Preparation method and application of vitamin D3 delivery system

Abstract

The invention provides a preparation method and application of a vitamin D3 delivery system, and relates to the field of nutrient substance delivery. According to the invention, the vitamin D3 is embedded by using alpha-cyclodextrin (alpha-CD) in an environment with the pH value of 7-11, so that the encapsulation efficiency of the alpha-CD and the water solubility and stability of VD3 are improved. The maximum encapsulation efficiency of alpha-CD can reach 89.27%, it is measured that the maximum solubility of alpha-CD-VD3 in water is 73 mg / mL, and the equivalent weight of VD3 is 11.8 mg / mL.

Description

Technical Field

[0001] The present invention relates to the field of nutrient delivery, and in particular to a preparation method and application of a vitamin D3 delivery system. Background Art

[0002] Vitamin D3 (VD3) has poor water solubility due to its hydrophobic structure and is susceptible to oxidation and decomposition in acidic environments, limiting its use in food and pharmaceuticals. Traditional delivery technologies, such as microencapsulation, are complex and costly. The addition of water-soluble antioxidants can also cause oxidation, and the oxidation products can even pose potential health risks.

[0003] β-cyclodextrin (β-CD) is widely used for vitamin D3 encapsulation due to its moderate molecular cavity size, strong inclusion capacity, and low cost. The hydrophobic cavity of β-CD forms an inclusion complex with the lipid-soluble structure of vitamin D3, which can improve the water solubility and stability of vitamin D3 to a certain extent. However, β-CD has limited water solubility, with a solubility of approximately 1.85 g / L at 25°C, and is susceptible to hydrolysis in acidic environments, resulting in premature release and degradation of the encapsulated vitamin D3 in gastric fluid, limiting its efficient delivery in liquid foods and pharmaceuticals. Furthermore, β-CD may have potential nephrotoxicity due to its potential to bind cholesterol.

[0004] Therefore, it is particularly important to develop a VD3 delivery system with strong water solubility, good stability, non-toxicity and high encapsulation efficiency. Summary of the Invention

[0005] In view of the technical problems existing in the prior art, the present invention aims to provide a preparation method and application of a vitamin D3 delivery system.

[0006] One of the objects of the present invention is to provide a method for preparing a vitamin D3 delivery system, comprising the following steps: S1, dissolving α-cyclodextrin in an aqueous solution with a pH of 7 to 11 to obtain an α-cyclodextrin solution; S2. dissolving vitamin D3 in anhydrous ethanol to obtain a vitamin D3 anhydrous ethanol solution; S3, adding the anhydrous ethanol solution of vitamin D3 to the α-cyclodextrin solution to react to obtain an α-cyclodextrin-vitamin D3 mixture solution; The molar ratio of the vitamin D3 to the α-cyclodextrin in the α-cyclodextrin-vitamin D3 mixture solution is 1:1; S4. The α-cyclodextrin-vitamin D3 mixture solution is freeze-dried, re-dissolved and centrifuged to obtain a precipitate, and the precipitate is freeze-dried again to obtain a vitamin D3 delivery system.

[0007] Preferably, the molar ratio of the vitamin D3 to the α-cyclodextrin in the α-cyclodextrin-vitamin D3 mixture solution is 1:1.

[0008] Preferably, the reaction time in step S3 is 18 to 36 hours.

[0009] A second object of the present invention is to provide an application of a vitamin D3 delivery system prepared by the preparation method as described above, characterized in that the vitamin D3 delivery system is used to prepare a calcium-vitamin D3 coordinated delivery system.

[0010] Preferably, the preparation method of the calcium-vitamin D3 coordinated delivery system comprises: dissolving the vitamin D3 delivery system in water to obtain an aqueous solution of the vitamin D3 delivery system; A casein phosphopeptide-calcium complex was prepared, and the casein phosphopeptide-calcium complex was added to an aqueous solution of a vitamin D3 delivery system. After stirring for 5 to 10 hours, a calcium-vitamin D3 synergistic delivery system consisting of a casein phosphopeptide-calcium-α-cyclodextrin-vitamin D3 complex was obtained.

[0011] Preferably, the mass ratio of the vitamin D3 delivery system to the casein phosphopeptide-calcium complex is 1:1.

[0012] Preferably, the method for preparing the casein phosphopeptide-calcium complex comprises: CaCl2 was added to the casein phosphopeptide aqueous solution, the pH was adjusted to 7, the reaction was carried out for 40 to 90 minutes, ethanol was added and the solution was centrifuged, and the precipitate was freeze-dried to obtain a casein phosphopeptide-calcium complex.

[0013] Preferably, the preparation method of the calcium-vitamin D3 coordinated delivery system further comprises: dissolving the vitamin D3 delivery system in water to obtain an aqueous solution of the vitamin D3 delivery system; dissolving calcium glutamate in water to obtain a calcium glutamate aqueous solution; The calcium glutamate aqueous solution is added to the vitamin D3 delivery system aqueous solution, lipase is added, and the mixture is stirred at 45-55° C. for 4-8 hours to obtain a calcium-vitamin D3 coordinated delivery system whose components are calcium glutamate-α-cyclodextrin-vitamin D3 chelate.

[0014] Preferably, the mass ratio of the sum of the mass of the vitamin D3 delivery system and calcium glutamate to the lipase is 100:5-9.

[0015] Preferably, the mass ratio of calcium glutamate and vitamin D3 delivery system is 1:1~4.

[0016] Preferably, the preparation method of the calcium glutamate complex comprises: Add CaCl2 to a glutamic acid aqueous solution at 70-80°C, adjust the pH to 7, react at 45-55°C for 40-90 minutes, add ethanol and centrifuge, and freeze-dry the precipitate to obtain calcium glutamate.

[0017] Beneficial effects of the present invention: This invention uses α-cyclodextrin (α-CD) to encapsulate vitamin D3 at a pH of 7 to 11, improving the encapsulation efficiency of α-CD and the water solubility and stability of VD3. The α-CD encapsulation efficiency can reach up to 89.27%, and the maximum solubility of α-CD-VD3 in water is measured to be 73 mg / mL, with a VD3 equivalent of 11.8 mg / mL.

[0018] It should be understood that the description of technical features, technical solutions, beneficial effects or similar language in this application does not imply that all features and advantages can be realized in any single embodiment. On the contrary, it is understood that the description of a feature or beneficial effect means that a specific technical feature, technical solution or beneficial effect is included in at least one embodiment. Therefore, the description of a technical feature, technical solution or beneficial effect in this specification does not necessarily refer to the same embodiment. Furthermore, the technical features, technical solutions and beneficial effects described in the present embodiment can also be combined in any appropriate manner. Those skilled in the art will understand that the embodiment can be implemented without one or more specific technical features, technical solutions or beneficial effects of a specific embodiment. In other embodiments, additional technical features and beneficial effects can also be identified in specific embodiments that do not embody all embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To test the encapsulation efficiency of α-CD-VD3; Figure 2 is the average particle size of the vitamin D3 delivery system at different salt ion concentrations; Figure 3 Zeta potential of the vitamin D3 delivery system at different salt ion concentrations; Figure 4 is the average particle size of the vitamin D3 delivery system at different pH values; Figure 5 Zeta potential of vitamin D3 delivery system at different pH; Figure 6 This is a graph showing the relationship between time and transmittance of the vitamin D3 delivery system; Figure 7 The in vitro release rate and glucose content of α-CD-VD3 inclusion complex vitamin D3 delivery system in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were investigated. Figure 8α-CD-VD3@CPP-Ca 2+ Average particle size at different salt ion concentrations; Figure 9 α-CD-VD3@CPP-Ca 2+ Zeta potential under different salt ion concentrations; Figure 10 α-CD-VD3@CPP-Ca 2+ Average particle size at different pH; Figure 11 α-CD-VD3@CPP-Ca 2+ Zeta potential at different pH; Figure 12 is the stability of α-CD-VD3 within 8 hours; Figure 13 CPP-Ca 2+ Stability within 8 hours; Figure 14 α-CD-VD3@CPP-Ca 2+ Stability of dispersion within 8 hours; Figure 15 α-CD-VD3, CPP-Ca 2+ and α-CD-VD3@CPP-Ca 2+ Overall stability index of the dispersion; Figure 16 α-CD-VD3@CPP-Ca 2+ In vitro Ca2+ expression of the complex in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) 2 + Release rate curve; Figure 17 α-CD-VD3@CPP-Ca 2+ In vitro VD3 release rate curves of the complex in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF); Figure 18 α-CD-VD3&L-Glu-Ca 2+ Average particle size at different salt ion concentrations; Figure 19 α-CD-VD3&L-Glu-Ca 2+ Zeta potential under different salt ion concentrations; Figure 20 α-CD-VD3&L-Glu-Ca 2+ Average particle size at different pH; Figure 21 α-CD-VD3&L-Glu-Ca 2+ Zeta potential at different pH; Figure 22 is the stability of α-CD-VD3 within 8 hours; Figure 23 L-Glu-Ca 2+ Stability within 8 hours; Figure 24 α-CD-VD3&L-Glu-Ca 2+ Stability of dispersion within 8 hours; Figure 25 α-CD-VD3, CPP-Ca 2+ and α-CD-VD3&L-Glu-Ca 2+ Overall stability index of the dispersion; Figure 26 α-CD-VD3&L-Glu-Ca 2+ In vitro Ca2+ expression of the complex in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) 2+ Release rate curve; Figure 27 α-CD-VD3&L-Glu-Ca 2+ In vitro VD3 release rate curves of the complex in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). DETAILED DESCRIPTION

[0020] According to the first aspect of the present invention, a method for preparing a vitamin D3 delivery system is provided, comprising the following steps: S1, dissolving α-cyclodextrin in an aqueous solution with a pH of 7 to 11 to obtain an α-cyclodextrin solution; S2. dissolving vitamin D3 in anhydrous ethanol to obtain a vitamin D3 anhydrous ethanol solution; S3, adding the anhydrous ethanol solution of vitamin D3 to the α-cyclodextrin solution to react to obtain an α-cyclodextrin-vitamin D3 mixture solution; S4. The α-cyclodextrin-vitamin D3 mixture solution is freeze-dried, re-dissolved and centrifuged to obtain a precipitate, and the precipitate is freeze-dried again to obtain a vitamin D3 delivery system.

[0021] In this invention, α-cyclodextrin is composed of seven glucose units, with an inner cavity diameter of approximately 0.45 to 0.6 nm, making it suitable for inclusion of small molecule drugs such as vitamin D3, short-chain fatty acids, and some antibiotics. Compared with β-CD, the size of the vitamin D3 molecule is more closely aligned with the inner cavity of α-CD, resulting in less steric hindrance and tighter binding during inclusion, resulting in higher encapsulation efficiency and stability than β-CD.

[0022] α-CD has a smaller hydrophobic region within its cavity, resulting in more concentrated hydrophobic interactions with small molecule drugs, enabling the formation of more stable inclusion complexes. The higher hydroxyl group density and smaller relative molecular mass of α-CD molecules, along with a higher proportion of hydroxyl groups, strengthen hydrogen bonding with drug molecules, further enhancing inclusion complex stability. In alkaline environments of pH 7-11, the dissociation degree of the hydroxyl groups in α-CD increases, leading to stronger ion-dipole interactions with the polar hydroxyl groups of vitamin D3. However, the larger internal cavity of β-CD results in a more random orientation of drug molecules during inclusion, resulting in slightly less stability.

[0023] Due to its smaller molecular weight and higher hydroxyl group ratio, α-CD has a solubility in water of approximately 14.5 g / 100 mL at 25°C, significantly higher than β-CD. Direct use in the preparation of high-concentration inclusion complex solutions avoids drug precipitation issues caused by low carrier solubility.

[0024] The α-cyclodextrin-encapsulated VD3 delivery system is suitable for aqueous injections, oral solutions, and other dosage forms, reducing the use of organic solvents and improving clinical safety. Unmodified α-CD and β-CD are both low-toxic carriers, but β-CD, especially unmodified β-CD, may bind to cholesterol at high concentrations, leading to cell membrane damage or kidney accumulation risk. Although modified β-CD is often used in drugs to reduce toxicity, natural α-CD is still safer. Due to its small molecular weight, α-CD can be degraded into glucose more quickly by digestive tract enzymes in the body and has a clear metabolic pathway, making it suitable for special populations such as children and elderly patients.

[0025] The surface of α-CD molecules is rich in hydroxyl groups. Under neutral or acidic conditions, these hydroxyl groups self-assemble into rod-shaped aggregates through intermolecular hydrogen bonding, resulting in the encapsulation of the hydrophobic cavities of individual α-CDs within the aggregates, reducing the number of cavities available for encapsulating VD3. When the pH rises to 7–11, the abundant hydroxide ions in the solution compete with the hydroxyl groups of α-CD to form hydrogen bonds, weakening the intermolecular hydrogen bonding and inhibiting the formation of rod-shaped aggregates. This results in a predominantly single molecule or short chain structure of α-CD, allowing VD3 to directly bind to the cavities of α-CD, reducing diffusion resistance. This inhibition of intermolecular hydrogen bonding exposes the hydrophobic cavities of α-CD from within the aggregates, significantly increasing the number of cavities available for encapsulating VD3 per unit volume.

[0026] In general, the present invention weakens the hydrogen bonds between α-CD molecules by setting the pH to 7-11, causing it to transform from an aggregated state to a dispersed state, significantly increasing the number of effective cavities and action sites that can be used to encapsulate VD3, and at the same time improving the stability of the inclusion complex by enhancing polar interactions.

[0027] In a preferred embodiment of the present invention, the pH of the aqueous solution is 9.

[0028] In the present invention, pH 9 is the optimal condition for balancing structural depolymerization and inclusion capacity.

[0029] In a preferred embodiment of the present invention, the molar ratio of vitamin D3 to α-cyclodextrin in the α-cyclodextrin-vitamin D3 mixture solution is 1:1.

[0030] In this invention, the inner cavity diameter of α-CD is approximately 0.45-0.6 nm, making it suitable for inclusion of small molecule drugs. The hydrophobic rings and long-chain hydrocarbon groups in the VD3 molecular structure are highly compatible with the inner cavity of α-CD. The 1:1 mass ratio allows the guest molecule VD3 to precisely embed into the hydrophobic cavity of the host molecule α-CD, forming a unimolecular inclusion complex and avoiding unfilled cavities or molecular aggregation caused by imbalanced ratios.

[0031] The hydroxyl groups in the outer cavity of α-CD can form hydrogen bonds with those of VD3, while the hydrophobic environment of the inner cavity stabilizes the nonpolar groups of VD3 through hydrophobic interactions. At a 1:1 ratio, these two forces are balanced, resulting in optimal inclusion complex stability. α-CD itself is highly water-soluble, and at a 1:1 molar ratio, VD3 is converted into a water-soluble complex through inclusion. To avoid solubility loss caused by an imbalance in the ratio, an excess of α-CD may cause the excess cyclodextrin to self-assemble into rod-like structures through intermolecular hydrogen bonding, reducing the number of free cavities. However, an excess of VD3 may cause unincluded VD3 to precipitate due to its lipid solubility, reducing system stability.

[0032] In a preferred embodiment of the present invention, the reaction time in step S3 is 18 to 36 hours.

[0033] In the present invention, the reaction time in step S3 is 18 to 36 hours to allow sufficient contact between vitamin D3 and α-cyclodextrin to achieve inclusion equilibrium, thereby avoiding low encapsulation efficiency due to insufficient time. Excessive reaction time may cause α-cyclodextrin to hydrolyze under alkaline conditions or vitamin D3 to degrade, reducing stability.

[0034] In a preferred embodiment of the present invention, the reaction time in step S3 is 24 hours.

[0035] In the present invention, the reaction time of step S3 is 24 hours, which can achieve the best encapsulation efficiency of 89.27% ​​and facilitate the connection of subsequent freeze-drying, centrifugation and other operations.

[0036] According to the second aspect of the present invention, an application of a vitamin D3 delivery system prepared by the preparation method is provided, wherein the vitamin D3 delivery system is used to prepare a calcium-vitamin D3 coordinated delivery system.

[0037] In a preferred embodiment of the present invention, the preparation method of the calcium-vitamin D3 coordinated delivery system comprises: dissolving the vitamin D3 delivery system in water to obtain an aqueous solution of the vitamin D3 delivery system; A casein phosphopeptide-calcium complex was prepared, and the casein phosphopeptide-calcium complex was added to an aqueous solution of a vitamin D3 delivery system. After stirring for 5 to 10 hours, a calcium-vitamin D3 synergistic delivery system consisting of a casein phosphopeptide-calcium-α-cyclodextrin-vitamin D3 complex was obtained.

[0038] In the present invention, the casein phosphopeptide-calcium-α-cyclodextrin-vitamin D3 complex (α-CD-VD3@CPP-Ca) was prepared by non-covalently binding the α-CD-VD3 inclusion complex vitamin D3 delivery system to the casein phosphopeptide-calcium complex. 2+ ). α-CD encapsulates VD3 to form a stable inclusion complex through hydrophobic interaction and hydrogen bonding, converting fat-soluble VD3 into a water-soluble complex, increasing its solubility to 73 mg / mL, with a VD3 equivalent of 11.8 mg / mL, preventing its premature release and degradation in gastric juice, with a gastric juice release rate of <5%, ensuring efficient release after reaching the intestine, with an intestinal juice release rate of >78%. Casein phosphopeptide binds to Ca through the phosphate group. 2+ The formation of soluble complexes prevents calcium precipitation in the alkaline environment of the intestine and promotes calcium transmembrane transport. 2+ The complex can efficiently release Ca 2+ and VD3, achieving simultaneous and stable delivery and synergistic absorption of VD3 and calcium, with safety, high efficiency and process convenience, and is suitable for functional foods, nutritional supplements and clinical nutritional preparations.

[0039] In a preferred embodiment of the present invention, the mass ratio of the vitamin D3 delivery system to the casein phosphopeptide-calcium complex is 1:1.

[0040] In the present invention, the mass ratio of α-CD-VD3 to CPP-Ca is 1:1. 2+ The molecular number matches that of the CPP, forming a stable quaternary complex α-CD-VD3@CPP-Ca through hydrogen bonds and van der Waals forces. 2+ At this time, the particle size of the complex is the smallest, the absolute value of the Zeta potential is the largest, the electrostatic repulsion and steric hindrance effect are the best, and the particle aggregation or dissociation can be avoided. When the ratio is unbalanced, the excess components are prone to cause turbidity in the system due to charge neutralization or cavity saturation, while the 1:1 ratio achieves dynamic equilibrium through non-covalent interaction, and shows good stability at different pH (1.5~9.0) and salt ion concentrations (0~200mM), which is suitable for complex physiological environments. The 1:1 ratio ensures that VD3 and Ca 2+ Synchronous and efficient release in simulated intestinal fluid (SIF) was achieved, with a VD3 release rate of 78.39% and Ca 2+ The release rate is 84.12%.

[0041] In a preferred embodiment of the present invention, the method for preparing the casein phosphopeptide-calcium complex comprises: CaCl2 was added to the casein phosphopeptide aqueous solution, the pH was adjusted to 7, the reaction was carried out at 45-55°C for 40-90 minutes, ethanol was added and the mixture was centrifuged, and the precipitate was freeze-dried to obtain a casein phosphopeptide-calcium complex.

[0042] In a preferred embodiment of the present invention, the preparation method of the calcium-vitamin D3 coordinated delivery system comprises: dissolving the vitamin D3 delivery system in water to obtain an aqueous solution of the vitamin D3 delivery system; dissolving calcium glutamate in water to obtain a calcium glutamate aqueous solution; The calcium glutamate aqueous solution is added to the vitamin D3 delivery system aqueous solution, lipase is added, and the mixture is stirred at 45-55° C. for 4-8 hours to obtain a calcium-vitamin D3 coordinated delivery system whose components are calcium glutamate-α-cyclodextrin-vitamin D3 chelate.

[0043] In the present invention, α-CD embeds VD3 through hydrophobic interaction to form a stable complex, thereby improving the water solubility of VD3 and preventing its oxidative decomposition in gastric acid. Calcium glutamate forms a chelate with α-CD-VD3 through ionic bonds, and its negatively charged glutamic acid group binds to the α-CD hydroxyl group through hydrogen bonds, which inhibits the calcium 2+ It forms a precipitate with phosphate and oxalate in gastrointestinal fluid, and on the other hand, maintains the dispersion of the system through electrostatic repulsion, so that calcium exists in the form of a soluble chelate. The two form a stable quaternary structure through covalent and non-covalent interactions, and the particle size fluctuation is less than 200nm under different pH and salt concentrations, ensuring stable coexistence in complex physiological environments. In simulated intestinal fluid (SIF), lipase catalyzes the cleavage of the covalent bond between α-CD-VD3 and calcium glutamate, achieving the cleavage of VD3 and Ca 2+ Synchronous rapid release. Vitamin D3 activates intestinal calcium binding protein (CaBP), promoting intestinal mucosal absorption of Ca 2+ Active transport of calcium glutamate releases Ca 2+ Since no precipitation is formed in advance, it can be directly absorbed by the intestines, avoiding the low bioavailability of traditional calcium supplements caused by precipitation.

[0044] In a preferred embodiment of the present invention, the mass ratio of the sum of the mass of the vitamin D3 delivery system and the calcium glutamate to the lipase is 100:5-9.

[0045] In a preferred embodiment of the present invention, the mass ratio of the calcium glutamate and vitamin D3 delivery system is 1:1-4.

[0046] In a preferred embodiment of the present invention, the preparation method of calcium glutamate complex comprises: Add CaCl2 to a glutamic acid aqueous solution at 70-80°C, adjust the pH to 7, react at 45-55°C for 40-90 minutes, add ethanol and centrifuge, and freeze-dry the precipitate to obtain calcium glutamate. In the embodiments of this application, words such as "exemplary" or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of this application should not be interpreted as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0047] Example 1 The preparation method of the vitamin D3 delivery system comprises the following steps: Sodium hydroxide aqueous solutions with pH values ​​of 7 to 11 were prepared respectively, and 9.72 mg of α-cyclodextrin was dissolved in 10 ml of ultrapure aqueous solution with pH value of 7, sodium hydroxide aqueous solution with pH value of 8, sodium hydroxide aqueous solution with pH value of 9, sodium hydroxide aqueous solution with pH value of 10, and sodium hydroxide aqueous solution with pH value of 11, respectively, to obtain 5 groups of α-cyclodextrin solutions with different pH values. Dissolve 3.84 mg of vitamin D3 in 10 ml of anhydrous ethanol to obtain a vitamin D3 anhydrous ethanol solution; According to the molar ratio of vitamin D3 to α-cyclodextrin of 1:1, the anhydrous ethanol solution of vitamin D3 was added to five groups of α-cyclodextrin solutions with different pH values ​​to obtain five groups of α-cyclodextrin-vitamin D3 mixture solutions; The five α-cyclodextrin-vitamin D3 mixture solutions were freeze-dried and then re-dissolved in anhydrous ethanol. The supernatants were then centrifuged at 10,000 rpm for 10 minutes. The precipitates were freeze-dried again to obtain five vitamin D3 delivery systems consisting of α-CD-VD3. The absorbance of the supernatant was measured at 267 nm using a UV-visible spectrophotometer (N4S, Shanghai Yidian Analytical Instrument Co., Ltd., China) to determine the VD3 content in the supernatant (Hashemi, Kaykhaii, Keikha, & Raisi).

[0048]

[0049] M1: total VD3 initially added (mg); M2: Total VD3 of supernatant (mg).

[0050] The encapsulation efficiency of 5 groups of vitamin D3 delivery systems was measured respectively. The test results are as follows: Figure 1As shown, under conditions of pH 7-11, the encapsulation efficiency of α-CD-VD3 first increases and then decreases with increasing pH. However, the encapsulation efficiency is highest at pH 9, reaching 89.27%. There is no significant difference in encapsulation efficiency at pH 7 and 8, and the encapsulation efficiency decreases with decreasing pH. This is because under alkaline conditions, hydrogen bonds between α-CD molecules increase, resulting in longer rod-like structures formed by self-assembly. This reduces the number of VD3 molecules embedded in the cavity end of the α-CD molecule, leading to a gradual decrease in the encapsulation efficiency of α-CD-VD3. The above analysis shows that pH can respond to the length of the rod-like structures formed by α-CD self-assembly, thereby affecting the encapsulation efficiency of α-CD encapsulated small molecules of active nutrients.

[0051] Ionic Strength Stability Analysis: The vitamin D3 delivery system prepared at pH 9 was subjected to ionic strength stability analysis using the following test method: The α-CD-VD3 inclusion complex solution was mixed with equal volumes of NaCl solutions of varying concentrations, resulting in final NaCl concentrations of 0, 25, 50, 100, 150, and 200 mM. The hydrodynamic diameter and zeta potential of the α-CD-VD3 inclusion complex solution were measured using a nanoparticle size analyzer and a zeta potential analyzer, which were used as indicators to analyze ionic strength stability. The instrument was equipped with a 50 mW high-performance solid-state laser operating at a wavelength of 671 nm.

[0052] The ionic strength stability test results of the vitamin D3 delivery system are as follows: Figure 2 and Figure 3 When the NaCl concentration is between 50mM and 150mM, the particle size and potential do not change significantly, indicating that the system has good stability within this ion concentration range.

[0053] pH stability analysis: The pH stability of the vitamin D3 delivery system prepared at pH 9 was analyzed using the following test method: The pH of the α-CD-VD3 inclusion complex solution was adjusted to 1.5, 3.0, 4.5, 6.0, 7.5, and 9.0 using 0.1 mol / L NaOH or HCl solutions. The pH stability of the α-CD-VD3 inclusion complex solution was analyzed using a nanoparticle size analyzer and zeta potential analyzer, using the hydrodynamic diameter and zeta potential as indicators. The instrument was equipped with a 50 mW high-performance solid-state laser operating at a wavelength of 671 nm.

[0054] The pH stability test results of the vitamin D3 delivery system are as follows: Figure 4 and Figure 5As shown in the figure, at pH 1.5, the average particle size of α-CD-VD3 was large, at 561.25 nm. This is likely due to the hydrolysis of α-CD into glucose under strong acidic conditions, which causes aggregation and leads to an increase in the average particle size. Subsequently, as the pH increases, the average particle size of α-CD-VD3 gradually decreases, with little fluctuation. The zeta potential is lowest at pH 4, at -0.51 mV. This is likely due to the low charge of α-CD-VD3, resulting in a low net charge on the surface of the α-CD-VD3 nanoparticles and a less stable α-CD-VD3 inclusion complex. As the pH increases, the potential of the α-CD-VD3 inclusion complex gradually increases, from -11.57 mV to -37.08 mV, indicating that the stability of α-CD-VD3 is gradually enhanced.

[0055] Dispersion stability test: The vitamin D3 delivery system prepared at pH 9 was subjected to dispersion stability analysis using the following test method: Carefully pour the freshly prepared α-CD-VD3 inclusion complex solution into a sample vial, taking care not to stick to the walls, to a height of 45 ± 2 mm. The sample's transmittance was measured at different time points using a dispersion stability analyzer (LAB expert, Turbiscan, France) to assess the dispersion stability of the sample. The sample vial was placed in the dispersion stability analyzer, and the sample was scanned every 30 minutes for a total of 16 measurements. The transmittance change of the sample solution over an 8-hour period was then calculated.

[0056] Figure 6 The figure shows the relationship between time and transmittance. As can be seen from the figure, the fluctuation of the transmittance of α-CD-VD3 is small, indicating that the dispersion stability of the α-CD-VD3 inclusion complex is good during the 8-hour storage process.

[0057] Depend on Figures 1 to 6 It can be seen that the prepared α-CD-VD3 inclusion complex vitamin D3 delivery system has good water solubility and good stability at different salt concentrations and different pH values.

[0058] The in vitro release rate and glucose content of the α-CD-VD3 inclusion complex vitamin D3 delivery system in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were tested using the following methods: To prepare simulated gastric fluid: Accurately weigh 0.6g NaHCO3, 1.1g KCl, and 3.1g NaCl, then dissolve them in ultrapure water and dilute to 1L. Adjust the pH of the solution to 2.0 with 1 mol / L HCl solution. Store at room temperature until ready to use. Dissolve 150.0mg of pepsin (3000-3500 U / mg) in the prepared solution to a final concentration of 1mg / mL. Store at 4°C and use the same day.

[0059] To prepare simulated intestinal fluid: Accurately weigh 0.65g KCl and 5.4g NaCl, then dissolve them in ultrapure water and dilute to 1L. Adjust the pH of the solution to 7.8 with 1mol / L NaOH solution. Accurately weigh 4.0g trypsin (>250U / mg) and dissolve them in ultrapure water and dilute to 100mL. Add 100mL of the prepared solution and mix thoroughly. Adjust the pH of the simulated intestinal fluid to 7.8 with 1mol / L NaOH solution. Add 1mL of saccharifying enzyme (100,000U / mL) and mix thoroughly. Store at 4°C and use the same day.

[0060] The in vitro release of VD3 from α-CD-VD3 samples in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was studied. 4 mL of α-CD-VD3 sample was mixed with 4 mL of SGF, and the pH of the solution was adjusted to 2.0 with 1 mol / L HCl. The mixture was incubated in SGF at 37°C and 100 rpm for 120 min. After the SGF incubation, 4 mL of SIF was added and mixed. The pH of the solution was adjusted to 7.8 with 1 mol / L NaOH solution, and the mixture was incubated in SIF at 37°C and 100 rpm for 120 min. Every 30 minutes, 1 mL of SGF / SIF was removed to measure the released VD3 and glucose content. The absorbance of the released VD3 was measured at 267 nm using a UV-visible spectrophotometer. The glucose content was determined using the 3,5-dinitrosalicylic acid method. First, we constructed a glucose standard curve using UV-visible spectroscopy. Then, we pipetted 1 mL of SGF / SIF into a 25 mL colorimetric tube, added 2 mL of DNS reagent, and heated each tube in a boiling water bath for 2 minutes. After cooling, the tubes were brought to a volume of 25 mL with ultrapure water. The absorbance was measured at 540 nm using a spectrophotometer and substituted into the glucose standard curve to determine the glucose content.

[0061]

[0062] M3: Total VD3 initially added (mg) M4: The total VD3 (mg) test results in SGF or SIF at each set time node are as follows: Figure 7 As shown, during incubation in SGF, the release rate of VD3 showed a slow upward trend, rising from 0% to 4.96%. The slow release rate of VD3 in SGF under strong acid conditions demonstrates the protective effect of α-CD on VD3. Under strong acid conditions, α-CD is broken down into glucose. We measured the glucose content in SGF and found that the glucose concentration gradually increased from 0 mg / mL to 0.33 mg / mL, indicating that the cyclodextrin molecular structure was disrupted, leading to a slow increase in the release rate of VD3. During incubation in SIF, the release rate of VD3 increased from 4.96% to 78.39%, indicating a burst release of VD3 from SGF to SIF. This phenomenon can be explained by the hydrolysis of α-CD in SGF, which partially disrupts the α-CD ring structure, but not enough to release VD3 molecules from the α-CD ring. Consequently, upon entry into SIF, the α-CD ring structure is completely destroyed by the action of glucoamylase, leading to the release of a large number of VD3 molecules. The VD3 release rate suddenly increased from 4.96% to 78.39%, thus achieving a burst release. During this period, the glucose content in SIF rapidly increased from 0.33 mg / mL to 0.97 mg / mL, indicating that α-CD is broken down into a large amount of glucose, leading to a rapid increase in the release rate of VD3.

[0063] Example 2 The preparation method of the calcium-vitamin D3 coordinated delivery system comprises: Dissolve 25 mg of α-CD-VD3 vitamin D3 delivery system in 25 ml of water to obtain an aqueous solution of the vitamin D3 delivery system; One gram of casein phosphopeptide was sonicated and dissolved in ultrapure water. CaCl₂ was then added to adjust the solution's pH to 7.0, and the solution was shaken at 50°C for 60 minutes. Subsequently, 10 volumes of 95% ethanol were added for precipitation. The solution was centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded. The collected precipitate was freeze-dried to obtain the casein phosphopeptide-calcium complex.

[0064] According to the mass ratio of casein phosphopeptide-calcium complex and vitamin D3 delivery system of 1:1, the casein phosphopeptide-calcium complex was added to the aqueous solution of the vitamin D3 delivery system, and stirred for 5 to 10 hours to obtain a calcium-vitamin D3 synergistic delivery system consisting of casein phosphopeptide-calcium-α-cyclodextrin-vitamin D3 complex.

[0065] The calcium-vitamin D3 synergistic delivery system (α-CD-VD3@CPP-Ca 2+) for ionic strength stability analysis, pH stability analysis, dispersion stability test, Ca 2+ in vitro release rate of vitamin D and in vitro release rate of vitamin D3.

[0066] like Figure 8 and Figure 9 As shown in Figure 2, with the increase of NaCl concentration, α-CD-VD3@CPP-Ca 2+ The particle size increases, but within the range of 0-150 mM, the growth is relatively slow, indicating that the system has good stability at different NaCl concentrations. The zeta potential decreases with increasing NaCl concentration. This is because under high ionic strength conditions, the electrolyte shields the surface charge of the nanoparticles, reducing electrostatic repulsion and thus the potential.

[0067] like Figure 10 and Figure 11 As shown, with the increase of pH, α-CD-VD3@CPP-Ca 2+ The particle size first increases and then decreases, reaching a peak at pH 4.5. This is because the pH is close to the isoelectric point of casein, causing nanoparticle aggregation and increased particle size. At this point, the zeta potential also reaches its minimum. Within the pH range of 6-9, the changes in particle size and zeta potential are relatively slow, indicating that the system exhibits good stability within this pH range.

[0068] Figure 12 、 Figure 13 、 Figure 14 and Figure 15 Shown α-CD-VD3, CPP-Ca 2+ and α-CD-VD3@CPP-Ca 2+ The stability and overall stability index (TSI) of the dispersion within 8 hours. The results show that the transmittance fluctuation of α-CD-VD3 is small and the TSI is low, which means that the solution changes little during the measurement period, the inclusion complex system is relatively stable, and the solution is clear and transparent; while CPP-Ca 2+ The transmittance of α-CD-VD3 fluctuated greatly, and the TSI value was high, indicating that the system changed greatly during the measurement, had poor stability, and the solution was turbid. 2+ When the complex is formed, the transmittance fluctuation is significantly reduced, the solution transparency is higher, and the TSI value is reduced, indicating that α-CD-VD3@CPP-Ca 2+ The composite exhibited good dispersion stability during 8 hours of storage.

[0069] Figure 16 and Figure 17demonstrated that α-CD-VD3@CPP-Ca 2+ In vitro Ca2+ expression of the complex in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) 2+ And VD3 release rate curve. Under SGF conditions, CPP-Ca 2+ Ca 2+ The release rate increased rapidly to 65.74% within 120 min and continued to rise under SIF conditions, reaching 81.42% at 240 min. This indicates that CPP can effectively promote the release of Ca 2+ Initial release in the stomach and further release in the intestine. 2+ Combined with α-CD-VD3 to form α-CD-VD3@CPP-Ca 2+ The release rate of the complex under SGF conditions was slightly lower than that of the CPP-Ca 2+ However, under SIF conditions, it showed a higher final release rate (84.12%). This indicates that the inclusion complexation of α-CD-VD3 contributes to the release of CPP-Ca 2+ More efficient release of Ca in the intestine 2+ Under SGF conditions, the VD3 release rate of α-CD-VD3 was low, but it increased significantly under SIF conditions, reaching 78.39% at 240 minutes. This is consistent with the lipid solubility of VD3, indicating that VD3 is mainly released in the intestine. α-CD-VD3@CPP-Ca2+ formed after α-CD-VD3 binds to CPP-Ca2+ 2+ The VD3 release rate of the complex under SGF conditions was similar to that of α-CD-VD3 alone, but the final release rate under SIF conditions was slightly higher. 2+ The presence of CPP helps to improve the release efficiency of VD3 in the intestine. 2+ Forms a stable complex to protect Ca 2+ After being released in the intestine, VD3 is converted into its active form to promote the intestinal absorption of Ca. 2+ In addition, VD3 is also involved in regulating Ca 2+ The homeostasis of α-CD-VD3@CPP-Ca 2+ The complex binds the Ca of the CPP 2+ The release capacity and the inclusion of α-CD on VD3 increase the Ca 2+ It has a synergistic effect on the bioavailability of vitamin D3.

[0070] Example 3 The preparation method of the vitamin D3 delivery system comprises the following steps: Dissolve 25 mg of α-CD-VD3 vitamin D3 delivery system in 25 ml of water to obtain an aqueous solution of the vitamin D3 delivery system; Dissolve 25 mg of calcium glutamate in 25 ml of water to obtain a calcium glutamate aqueous solution; According to the mass ratio of calcium glutamate and vitamin D3 delivery system of 1:1, the calcium glutamate aqueous solution was added to the vitamin D3 delivery system aqueous solution, 9% lipase was added, and stirred at 50°C for 6 hours. The solvent was removed by freeze-drying to obtain a calcium-vitamin D3 synergistic delivery system whose components were calcium glutamate-α-cyclodextrin-vitamin D3 chelate.

[0071] The calcium-vitamin D3 co-delivery system composed of calcium glutamate-α-cyclodextrin-vitamin D3 chelate was subjected to ionic strength stability analysis, pH stability analysis, dispersion stability test, Ca stability in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) and the like. 2+ in vitro release rate of vitamin D and in vitro release rate of vitamin D3.

[0072] like Figure 18 and Figure 19 As shown, with the increase of NaCl concentration, α-CD-VD3&L-Glu-Ca 2+ The particle size increases, but within the range of 0-150 mM, the growth is relatively slow, indicating that the system has good stability at different NaCl concentrations. The zeta potential decreases with increasing NaCl concentration. This is because under high ionic strength conditions, the electrolyte shields the surface charge of the nanoparticles, reducing electrostatic repulsion and thus the potential.

[0073] like Figure 20 and Figure 21 As shown, with the increase of pH, α-CD-VD3&L-Glu-Ca 2+ The particle size first increases and then decreases, reaching a peak at pH 3. This is because the pH is close to the isoelectric point of glutamate, causing the nanoparticles to aggregate and increase in size. At this point, the zeta potential also reaches its minimum. Within the pH range of 4.5 to 9, the changes in particle size and zeta potential are relatively slow, indicating that the system exhibits good stability within this pH range.

[0074] Figure 22 、 Figure 23 、 Figure 24 and Figure 25 Shows α-CD-VD3, L-Glu-Ca 2+ and α-CD-VD3&L-Glu-Ca 2+ The stability and overall stability index (TSI) of the dispersion within 8 hours showed that α-CD-VD3 and L-Glu-Ca2+ The transmittance fluctuation is small and the TSI is low, which means that the solution changes little during the measurement, the system is relatively stable, and the solution is clear and transparent. 2+ When the complex is formed, its transmittance still maintains a small fluctuation, the solution transparency is high, and the TSI value is low, indicating that α-CD-VD3&L-Glu-Ca 2+ The composite exhibited good dispersion stability during 8 hours of storage.

[0075] Figure 26 and Figure 27 Shows α-CD-VD3&L-Glu-Ca 2+ In vitro Ca2+ expression in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) 2+ And VD3 release rate curve. Under SGF conditions, L-Glu-Ca 2+ Ca 2+ The release rate increased rapidly to 64.07% within 120 min and continued to rise under SIF conditions, reaching 69.05% at 240 min. 2+ Combines with α-CD-VD3 to form α-CD-VD3&L-Glu-Ca 2+ The release rate of the complex under SGF conditions was slightly lower than that of L-Glu-Ca alone. 2+ , but showed a higher final release rate (73.81%) under SIF conditions. The VD3 release rate of α-CD-VD3 was low under SGF conditions, but increased significantly under SIF conditions, reaching 78.392% at 240 minutes. 2+ α-CD-VD3&L-Glu-Ca 2+ The VD3 release rate of the complex under SGF conditions was similar to that of α-CD-VD3 alone, but the final release rate under SIF conditions was slightly higher (80.39). This means that Ca 2+ The presence of helps improve the release efficiency of vitamin D3 in the intestine. This system can protect vitamin D3 from stably passing through the acidic environment of the stomach and quickly releasing it in the small intestine, while also improving the release of calcium.

[0076] The above description is only a specific implementation method of the present application, but the protection scope of the present application is not limited thereto. Any changes or replacements within the technical scope disclosed in the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a vitamin D3 delivery system, characterized in that: The following steps are involved: S1, dissolving α-cyclodextrin in an aqueous solution with a pH of 7 to 11 to obtain an α-cyclodextrin solution; S2. dissolving vitamin D3 in anhydrous ethanol to obtain a vitamin D3 anhydrous ethanol solution; S3, adding the anhydrous ethanol solution of vitamin D3 to the α-cyclodextrin solution to react to obtain an α-cyclodextrin-vitamin D3 mixture solution; S4. The α-cyclodextrin-vitamin D3 mixture solution is freeze-dried, re-dissolved and centrifuged to obtain a precipitate, and the precipitate is freeze-dried again to obtain a vitamin D3 delivery system.

2. The method for preparing the vitamin D3 delivery system according to claim 1, wherein The molar ratio of the vitamin D3 to the α-cyclodextrin in the α-cyclodextrin-vitamin D3 mixture solution is 1:

1.

3. An application of the vitamin D3 delivery system prepared by the preparation method according to any one of claims 1 to 2, characterized in that: The vitamin D3 delivery system is used to prepare a calcium-vitamin D3 coordinated delivery system.

4. The use according to claim 3, characterized in that The preparation method of the calcium-vitamin D3 coordinated delivery system comprises: dissolving the vitamin D3 delivery system in water to obtain an aqueous solution of the vitamin D3 delivery system; A casein phosphopeptide-calcium complex was prepared, and the casein phosphopeptide-calcium complex was added to an aqueous solution of a vitamin D3 delivery system. After stirring for 5 to 10 hours, a calcium-vitamin D3 synergistic delivery system consisting of a casein phosphopeptide-calcium-α-cyclodextrin-vitamin D3 complex was obtained.

5. The use according to claim 4, characterized in that The mass ratio of the vitamin D3 delivery system to the casein phosphopeptide-calcium complex is 1:

1.

6. The use according to claim 5, characterized in that The preparation method of the casein phosphopeptide-calcium complex comprises: CaCl2 was added to the casein phosphopeptide aqueous solution, the pH was adjusted to 7, the reaction was carried out for 40 to 90 minutes, ethanol was added and the solution was centrifuged, and the precipitate was freeze-dried to obtain a casein phosphopeptide-calcium complex.

7. The use according to claim 3, characterized in that The preparation method of the calcium-vitamin D3 coordinated delivery system comprises: dissolving the vitamin D3 delivery system in water to obtain an aqueous solution of the vitamin D3 delivery system; dissolving calcium glutamate in water to obtain a calcium glutamate aqueous solution; The calcium glutamate aqueous solution is added to the vitamin D3 delivery system aqueous solution, lipase is added, and the mixture is stirred at 45-55° C. for 4-8 hours to obtain a calcium-vitamin D3 coordinated delivery system whose components are calcium glutamate-α-cyclodextrin-vitamin D3 chelate.

8. The use according to claim 7, characterized in that The mass ratio of the sum of the mass of the vitamin D3 delivery system and the calcium glutamate to the lipase is 100:5-9.

9. The use according to claim 7, characterized in that The mass ratio of the calcium glutamate and the vitamin D3 delivery system is 1:1-4.

10. The use according to claim 7, characterized in that The preparation method of calcium glutamate comprises: adding CaCl2 to a glutamic acid aqueous solution at 70-80°C, adjusting the pH to 7, reacting at 45-55°C for 40-90 minutes, adding ethanol and centrifuging, and lyophilizing the precipitate to obtain calcium glutamate.

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