Soft capsule containing calcium, vitamin D and vitamin K and preparation process of soft capsule

Abstract

The invention relates to the technical field of biological medicine preparations, and discloses a calcium, vitamin D and vitamin K. A content composition of the soft capsule comprises a microgel skeleton composed of amphiphilic polypeptide calcium ions and vitamin oil drops, the hydrophobic end of amphiphilic polypeptide adsorbs the oil drops, and the hydrophobic end of the amphiphilic polypeptide adsorbs the vitamin oil drops. A hydrophilic end of the composition is bridged by calcium ions to form a network, the composition also comprises a histidine-enriched polypeptide which can form a protective shielding layer on the surface of a skeleton under acidic pH, the calcium ions are converted from a chemical destroyer to a structural stabilizer, and a static synergistic system is constructed, so that the contact degradation of calcium and vitamins is eliminated, and the stability of the composition is improved. The performance contradiction between the high calcium content and the vitamin stability is avoided, and the endophytic environmental adaptability of the composition is improved.

Description

Technical Field

[0001] This invention relates to a calcium, vitamin D, and vitamin K soft capsule and its preparation process, belonging to the field of biopharmaceutical formulation technology. Background Technology

[0002] Currently, in liquid formulations containing both aqueous and oil phases, a fundamental physicochemical principle is that polyvalent cations, especially divalent calcium ions, can catalyze lipid oxidation or saponification through interfacial bridging, causing oil droplet aggregation, content stratification, turbidity, and degradation of fat-soluble active ingredients. Therefore, in this field, when encapsulating an aqueous solution of calcium salts and an oil phase containing fat-soluble vitamins in a unified dosage form, the accepted technical approach is to use emulsifiers for homogenization to form a macroscopically uniform and stable emulsion system. This method can ensure the uniform distribution of each active ingredient in the initial state of the product.

[0003] However, while emulsification technology achieves uniform component distribution, it also generates a huge oil-water interface area. This provides sufficient conditions for the aforementioned calcium ion-mediated degradation reaction. Over time, microscopic ion diffusion and interfacial reactions continue to occur. This phenomenon is determined by the characteristics of the emulsification method itself and has long been regarded as an inherent limitation accompanying the achievement of component uniformity. To delay this process, the improvement ideas of those skilled in the art usually focus on using stronger emulsifiers or adding chelating agents to reduce the concentration of free calcium ions. However, these improvements do not change the basic structure of the emulsion system. While increasing the complexity of the formulation, they still fail to physically block the final contact between calcium ions and fat-soluble vitamins at the interface.

[0004] Specifically, existing technologies have the following shortcomings: 1. The need for stable preservation of active ingredients and the need for homogeneous mixing are mutually restrictive within the same emulsified system. The stability of the system depends on the assistance of external additives rather than its own intrinsic structure. 2. There is an inherent contradiction between the functionality of calcium ions as nutrients and their chemical activity as catalysts for degradation reactions. In existing systems, inhibiting the latter's activity usually also affects the bioavailability of the former as a nutrient. 3. There is a direct correlation between the long-term stability of the product and its initial homogeneity. Pursuing a higher degree of homogenization objectively provides a larger reaction interface for degradation reactions, which may accelerate the long-term loss of active ingredients. Therefore, how to construct an internal structure for a composition in which calcium ions exist as structural units in a physically fixed manner, serving as a sufficient nutrient while preventing them from reacting with fat-soluble vitamins in the oil phase, thus eliminating the mutual restriction between high calcium supplementation and the maintenance of high-stability vitamin activity, is the technical problem to be solved by this invention. Summary of the Invention

[0005] This invention provides a calcium, vitamin D, and vitamin K soft capsule. Its main purpose is to solve the problem that in existing liquid formulations, the nutritional function of calcium ions and their chemical destructive effect on fat-soluble vitamins are inherently contradictory, and that traditional emulsification methods cannot fundamentally prevent the contact and degradation of the two.

[0006] To achieve the above objectives, the present invention provides a calcium, vitamin D, and vitamin K soft capsule, wherein the contents of the soft capsule are a composition comprising: Oil droplets containing vitamins D and K; The first amphiphilic polypeptide comprises a polyanionic head consisting of 8 to 12 glutamic acid residues and aspartic acid residues and a hydrophobic tail consisting of 12 to 20 amino acid residues selected from at least one of leucine, isoleucine, and valine. The second type of histidine-enriched cationic shielding peptide; And calcium ions; the hydrophobic tail of the first amphiphilic peptide adsorbs onto the surface of the oil droplet, and its polyanionic head connects two first amphiphilic peptides from different oil droplet surfaces via calcium ions as bridging nodes, thereby forming a three-dimensional microgel framework and embedding the oil droplet therein; when the composition is under acidic pH conditions, the histidine-rich cation-shielding peptide becomes positively charged due to the protonation of its histidine residues, and spontaneously adsorbs onto the surface of the microgel framework via electrostatic attraction to form a cation-shielding layer; the composition at pH 6.8 has a storage modulus of The range is 10 Pa to 1000 Pa, and its Fourier transform infrared spectrum or X-ray photoelectron spectrum shows a peak shift from carboxylate group to calcium ion coordination state.

[0007] Preferably, in the composition, the total molar ratio of calcium ions to carboxylate ions in the polyanionic head of the first amphiphilic polypeptide is 0.5:1 to 1.5:1; and the mass ratio of oil droplets to the aqueous phase containing the first amphiphilic polypeptide and the second histidine-enriched cationic shielding peptide is (0.5-10):1 based on the total weight of the composition.

[0008] Preferably, the first amphiphilic polypeptide contains an amino acid sequence at the junction of the polyanionic head and the hydrophobic tail that can be specifically cleaved by trypsin or chymotrypsin.

[0009] Preferably, the amino acid sequence that can be specifically cleaved contains arginine residues or lysine residues; when the first amphiphilic polypeptide is cleaved, the independent polyanionic head and independent hydrophobic tail generated by its breakage form a self-molecular emulsion system in situ around the oil droplet.

[0010] Preferably, the composition further comprises a water-soluble sacrificial polypeptide containing one or more sites that can be cleaved by trypsin or chymotrypsin, and the sacrificial polypeptide has an affinity for trypsin or chymotrypsin equal to or greater than that for the first amphiphilic polypeptide.

[0011] Preferably, the initial degradation rate of the microgel backbone upon encountering trypsin or chymotrypsin. The initial concentration of the sacrificial peptide The regulation of these relationships satisfies the following: ,in, and To characterize the composition system under preset enzyme concentration and temperature conditions, using objective physicochemical constants that can be determined by enzyme kinetic experiments. The intrinsic maximum degradation rate of the microgel framework, The coefficient of competitive inhibition of the enzyme by the sacrificial polypeptide is denoted as .

[0012] Preferably, the polyanionic head of the first amphiphilic polypeptide, after being cleaved by trypsin or chymotrypsin, forms a fragment that can act as a competitive inhibitor of the trypsin or chymotrypsin.

[0013] Preferably, the amino acid sequence of the polyanionic head further includes at least one arginine residue or lysine residue at the end furthest from the junction, so that the fragment forms a negative feedback inhibition on the active site of the enzyme in the enzymatic hydrolysis environment.

[0014] Preferably, in the second type of histidine-enriched cation-shielding peptide, the molar fraction of histidine residues is not less than 30%; when the composition is transferred from an environment with a pH of 1.5 to 3.5 to an environment with a pH higher than 6.0, the cation-shielding layer automatically dissociates from the surface of the microgel framework due to the deprotonation of histidine residues. The composition is measured by dynamic light scattering method, and the peak volume distribution of oil droplets embedded in the three-dimensional microgel framework is 100 nm to 800 nm.

[0015] A process for preparing the contents composition of a calcium, vitamin D, and vitamin K soft capsule includes the following steps: Vitamin D and vitamin K are dissolved in the pharmaceutical oil phase to obtain the oil phase; A first amphiphilic polypeptide comprising a polyanionic head consisting of 8 to 12 glutamic acid residues and aspartic acid residues and a hydrophobic tail consisting of 12 to 20 amino acid residues selected from at least one of leucine, isoleucine, and valine, and a second histidine-enriched cation-shielding peptide, are dissolved together in an aqueous phase containing calcium salts to obtain an aqueous phase. Under gentle stirring conditions, the oil phase is dispersed in the aqueous phase, allowing the hydrophobic tail of the first amphiphilic polypeptide to spontaneously adsorb onto the oil droplet interface formed between the oil and aqueous phases; the gentle stirring conditions are achieved by controlling the shear rate at 100... Up to 1000 The operation is carried out within a certain range, with the aim of dispersing the oil phase into micron-sized oil droplets while avoiding the formation of a high-energy-consuming dense emulsion system, thereby providing favorable conditions for the subsequent spontaneous adsorption and ion bridging of peptides.

[0016] Furthermore, calcium ions in the aqueous phase are used to ion bridge the polyanionic heads of the first type of amphiphilic polypeptide adsorbed on different oil droplet interfaces, thereby spontaneously forming a three-dimensional microgel framework that embeds and fixes the oil droplets, thus obtaining the composition.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. The present invention provides a composition in which the hydrophobic tail of an amphiphilic polypeptide is adsorbed onto the surface of an oil droplet, while its hydrophilic head, under the bridging effect of calcium ions, connects polypeptides from different oil droplet surfaces, forming a three-dimensional network framework between the oil droplets. In this framework, calcium ions, as nutrients, also act as structural nodes, transforming ions that originally move freely in the liquid phase and have a destructive effect on the oil-water interface into structural components fixed in specific spatial positions. This physically locks the vitamin oil droplets into the network pores, thereby constructing a static synergistic system in which active ingredients coexist in an orderly manner, avoiding the problem of continuous contact degradation caused by the large interfacial area and free movement of ions in traditional emulsification methods.

[0018] 2. The composition also contains a polypeptide rich in histidine residues. The histidine side chain of the polypeptide is protonated and becomes positively charged in the acidic environment of the stomach, allowing it to automatically adsorb onto the surface of the negatively charged microgel backbone via electrostatic attraction, forming a temporary cationic shielding layer to prevent gastric acid from damaging the calcium ion bridge bonds inside the backbone. When the composition enters the near-neutral environment of the intestine, the histidine side chain of the polypeptide is deprotonated and returns to electroneutrality, allowing it to automatically detach from the backbone surface and restore the original state of the microgel backbone, which is easily accessible to digestive enzymes. The entire process is triggered by the direct interaction between the composition's own components and the chemical environment of different sections of the digestive tract, enabling autonomous recognition and adaptive structural adjustment to specific physiological environments without external coating.

[0019] 3. The amphiphilic polypeptide contains an amino acid sequence at the junction of its hydrophilic head and hydrophobic tail that can be specifically cleaved by intestinal proteases. When the microgel backbone is acted upon by proteases in the small intestine, its structural disintegration is a pre-programmed cleavage process. Each amphiphilic polypeptide molecule is precisely cleaved, generating an independent hydrophilic polypeptide fragment and an independent hydrophobic polypeptide fragment in situ. These released fragments immediately form a high-concentration self-emulsifying system around the vitamin oil droplets, actively dispersing the oil droplets into easily absorbed microparticles. This allows the core component, which originally provided structural support, to transform into a functional component that promotes the absorption of the active ingredient it encapsulates at the end of its life cycle. Furthermore, during the preparation of the composition... A water-soluble sacrificial peptide is added to the aqueous phase. This sacrificial peptide contains protease cleavage sites with the same or higher affinity as the amphiphilic peptide. When the composition encounters proteases in the small intestine, the freely dispersed and easily accessible sacrificial peptide in the aqueous phase will be preferentially degraded, consuming some of the enzyme's activity. This creates a time delay for the degradation of the larger and more complex microgel framework. By adjusting the initial concentration of this sacrificial peptide in the formulation, the length of this delay can be directly set, thereby regulating the overall degradation rate of the subsequent microgel framework. This allows the release rhythm of the active ingredient to adapt to different absorption needs, providing an intrinsic kinetic control pathway determined by the formulation itself for the performance consistency of oral formulations across different individuals. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the multi-level environmental response and programmed degradation mechanism of the composition of the present invention; Figure 2 This is a comparative graph showing the adaptive behavior of the release kinetics of the composition of the present invention to changes in enzyme concentration. Figure 3 This is a flowchart illustrating the sequential in vivo deployment and functional release of the soft capsules of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be noted that the embodiments described herein are only for explaining this invention and are not intended to limit it.

[0022] This invention discloses a composition for the contents of calcium-vitamin D and vitamin K soft capsules. It establishes a delivery system synergistically utilizing a calcium-bridged microgel framework, a pH-responsive cation shielding layer, and a programmed degradation and kinetic regulation mechanism. The calcium-bridged microgel framework forms the structural basis of the entire composition, addressing the chemical destructive effects of high concentrations of free calcium ions on fat-soluble vitamins in liquid formulations, and the technical problem of traditional emulsification methods providing a large reaction interface for such destructive contact. The framework comprises oil droplets encapsulating vitamin D and vitamin K, calcium ions, and a first amphiphilic polypeptide. The molecular structure of the first amphiphilic polypeptide includes a polyanionic head composed of 8 to 12 glutamic acid and aspartic acid residues, and a hydrophobic tail composed of 12 to 20 amino acid residues selected from at least one of leucine, isoleucine, and valine. In the preparation process of the composition, by dispersing the oil phase containing vitamins in an aqueous phase containing the first amphiphilic polypeptide and calcium salts, the hydrophobic tail of the polypeptide is hydrophobically affected. Spontaneously adsorbed onto the surface of oil droplets, while their hydrophilic polyanionic heads extend into the aqueous phase, calcium ions in the aqueous phase act as structural bridging nodes. Each divalent calcium ion can simultaneously form ionic bonds with the polyanionic heads of the first amphiphilic polypeptide from two different oil droplet surfaces, thus crosslinking the originally independent oil droplet units into a macroscopic three-dimensional microgel framework. The oil droplets are physically embedded in the pores of this framework. Determining the molar ratio of calcium ions to the total carboxyl groups in the polyanionic heads of the first amphiphilic polypeptide is crucial for forming a stable gel structure. When this molar ratio is below 0.5:1, the number of calcium ions is insufficient to form an effective crosslinking network, resulting in low gel strength. When this molar ratio is above 1.5:1, excessive free calcium ions exist in the aqueous phase, potentially triggering a renewed risk of chemical damage to vitamins. Therefore, setting this molar ratio within a working window of 0.5:1 to 1.5:1 yields a gel framework with defined physical stability. This framework, at pH 6.8, exhibits a storage modulus of [missing value]. The system was calibrated to range from 10 Pa to 1000 Pa. This parameter was measured using a rheometer. The system below 10 Pa exhibited fluid characteristics and could not effectively fix the oil droplets. The system above 1000 Pa was too rigid and not conducive to subsequent disintegration in the intestine. Thus, a static system was constructed that transformed calcium ions from chemical destroyers into structural stabilizers, thereby avoiding contact degradation of vitamins by calcium ions from a physical structure perspective.

[0023] Based on the calcium-bridged microgel framework, since this framework relies on ionic bonds for stability, when it enters the highly acidic environment of the stomach (pH 1.5-3.5), a large number of hydrogen ions protonate the carboxyl groups at the polyanion head, leading to ionic bond breakage and premature framework disintegration. To address this issue, a pH-responsive cation shielding layer is introduced into the system. The core component of this shielding layer is a second type of histidine-rich cation shielding peptide, in which the molar fraction of histidine residues is not less than 30%, and the pKa value of the imidazole ring on the histidine side chain is approximately 6.0. When the pH in the stomach is much lower than 6.0, the imidazole ring undergoes protonation. The peptide chain is deprotonated to acquire a net positive charge. A histidine content of at least 30% ensures sufficient positive charge density under gastric acid conditions, allowing the peptide to automatically adsorb onto the negatively charged microgel framework surface via electrostatic attraction, forming a dense cationic shielding layer. This shielding layer physically prevents hydrogen ions from gastric acid from penetrating inward and provides additional electrostatic reinforcement to the original framework. When the composition enters the small intestine environment (pH above 6.0), the histidine side chain deprotonates, the shielding peptide returns to electroneutrality, the electrostatic attraction disappears, and it can automatically dissociate from the framework surface. This process enables the composition to maintain structural stability in the acidic gastric environment and automatically deprotects itself upon entering the near-neutral intestinal environment, ensuring the integrity of the core structure until reaching the target absorption site. Once the composition is deprotected in the small intestine, a programmed degradation and kinetic regulation mechanism is configured to achieve efficient release and absorption of the active ingredient. First, the amino acid sequence of the first amphiphilic polypeptide is designed, and an amino acid sequence that can be specifically cleaved by trypsin or chymotrypsin in the small intestine, such as arginine, is inserted at the junction of its polyanionic head and hydrophobic tail. The sequence of lysine residues, or when the microgel backbone encounters trypsin in the small intestine, the enzyme will cleave at the preset site, causing each first amphiphilic polypeptide molecule to be simultaneously cleaved into an independent polyanionic head fragment and an independent hydrophobic tail fragment. These fragments, which are released in situ at high concentrations around the vitamin oil droplets, immediately act as a self-emulsifying system, dispersing large oil droplets into tiny micelles that are easily absorbed by intestinal epithelial cells. This design allows the degradation products of the first amphiphilic polypeptide, after completing its structural support function, to then promote the dispersion and absorption of the active ingredient.

[0024] To address the issue of uneven release rates potentially caused by differences in enzyme activity among individuals, this mechanism also introduces a kinetic regulation step. Specifically, the composition includes a water-soluble sacrificial peptide with one or more cleavage sites by trypsin or chymotrypsin, exhibiting an affinity for the enzyme equal to or greater than that of the first amphiphilic peptide. Upon entering the small intestine, the readily accessible sacrificial peptide, dispersed in the aqueous phase, acts as a competitive substrate and is preferentially degraded by the enzyme. This objectively consumes some of the enzyme's activity and creates a time delay for the degradation of the more complex microgel framework, thus slowing down the initial degradation rate. Initial concentration of sacrificial peptide The regulation of these relationships satisfies the following: ,in, This refers to the inherent maximum degradation rate of the microgel backbone under preset enzyme concentrations and temperatures, without sacrificial peptides. The two parameters, namely the competitive inhibition coefficient of the sacrificial peptide on the enzyme, are physicochemical constants that can be determined by standard in vitro enzyme kinetic experiments. The initial concentration of the sacrificial peptide in the formulation is adjusted accordingly. This allows for the preset release rhythm of the active ingredient. As a further optimization of kinetic regulation, to achieve adaptive adjustment to individual physiological differences, the system also introduces a negative feedback inhibition mechanism. This mechanism is achieved by redesigning the sequence of the polyanionic head of the first amphiphilic polypeptide. Specifically, at its end away from the enzyme cleavage junction, it contains at least one arginine or lysine residue. When the polypeptide is cleaved by the enzyme, the released polyanionic head fragment, due to the presence of its positively charged arginine or lysine residue at its end, can act as a competitive inhibitor of the trypsin or chymotrypsin. The mechanism is that when an individual's enzyme activity is too high, the skeletal degradation rate is fast, which instantaneously produces a high concentration of inhibitory fragments locally. These fragments bind to the enzyme's active site, thereby reducing the enzyme's catalytic efficiency and forming a negative feedback loop, which automatically slows down the subsequent degradation rate. Conversely, when the enzyme activity is low, the concentration of the inhibitory fragments produced is low, and the inhibitory effect on the enzyme is weak, thus allowing the degradation process to proceed normally. This forms a negative feedback regulation mechanism that can respond to the level of enzyme activity in the environment, enabling the degradation rate of the composition to tend towards a steady state among different physiological individuals, thereby improving the uniformity of the oral formulation's performance.

[0025] Example 1: In an accelerated aging test to evaluate the long-term stability of an oral liquid formulation, a prepared soft capsule contents composition containing calcium gluconate and oil droplets containing dissolved vitamin D and vitamin K was placed at a temperature of 40°C. The product was stored for 6 months at a relative humidity of 75%, simulating the storage environment during its 24-month shelf life. In the initial stage of the test, the composition exhibited a uniform gel-like structure. The calcium ions inside served as structural nodes forming the calcium-bridged microgel framework. The oil droplets encapsulating the vitamins were physically isolated from the first amphiphilic polypeptide molecular layer. This structure inhibited the oil-phase saponification or fat-soluble vitamin oxidative degradation reactions that are usually catalyzed by calcium ions in high-temperature and high-humidity environments. Throughout the entire 6-month accelerated aging cycle, the composition maintained its initial macroscopically uniform gel morphology, without oil droplet aggregation, flocculation, or insoluble precipitation.

[0026] After the experiment, a portion of the aged samples were subjected to in vitro simulated digestion. First, the samples were introduced into simulated gastric fluid at pH 1.5. The second free histidine-rich cation-shielding peptide in the composition, due to the protonation of its histidine residues, became positively charged and adsorbed onto the surface of the microgel backbone, forming a cation-shielding layer. This layer protected the calcium-carboxyl ion bonds that maintain the backbone's stability, preventing them from being damaged by the strong acid environment. Subsequently, the system was transferred to simulated intestinal fluid at pH 6.8 containing trypsin. The cation-shielding layer dissociated from the backbone surface due to the deprotonation of histidine residues. The exposed microgel backbone was acted upon by trypsin. Because the specific linkages of the first amphiphilic peptide contained pre-defined cleavage sites, the backbone disintegrated in a programmed cleavage manner, and its degradation products, namely independent polyanionic peptides... The microgel consists of a head and an independent hydrophobic tail, forming a self-emulsifying system in situ around the oil droplet. Quantitative analysis of the aged sample using high-performance liquid chromatography (HPLC) showed that the contents of vitamin D and vitamin K remained above 95% of their initial values. Furthermore, particle size distribution analysis of the in vitro digested sample using dynamic light scattering revealed that it could disperse oil droplets into particles with an average diameter ranging from 100 nm to 800 nm in a simulated intestinal environment. These experimental results indicate that the calcium-bridged microgel framework maintained the physical and chemical stability of the contents under accelerated aging conditions. The pH-responsive cation shielding layer further ensured the integrity of the framework in simulated gastric juice. Finally, a programmed degradation mechanism triggered structural disintegration and effective dispersion of the contents in simulated intestinal juice.

[0027] Example 2: To quantitatively verify the technical effect of the composition in terms of physical stability and protection of active ingredients, this example established a set of control experiments, which aimed to compare the performance of the sample group using the scheme of the present invention with the control group using the prior art that lacks key components or has key ratio parameters that are out of range, under accelerated aging conditions.

[0028] Five sample groups were set up for the experiment. The sample group of this invention was a complete composition prepared according to a specific embodiment, with a total molar ratio of calcium ions to carboxylate groups in the polyanionic head of the first amphiphilic polypeptide of 1:1. Control group 1 was a conventional emulsification system with the same active ingredients and oil-water ratio as the sample group of this invention, but without functional polypeptides, using lecithin as an emulsifier and prepared by a high-pressure homogenizer. Control group 2 was prepared without calcium ions based on the formulation of the sample group of this invention. Control groups 3 and 4 were prepared based on the formulation of the sample group of this invention, with the total molar ratio of calcium ions to carboxylate groups set to 0.3:1 and 2.0:1, respectively. After preparation, all sample groups were placed at a temperature of 40°C. Accelerated aging tests were conducted for 3 months in a stability test chamber with a relative humidity of 75%, and samples were taken at the end of months 0, 1, 2, and 3 to test its physical properties and energy storage modulus. The retention rates of vitamin D and vitamin K content; physical properties during the experiment were observed visually, including storage modulus. Using a rotational rheometer at 25 The vitamin content was determined by high-performance liquid chromatography (HPLC) under conditions of 1 Hz, and the key data are recorded in Table 1.

[0029] Table 1: Changes in performance indicators of each sample group during accelerated aging test.

[0030] Table 1 shows that control group 1 rapidly destabilized under accelerated conditions, exhibiting oil-water separation and a significant decrease in vitamin content; control group 2, lacking calcium ions, failed to form an effective gel network, resulting in physical instability and rapid vitamin degradation; control group 3, due to insufficient calcium ion crosslinking density, formed a gel with low strength, gradually collapsing and delaminating during storage; control group 4, although forming a stable gel structure, experienced faster vitamin degradation than the sample group of this invention, because excessive free calcium ions in the aqueous phase chemically damaged the vitamins. The experimental results indicate that only the sample group of this invention maintained stable physical properties and storage modulus throughout the entire experimental period. All remained stable, and the vitamin content retention rate was significantly higher than that of all control groups.

[0031] Example 3: This example combines Figures 1 to 3 A description of a calcium, vitamin D, and vitamin K soft capsule and its preparation process, such as... Figure 1As shown, the framework physically encapsulates and isolates vitamin oil droplets. Upon entering the digestive tract, the system first enters a framework stabilization phase. At this stage, the pH-responsive cation shielding layer protonates and becomes positively charged under the acidic pH of the stomach, spontaneously adsorbing onto the framework surface to form a protective layer, thus maintaining the framework's stability in a strongly acidic environment. Subsequently, in the intestinal environment, the system enters a programmed degradation and self-emulsification phase. The shielding layer automatically dissociates, and the framework undergoes programmed degradation. Its products form a self-molecular emulsion system in situ around the oil droplets. This process is precisely regulated by a programmed degradation and kinetic control mechanism. This mechanism achieves precise pre-setting and adaptive adjustment of the release rate through specific enzymatic cleavage, sacrificial peptide competition, and product negative feedback, ultimately achieving the goal of efficient absorption of active ingredients and dispersing vitamin oil droplets into nanoparticles, thereby improving bioavailability.

[0032] like Figure 2 As shown in the figure, the logarithm of trypsin concentration (mg / mL) is used as the x-axis, and the main release time (T50% min) of the contents is used as the y-axis. The performance curves of sample group A with negative feedback and control group B without negative feedback are compared. The results in the figure show that the release time of control group B is highly sensitive to changes in enzyme concentration. Its T50% decreases sharply as the enzyme concentration increases from 0.02 mg / mL to 0.5 mg / mL. In contrast, the release time of sample group A of the present invention shows significantly low sensitivity within the same enzyme concentration range. The slope of its response curve is much smaller than that of control group B. This indicates that the present invention effectively achieves steady-state control of the release rate at different physiological enzyme activity levels through a negative feedback inhibition mechanism.

[0033] like Figure 3 As shown, the process begins with the self-assembly of oil-phase raw materials, aqueous-phase raw materials, and functional peptides into a static synergistic system via a microgel framework, which is then encapsulated into a finished soft capsule. After oral administration, the soft capsule enters the stomach, where a pH-responsive shielding mechanism is activated. The cationic shielding peptides become positively charged in the acidic environment of the stomach and adsorb onto the surface of the framework, forming a physical protective layer to resist the erosion of gastric acid. Subsequently, when it enters the small intestine, the shielding layer detaches due to the increase in pH, exposing the framework and triggering programmed degradation. Small intestinal proteases specifically cleave the framework at preset sites, causing the framework structure to disintegrate. The peptide fragments generated by degradation then undergo self-emulsification, forming an efficient emulsification system in situ around the oil droplets, dispersing the oil droplets into easily absorbed nanoparticles. The entire degradation and release process is also regulated by a kinetic control mechanism, with the degradation rate jointly regulated by sacrificial peptides and negative feedback inhibition. Ultimately, this achieves a stable and predictable release of active ingredients and the release and absorption of nutrients, enabling the nano-sized vitamin particles and calcium ions to be efficiently absorbed by intestinal epithelial cells, thereby improving bioavailability.

[0034] Example 4: This example discloses a method for determining the initial concentration of sacrificial peptides. Standardized engineering calibration procedures to achieve the initial degradation rate of the microgel framework. The parameters are pre-tunable; in a specific formulation development task, the technical objective is to ensure that the microgel backbone of the composition undergoes significant degradation at approximately 30 minutes after entering a simulated small intestinal environment, wherein the storage modulus is [missing information]. Significant degradation is defined as a decrease to 50% of the initial value. To achieve this, the concentration of the sacrificial peptide was determined using the following experimental procedure. The initial conditions used in the experiment were: a reaction vessel equipped with a magnetic stirrer and temperature control device, containing a solution with a pH of 6.8 and a temperature maintained at 37°C. Simulated intestinal fluid with a trypsin concentration of 0.1 mg / mL; a device capable of real-time monitoring of sample storage modulus. The rotational rheometer has its measurement frequency set to 1 Hz.

[0035] First, a baseline composition sample without sacrificial peptides was prepared and placed on a rheometer until it reached 37 °C. After thermal equilibrium is reached, trypsin solution is injected and timing is started to continuously monitor its storage modulus. Records of changes over time The time required for the initial value to decrease to 50% is denoted as the baseline degradation time. The results were measured under the experimental conditions. The time was 5.2 minutes, which characterizes the intrinsic degradation kinetics of the microgel framework in the absence of competitive inhibition, and is related to the formula... constants in Related; secondly, a series of initial concentrations of sacrificial peptides were prepared. Except for the differences, the test composition sample is completely identical to the reference sample in all other components. The concentration gradients were set at 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL. The same enzymatic digestion and rheological monitoring procedures were repeated for each of the above test samples, and the concentrations were recorded. The time required for the value to decrease to 50% of its initial value is recorded as the total degradation time. And calculate the time delay introduced by the sacrificial peptide. The time delay obtained With the corresponding initial concentration of sacrificial peptide Perform function fitting to establish a calibration relationship under this experimental system, and calculate based on this calibration relationship when the target time delay is... At minute, the corresponding initial concentration of sacrificial peptide The concentration is 0.36 mg / mL, which is the concentration required to achieve the preset technical target.

[0036] Example 5: In the industrial production of the composition, to ensure consistent functional properties of the first amphiphilic peptide raw material across different batches, a standardized raw material function verification procedure needs to be performed. This procedure is carried out in a constant-temperature stirred reactor, using a batch of standard oil phase and standard calcium ion aqueous solution, respectively mixed with a known qualified reference batch of peptide and a batch of peptide to be tested, and two small-scale samples are prepared under the same stirring rate and time. Subsequently, a rotational rheometer is used at 25°C. The storage modulus of the two samples was measured under the condition of 1 Hz. The acceptance criterion for the batch of peptides to be tested is the storage modulus of the sample formed by them. The value, compared with the value of the reference batch sample, deviates within ±10%.

[0037] Accordingly, before each batch of final content composition is prepared and enters the soft capsule encapsulation process, a batch release test is performed to confirm the formation of the calcium-bridged microgel framework structure. This test is conducted by detecting the sample using Fourier transform infrared spectroscopy, analyzing the carboxylate functional groups in the 1550-1650°C range. The symmetrical stretching vibration peaks in the band are used to determine the coordination state with calcium ions. The standard for batch release is that, compared with the semi-finished product without added calcium ions, the spectrum of the final composition sample shows a clearly identifiable peak shift caused by the ionic bonding between carboxylate and calcium ions.

[0038] Example 6: This example discloses a standardized procedure for establishing a baseline model of the kinetic response of a composition at different enzyme activity levels. The test results of this procedure are used as a control benchmark to ensure that products from different production batches have adaptive release kinetics. In an offline optimization test designed to characterize the robustness of the formulation, the objective function is to minimize the release time. To test the fluctuation range of different trypsin concentrations, two sample groups were set up. Sample group A of the present invention was prepared according to the above, and its first amphiphilic polypeptide contained an arginine residue at the hydrophilic head end of the complete composition. The formulation of control group B was the same as that of sample group A of the present invention, but the first amphiphilic polypeptide used in it did not contain an arginine residue at the hydrophilic head end, and therefore did not have the function of inhibiting degradation products.

[0039] The experiment was conducted on a testing platform consisting of a temperature-controlled reaction vessel and a rotational rheometer. Sample A and control group B were placed in three pre-set simulated intestinal fluid environments with different trypsin concentrations: 0.02 mg / mL, 0.1 mg / mL, and 0.5 mg / mL, respectively, to cover the physiological range of enzyme activity fluctuations. The test was conducted at 37°C. Below, the storage modulus of each sample group at various enzyme concentrations is recorded. Time required for the value to drop to 50% from the initial value This time was used to characterize the primary release time of the contents; the experimental results showed that the release time of control group B was... It exhibits high sensitivity to changes in enzyme concentration, and the measured values ​​at enzyme concentrations of 0.02 mg / mL, 0.1 mg / mL, and 0.5 mg / mL are [data missing]. The release times were 95.2 minutes, 15.8 minutes, and 3.1 minutes, respectively; these were the release times measured for sample group A of this invention under the same three enzyme concentration gradients. The release times were 48.5 minutes, 30.1 minutes, and 19.8 minutes, respectively. The slope of the response curves for sample A was much smaller than that for control group B. Calculations show that the release time of sample A in this invention is... The variance within the tested enzyme concentration range was one order of magnitude lower than that of control group B. This set of data established a kinetic response baseline model for sample group A of the present invention. Its characteristic is the low sensitivity of release time to fluctuations in external enzyme concentration. This model was used for quality consistency evaluation of subsequent production batches.

[0040] Example 7: This example provides a specific component ratio and preparation process for a content composition. In a 1000g total formulation mass, it contains 300.0g of oil phase component and 700.0g of aqueous phase component. The oil phase component consists of 299.8g of pharmaceutical-grade soybean oil and 0.1g of vitamin C. (Cholecalciferol) and 0.1g vitamin (Menadione-7) consists of an aqueous phase comprising 15.0 g of a first amphiphilic peptide (whose polyanionic head consists of 10 glutamic acid residues and its hydrophobic tail consists of 16 leucine residues, linked by a lysine residue), 4.0 g of a second histidine-rich cation-shielding peptide (whose total amino acid sequence contains 40% histidine residues), 0.36 g of a water-soluble sacrificial peptide, 21.4 g of calcium gluconate (the amount of which results in a 1:1 molar ratio of calcium ions to the total carboxyl group of the first amphiphilic peptide), and 659.24 g of water for injection.

[0041] Its preparation process is as follows: First, at 25 In an environment where vitamins With vitamins Add to soybean oil and stir at 200 rpm for 30 minutes to obtain a clear oil phase; simultaneously, at 25... Under certain conditions, all aqueous phase components were added to water for injection and stirred at 300 rpm for 60 minutes to obtain a clear aqueous phase; subsequently, the obtained oil phase was subjected to a shear rate of... Under these conditions, the mixture was slowly injected into the aqueous phase and sheared continuously for 10 minutes to form a primary dispersion system; finally, the shearing was stopped, and the dispersion system was incubated at 25°C. After standing for 2 hours, the system spontaneously formed a three-dimensional microgel framework, yielding the final content composition. Characterization of the prepared composition showed that at 25... Its storage modulus at pH 6.8 The Pa was 360; Fourier transform infrared spectroscopy results showed that the carboxyl group was at... The symmetric stretching vibration peak at that point shifted towards higher wavenumbers compared to the system without added calcium gluconate. The movement is attributed to the coordination of the carboxylate group to the calcium ion.

[0042] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A calcium, vitamin D, and vitamin K soft capsule, characterized in that, The contents of the soft capsules are a composition comprising: Oil droplets containing vitamins D and K; The first amphiphilic polypeptide comprises a polyanionic head consisting of 8 to 12 glutamic acid residues and aspartic acid residues and a hydrophobic tail consisting of 12 to 20 amino acid residues selected from at least one of leucine, isoleucine, and valine. The second type of histidine-enriched cationic shielding peptide; And calcium ions; the hydrophobic tail of the first amphiphilic polypeptide is adsorbed onto the surface of the oil droplet, and its polyanionic head connects two first amphiphilic polypeptides from different oil droplet surfaces via calcium ions as bridging nodes, thereby forming a three-dimensional microgel framework and embedding the oil droplet therein; When the composition is under acidic pH conditions, the histidine-rich cation-shielding peptides become positively charged due to the protonation of their histidine residues and spontaneously adsorb onto the surface of the microgel framework via electrostatic attraction to form a cation-shielding layer; the storage modulus of the composition at pH 6.8 is... The range is 10 Pa to 1000 Pa, and its Fourier transform infrared spectrum or X-ray photoelectron spectrum shows a peak shift from carboxylate group to calcium ion coordination state.

2. The calcium, vitamin D, and vitamin K soft capsule according to claim 1, characterized in that, In the composition, the total molar ratio of calcium ions to carboxylate ions in the polyanionic head of the first amphiphilic polypeptide is 0.5:1 to 1.5:1; and the mass ratio of oil droplets to the aqueous phase containing the first amphiphilic polypeptide and the second histidine-enriched cation-shielding peptide is (0.5-10):1 based on the total weight of the composition.

3. A calcium, vitamin D, and vitamin K soft capsule according to claim 1, characterized in that, The first amphiphilic polypeptide contains an amino acid sequence at the junction of the polyanionic head and the hydrophobic tail that can be specifically cleaved by trypsin or chymotrypsin.

4. A calcium, vitamin D, and vitamin K soft capsule according to claim 3, characterized in that, The amino acid sequence that can be specifically cleaved contains arginine residues or lysine residues; when the first amphiphilic polypeptide is cleaved, the independent polyanionic head and independent hydrophobic tail generated by its breakage form a self-molecular emulsion system in situ around the oil droplet.

5. A calcium, vitamin D, and vitamin K soft capsule according to claim 1, characterized in that, The composition also includes a water-soluble sacrificial polypeptide containing one or more sites that can be cleaved by trypsin or chymotrypsin, and the sacrificial polypeptide has an affinity for trypsin or chymotrypsin equal to or greater than that for the first amphiphilic polypeptide.

6. A calcium, vitamin D, and vitamin K soft capsule according to claim 5, characterized in that, Initial degradation rate of the microgel framework upon encountering trypsin or chymotrypsin The initial concentration of the sacrificial peptide The regulation of these relationships satisfies the following: ,in, and To characterize the composition system under preset enzyme concentration and temperature conditions, using objective physicochemical constants that can be determined by enzyme kinetic experiments. The intrinsic maximum degradation rate of the microgel framework, The coefficient of competitive inhibition of the enzyme by the sacrificial polypeptide is denoted as .

7. A calcium, vitamin D, and vitamin K soft capsule according to claim 3, characterized in that, The first type of amphiphilic polypeptide's polyanionic head, after being cleaved by trypsin or chymotrypsin, forms a fragment that can act as a competitive inhibitor of the trypsin or chymotrypsin.

8. A calcium, vitamin D, and vitamin K soft capsule according to claim 7, characterized in that, The amino acid sequence of the polyanion head also contains at least one arginine or lysine residue at the end furthest from the junction, which causes the fragment to form a negative feedback inhibition on the active site of the enzyme in the enzymatic hydrolysis environment.

9. A calcium, vitamin D, and vitamin K soft capsule according to claim 1, characterized in that, In the second type of histidine-enriched cation-shielding peptide, the molar fraction of histidine residues is not less than 30%. When the composition is transferred from an environment with a pH of 1.5 to 3.5 to an environment with a pH higher than 6.0, the cation-shielding layer automatically dissociates from the surface of the microgel framework due to the deprotonation of histidine residues. The composition was measured by dynamic light scattering method, and the peak volume distribution of oil droplets embedded in the three-dimensional microgel framework was 100 nm to 800 nm.

10. The preparation process of the content composition of a calcium, vitamin D, and vitamin K soft capsule according to claim 1, characterized in that, Includes the following steps: Vitamin D and vitamin K are dissolved in the pharmaceutical oil phase to obtain the oil phase; A first amphiphilic polypeptide comprising a polyanionic head consisting of 8 to 12 glutamic acid residues and aspartic acid residues and a hydrophobic tail consisting of 12 to 20 amino acid residues selected from at least one of leucine, isoleucine, and valine, and a second histidine-enriched cation-shielding peptide, are dissolved together in an aqueous phase containing calcium salts to obtain an aqueous phase. Under gentle stirring conditions, the oil phase is dispersed in the aqueous phase, so that the hydrophobic tail of the first amphiphilic polypeptide is spontaneously adsorbed at the oil droplet interface formed by the oil phase and the aqueous phase. Furthermore, calcium ions in the aqueous phase are used to ion bridge the polyanionic heads of the first type of amphiphilic polypeptide adsorbed on different oil droplet interfaces, thereby spontaneously forming a three-dimensional microgel framework that embeds and fixes the oil droplets, thus obtaining the composition.

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