE with different oil-water ratios are respectively.

[0065] The GLCN-Mg PE emulsion system exhibits significant temperature-dependent stability characteristics. Under freezing conditions (-20°C), the mechanical stress generated by water crystallization forces the oil droplets to compress and deform, resulting in irreversible phase separation after thawing. The system maintains good stability in the range of 4-25°C, but deteriorates significantly when the temperature rises above 50°C, and the emulsification index (EI) returns to zero completely when the temperature reaches 80°C. The thermal degradation mechanism reveals the attenuation of the GLCN-Mg interface adsorption capacity and the thermal motion dissociation of the network structure of the complex. Figure 8 As shown, GLCN-Mg PE prepared at different homogenization speeds exhibited significant differences in thermal stability. When the homogenization speed was in the low range of 5000-10000 rpm, the EI values ​​of the emulsions at all temperatures were less than 10%, and the imperfect interfacial coverage directly led to its thermal stability defects. Under the process conditions of 20000 rpm, the system maintained high EI values ​​of 93.73±0.18% and 87.07±0.42% at 4°C and 25°C, respectively, which was attributed to the uniform distribution of emulsion droplets and the formation of a dense interfacial film. When the homogenization speed was increased to 25000 rpm, excessive shearing caused the interfacial film to rupture, resulting in a sharp drop in the EI value to 7.33±0.22% at 50°C. Figure 9 The effect of GLCN-Mg concentration on the thermal stability of the system was revealed. When the concentration was 0.3%, the EI values ​​at all temperatures were less than 10%, indicating that too low a GLCN-Mg concentration could not form a continuous interfacial protective layer. At the optimal concentration of 1.10%, the system had EI values ​​of 76.00±0.13% and 73.10±0.27% at 4°C and 25°C, respectively. This indicates that the appropriate amount of GLCN-Mg completely constructed the oil-water interfacial film. When the concentration increased to 1.3%, the excessive GLCN-Mg caused interfacial crowding, resulting in defects in the emulsion structure, and the EI value at 25°C dropped back to 69.03±0.24%. Figure 10The effect of the oil-water ratio on thermal stability was demonstrated. When the oil-water ratio was 3:7, the EI values ​​at all temperatures were below 5%, and the low-oil phase system became unstable due to weak interfacial bonding. Within moderate oil-water ratios (6:4 and 7:3), the relatively dense emulsion stacking structure endowed the system with excellent thermal stability, with EI values ​​reaching 78.73±0.22% and 79.17±0.22% at 4°C, respectively. However, when the oil phase ratio increased to 8:2, phase transition phenomena and interfacial film dissociation combined to cause system instability.

[0066] Determination of storage stability of GLCN-Mg PE Carefully weigh the prepared GLCN-Mg PE and seal them in transparent glass sample bottles of equal mass. Then store these samples in the dark at room temperature for 30 days, observe the condition of the emulsion and evaluate its storage stability, such as Figures 11-13 Shown are the appearance of GLCN-Mg PE at different homogenizer speeds after storage at room temperature for 30 days, the appearance of GLCN-Mg PE with different GLCN-Mg mass volume fractions after storage at room temperature for 30 days, and the appearance of GLCN-Mg PE with different oil-water ratios after storage at room temperature for 30 days.

[0067] The freshly prepared GLCN-Mg PE emulsion system exhibited high initial stability. However, during 30 days of storage, the emulsion coalesced and settled under gravity, leading to phase separation. This destabilization gradually intensified over time, a mechanism attributed to the combined effects of the density difference between the oil and water phases and the progressive degradation of the interfacial film. Figure 11 The study revealed the impact of homogenization speed on storage stability. Emulsions prepared at low speeds of 5,000-10,000 rpm experienced severe phase separation within a short period of time due to insufficient GLCN-Mg coverage at the interface. However, homogenization at 20,000 rpm resulted in uniform droplets and a strong interfacial membrane, which prevented the emulsion from experiencing significant instability during 30 days of storage. In contrast, a speed of 25,000 rpm significantly accelerated system instability due to membrane rupture and droplet aggregation. Figure 12 The effect of GLCN-Mg concentration on storage stability was demonstrated. When the concentration was 0.3%, the phenomenon of emulsion stratification and water phase precipitation confirmed that the oil-water interface bonding force was relatively weak. When the concentration was ≥0.5%, the formation of a continuous interfacial protective layer significantly improved its storage stability. When the GLCN-Mg concentration was in the range of 0.50-1.30%, a dense interfacial film with high mechanical strength could be formed on the surface of the emulsion droplets, and there was no significant difference in the degree of phase separation after 30 days of storage. Figure 13The study demonstrated the influence of the oil-water ratio on the storage stability of the emulsion. At an oil-water ratio of 3:7, the system experienced severe phase separation due to sparse droplet distribution and weak interfacial bonding. The optimal oil-water ratio of 7:3 effectively resisted gravitational settling through the dense droplet stacking structure, achieving optimal storage stability. When the oil-phase ratio continued to increase to 8:2, the excess oil phase triggered phase transitions and uneven interfacial film distribution, reducing the system's storage stability.

[0068] In vitro simulated gastrointestinal digestion 0.1 g of GLCN-Mg was emulsified and mixed with 1 ml of simulated gastric fluid (5 g of pepsin in 5000 ml of 0.1 mol / L HCl). The mixture was placed in a dialysis bag (100 Da MWCO) and digested in 100 ml of simulated gastric fluid at 37°C for 2 hours. After gastric digestion, 1 / 10 of the solution was transferred to a new dialysis bag, mixed with 1 ml of simulated intestinal fluid (1.2 g of bile salts and 0.2 g of pancreatic enzyme in 100 ml of 0.1 mol / L NaHCO3), and digested in 100 ml of simulated intestinal fluid at 37°C for another 2 hours. The pH of the solution was adjusted to 12-14 with NaOH to precipitate Mg(OH)2. Ca was added. 2+ Indicator, titrate the solution with EDTA to determine Ca 2+ Then adjust the pH to 8-10 and titrate the total Ca using chrome black T as an indicator. 2+ and Mg 2+ By subtracting Ca from the total titrated volume 2+ Titration volume to calculate Mg 2+ Mg content 2+ Calculation of release rate: Release rate = W2 / W1×100% Where: W1——Mg in the dialysis bag before digestion 2+ content; W2——Mg in the beaker after digestion 2+ content.

[0069] like Figures 14-16 As shown, the gastrointestinal digestibility of GLCN-Mg PE at different homogenizer speeds, the gastrointestinal digestibility of GLCN-Mg PE with different GLCN-Mg mass volume fractions, and the gastrointestinal digestibility of GLCN-Mg PE with different oil-water ratios.

[0070] In vitro simulated gastrointestinal digestion experiments showed that the emulsion system showed a phased Mg 2+ Release mechanism. At the end of gastrointestinal digestion, Mg 2+The cumulative release rate was as high as 80.42±1.94%. Under the process conditions of 0.90% GLCN-Mg addition and 20000rpm homogenization speed, the Mg in the gastric phase was significantly increased. 2+ The release rate can reach 65.42±1.11%. However, when the homogenization speed is increased to 25000rpm, the excessive mechanical shear force causes the emulsion structure to collapse, resulting in the release of Mg in the stomach phase. 2+ The release rate was significantly reduced to 38.33±5.56%. In addition, when the concentration of GLCN-Mg exceeded 0.90%, the structural defects at the oil-water interface were not conducive to the release of Mg. 2+ During the gastric digestion stage, the amino and hydroxyl groups in the GLCN-Mg coordination structure are protonated by acidic hydrolysis, which effectively neutralizes the electrostatic repulsion and triggers the rapid aggregation of particles, thereby accelerating the release of Mg. 2+ Diffusion process to gastric juice. At this time, Mg 2+ The release of Mg is mainly regulated by gastric acid at the interface. 2+ The release rate in the intestinal phase reached 80.42±1.94%. Lipase mediated the cleavage of the ester bond to generate amphiphilic metabolites such as free fatty acids (FFA) and monoglycerides (MAG). These surfactants dissociated GLCN-Mg from the oil-water interface through competitive substitution, and then formed Mg-loaded 2+ The lipid matrix moderately maintains the stability of the emulsion in the gastric phase and achieves complete dissociation in the intestinal phase. However, when the oil phase ratio is increased to 8:2, the phase transition and overcrowding of the droplets caused by excessive oil significantly hinder the enzymatic hydrolysis process, resulting in Mg 2+ The release rate in the intestine dropped to 57.08±0.56%.

[0071] The above description is only a preferred embodiment of the present invention and does not constitute any formal limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex, characterized in that: The following steps are involved: Preparation of D-glucosamine-magnesium complex; uniformly dispersing the D-glucosamine-magnesium complex in water to form a complex dispersion; The oil component is added to the complex dispersion and homogenized to obtain a Pickering emulsion stabilized by the D-glucosamine-magnesium complex.

2. The method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex according to claim 1, wherein: The molar ratio of D-glucosamine to magnesium in the D-glucosamine-magnesium complex is 1:

1.

3. The method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex according to claim 1, wherein: The mass volume fraction of the D-glucosamine-magnesium complex in the complex dispersion is 0.3-1.3%.

4. The method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex according to claim 1, wherein: The oil component includes vegetable oil and / or biological oil; The vegetable oil includes one or more of soybean oil, olive oil or almond oil; The biological oil includes one or more of fish oil, beef tallow or sheep oil.

5. The method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex according to claim 1, wherein: The volume ratio of the oil component to the complex dispersion is 3-8:2-7.

6. The method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex according to claim 1, wherein: The rotation speed of the homogenization process is 5000~25000rpm.

7. The method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex according to claim 5, wherein: The homogenization time is 2 to 4 minutes.

8. A Pickering emulsion prepared by the method for preparing a Pickering emulsion stabilized by a D-glucosamine-magnesium complex according to any one of claims 1 to 7.

9. The Pickering emulsion according to claim 8, wherein The raw materials of the Pickering emulsion also include auxiliary functional ingredients; the auxiliary functional ingredients include one or more of hyaluronic acid, hyaluronic acid, vitamin C, chondroitin sulfate, curcumin, resveratrol, chlorogenic acid or asiaticoside.

10. Use of the Pickering emulsion according to any one of claims 8 to 9 in the preparation of medicines for osteoarthritis.

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