A liquid calcium preparation with high solubility and high absorbability, its preparation method and application

Abstract

This invention proposes a liquid calcium preparation with high solubility and high absorption rate, its preparation method, and its application, belonging to the field of pharmaceutical technology. Food-grade calcium citrate is ultra-finely pulverized, ultrasonically treated, and loaded into milk exosomes. This mixture is then combined with liposomes loaded with vitamin K2 and vitamin D3, ultrasonically treated, and incubated to obtain a fusion compound. This fusion compound is then mixed with calcium-chelated collagen peptides, homogenized under ultra-high pressure, filtered, sterilized, and filled to obtain a liquid calcium preparation with high solubility and high absorption rate. This invention, through improvements in production process and formulation, prepares a liquid calcium preparation product with high solubility and high absorption rate that can maintain long-term stability, improving the absorption rate of calcium supplements and thus enhancing the calcium supplementation effect, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a liquid calcium preparation with high solubility and high absorption rate, its preparation method, and its application. Background Technology

[0002] Calcium is an essential component of the human body, being the most abundant inorganic element. It is not only a major component of bones and teeth but also widely participates in various physiological activities within cells and blood. Insufficient calcium intake can increase the risk of poor bone development in children and osteoporosis in the elderly. Besides insufficient calcium intake, low calcium absorption and utilization rates are also a significant factor contributing to calcium deficiency among Chinese residents. Factors affecting calcium absorption are complex, including gender, age, disease, and diet; however, the particle size of calcium compounds plays a crucial role in influencing intestinal absorption and bioavailability.

[0003] Chinese invention patent document CN120398667A discloses a method for preparing ultrafine calcium citrate, including the chemical synthesis of calcium citrate, drying, and pulverization. This method mainly solves the chemical synthesis scheme of ultrafine calcium citrate, but does not verify the application of calcium citrate in actual liquid nutritional products.

[0004] Chinese invention patent document CN120514127A discloses a method for preparing a high-calcium and zinc dietary nutrient solution that promotes bone development. It includes the formulation of a compound formula of high-calcium nutrient solution. It only verifies that the stability of vitamin D is improved by microencapsulation and that different component formulations improve calcium absorption rate. It does not verify the effect of the particle size of calcium nutrients on calcium absorption and utilization.

[0005] Therefore, there is a need for a liquid nutrient calcium composition that can stably maintain a micron-sized particle size in liquid products for a long time and has high solubility and high absorption rate. Summary of the Invention

[0006] The purpose of this invention is to propose a liquid calcium preparation with high solubility and high absorption rate, its preparation method, and its application. Through improvements in production process and formulation, a liquid calcium preparation product with high solubility and high absorption rate that can exist stably for a long time has been prepared, which improves the absorption rate of calcium supplements and thus enhances the calcium supplementation effect of the product, showing good application prospects.

[0007] The technical solution of this invention is implemented as follows: This invention provides a method for preparing a liquid calcium preparation with high solubility and high absorption rate. Food-grade calcium citrate is ultra-finely pulverized, ultrasonically treated, loaded into milk exosomes, mixed with liposomes loaded with vitamin K2 and vitamin D3, ultrasonically treated, and incubated to obtain a fusion. The fusion is then mixed with collagen peptides chelated with calcium, homogenized under ultra-high pressure, filtered, sterilized, and filled to obtain a liquid calcium preparation with high solubility and high absorption rate.

[0008] As a further improvement to the present invention, the following steps are included: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant and ultrafine grinding is carried out under low temperature conditions. After grinding, it is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take calcium citrate that has been crushed and sieved, add a certain amount of stabilizer, stir and mix it in purified water, and then ultrasonically disperse it to obtain a calcium citrate dispersion emulsion. S3. Exosome loading: Milk exosomes were added to a calcium citrate dispersion emulsion and sonicated on ice to obtain calcium-loaded exosomes; S4. Preparation of vitamin K2 / vitamin D3 complex liposomes: Vitamin K2 and vitamin D3 were added to ethanol and stirred until homogeneous. Cholesterol, soybean lecithin and Tween were added. The solvent was removed by rotary evaporation under reduced pressure to form a film. The film was hydrated with purified water and sonicated to obtain vitamin K2 / vitamin D3 complex liposomes. S5. Preparation of fusion body: Calcium-loaded exosomes and vitamin K2 / vitamin D3 complex liposomes were mixed, sonicated, and incubated to obtain the fusion body; S6. Preparation of peptide calcium: Collagen peptides are added to water, calcium is added, stirred and chelated, dialyzed, and freeze-dried to obtain peptide calcium; S7. Preparation of liquid calcium preparation with high solubility and high absorption rate: Add peptide calcium to the fusion body, perform ultra-high pressure homogenization, filter, sterilize, and fill to obtain liquid calcium preparation with high solubility and high absorption rate.

[0009] As a further improvement of the present invention, the amount of anhydrous ethanol added in step S1 is 8%-12% of the mass of food-grade calcium citrate, the temperature of the low-temperature ultrafine pulverization is 0-5℃, and the low-temperature ultrafine pulverization is carried out using any one of a low-temperature airflow ultrafine pulverizer, a low-temperature impact mill, or a low-temperature vibration ultrafine pulverizer.

[0010] As a further improvement of the present invention, the stabilizer in step S2 includes one or more of xanthan gum, gellan gum, and carrageenan, the mass ratio of calcium citrate to stabilizer is 10:1-2, the mass ratio of calcium citrate to purified water is 1:150-200, and the ultrasonic dispersion power is 60-80W for 40-60min.

[0011] As a further improvement of the present invention, the mass ratio of milk exosomes and calcium citrate dispersion emulsion in step S3 is 2-3:150-200, the ultrasonic power of the ultrasonic co-incubation on ice is 30-50W, and the incubation time is 15-35min.

[0012] As a further improvement of the present invention, the mass ratio of vitamin K2, vitamin D3, cholesterol, soybean lecithin and Tween in step S4 is 0.4-0.7:1-2:5-7:20-40:2-3, and the Tween is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85.

[0013] As a further improvement of the present invention, the volume ratio of calcium-loaded exosomes and vitamin K2 / vitamin D3 complex liposomes in step S5 is 1-1.5:1, the power of the ultrasonic treatment is 200-300W, the time is 1-3min, the incubation temperature is 36-38℃, and the time is 0.5-1.5h.

[0014] As a further improvement of the present invention, the mass ratio of collagen peptide to calcium agent in step S6 is 10:1-2, and the calcium agent is calcium chloride, calcium sulfate or calcium nitrate; the mass-volume ratio of peptide calcium to fusion body in step S7 is 10-15:100g / mL, the pressure of ultra-high pressure homogenization is 100-120MPa, the flow rate is 100-200mL / min, and the number of treatments is 1-3 times.

[0015] This invention further protects a liquid calcium preparation with high solubility and high absorption rate obtained by the above-described preparation method.

[0016] This invention further protects the use of the above-mentioned liquid calcium preparation with high solubility and high absorption rate in the preparation of medicaments for the prevention and / or treatment of osteoporosis.

[0017] The present invention has the following beneficial effects: In this invention, the phospholipid bilayer of exosomes protects calcium ions from gastric acid degradation, enabling targeted intestinal delivery. Because exosomes naturally cross biological barriers, they enhance calcium bioavailability. Liposomes have a high encapsulation rate for vitamin D3 and vitamin K2, simultaneously delivering fat-soluble components and preventing oxidative degradation. Vitamin D3 promotes the synthesis of calcium-binding proteins, and vitamin K2 activates osteocalcin, directing free calcium into bones, effectively preventing calcium from entering blood vessels and avoiding arterial calcification, thus effectively protecting the cardiovascular system. Simultaneously, it significantly increases calcium content in bones, promoting calcium absorption. Furthermore, by fusing exosomes and liposomes, the spatial distance between calcium and vitamins at the absorption sites is shortened, facilitating synergistic metabolism and greatly improving efficacy.

[0018] Furthermore, this invention utilizes collagen peptide-chelated calcium. The peptide calcium binds to calcium ions through coordination bonds, significantly increasing its calcium loading capacity. As a supplementary phase, it can compensate for the insufficient calcium loading capacity of exosomes and also possesses good water solubility and absorbability. While fusion-type calcium enables rapid calcium release, peptide calcium allows for sustained calcium release, avoiding absorption saturation caused by a single high-concentration calcium intake.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. Significantly improves calcium bioavailability By using low-temperature ultrafine grinding and ultrasonic treatment, the particle size of calcium citrate was reduced to the micron level with Dv90≤7 μm, which greatly increased the specific surface area of ​​the particles, thereby improving its solubility and dispersibility in gastrointestinal fluid. Through the complex of fusion body and peptide calcium, it promoted the release and transmembrane transport of calcium ions, coordinated the burst release and sustained release of calcium, increased bone calcium concentration, and significantly promoted bone growth in experimental animals, confirming that it has a higher calcium absorption and utilization rate.

[0020] 2. Excellent physical stability and predictability of shelf life The use of ultra-high pressure homogenization dispersion technology forms a uniform and stable dispersion system, effectively preventing particle sedimentation and aggregation, and possessing good long-term stability, which is conducive to the commercial storage and circulation of products.

[0021] 3. Gentle processing, preserving nutrients The entire process employs non-thermal processing technologies such as low-temperature ultrafine grinding, ultrasonic treatment, liposome and exosome preparation, and ultra-high pressure homogenization to minimize the damage to heat-sensitive nutrients.

[0022] 4. Possesses promising prospects for industrial application. The preparation method features clear process steps and controllable parameters. All selected raw materials are food-grade and compliant, facilitating large-scale production. The resulting liquid calcium preparation can be directly used as a medicine for the prevention or treatment of osteoporosis, demonstrating strong market adaptability and promotional value. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Milk exosomes have an average particle size of 60-100 nm; collagen peptides have an average molecular weight of 1000-2000 Da.

[0025] Example 1 This embodiment describes a method for preparing a liquid calcium preparation with high solubility and high absorption rate, comprising the following steps: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant. The amount of anhydrous ethanol added is 8% of the mass of food-grade calcium citrate. Ultrafine grinding is carried out using a low-temperature airflow ultrafine grinder at 0℃. After grinding, the powder is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take 1g of calcium citrate that has been pulverized and sieved, add 0.1g of xanthan gum, stir and mix in 150mL of purified water, and ultrasonically disperse at 60W for 40min to obtain calcium citrate dispersion emulsion. S3. Exosome loading: 2g of milk exosomes were added to 150g of calcium citrate dispersion emulsion and incubated on ice with sonication at a power of 30W for 15min to obtain calcium-loaded exosomes; S4. Preparation of Vitamin K2 / Vitamin D3 Complex Liposomes: 0.4g of Vitamin K2 and 1g of Vitamin D3 were added to 100mL of ethanol and stirred until homogeneous. 5g of cholesterol, 2g of soybean lecithin and 2g of Tween-60 were added. The solvent was removed by rotary evaporation under reduced pressure to form a film. 100mL of purified water was added for hydration for 60min. The film was then sonicated at 100W for 20min to obtain Vitamin K2 / Vitamin D3 complex liposomes. S5. Preparation of fusion body: 100 mL of calcium-loaded exosomes and 100 mL of vitamin K2 / vitamin D3 complex liposomes were mixed, sonicated at 200 W for 3 min, and incubated at 36 °C for 1.5 h to obtain the fusion body; S6. Preparation of peptide calcium: 10g of collagen peptide was added to 200mL of water, 1g of calcium chloride was added, and the mixture was stirred and chelated for 30min. The mixture was dialyzed for 24h using a dialysis bag with a pore size of 500Da. The unpermeated liquid was freeze-dried to obtain peptide calcium. S7. Preparation of liquid calcium preparation with high solubility and high absorption rate: 10g of peptide calcium was added to 100mL of fusion medium and subjected to ultra-high pressure homogenization treatment at a pressure of 100MPa and a flow rate of 100mL / min for 3 times. After filtration, sterilization, and filling, a liquid calcium preparation with high solubility and high absorption rate was obtained.

[0026] Example 2 This embodiment describes a method for preparing a liquid calcium preparation with high solubility and high absorption rate, comprising the following steps: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant. The amount of anhydrous ethanol added is 12% of the mass of food-grade calcium citrate. Ultrafine grinding is carried out at 5°C using a low-temperature vibrating ultrafine grinder. After grinding, the powder is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take 1g of calcium citrate that has been crushed and sieved, add 0.2g of gellan gum, stir and mix in 200mL of purified water, and ultrasonically disperse at 80W for 60min to obtain calcium citrate dispersion emulsion. S3. Exosome loading: 3g of milk exosomes were added to 200g of calcium citrate dispersion emulsion and incubated on ice with ultrasonic power of 50W for 35min to obtain calcium-loaded exosomes; S4. Preparation of Vitamin K2 / Vitamin D3 Complex Liposomes: 0.7g of Vitamin K2 and 2g of Vitamin D3 were added to 100mL of ethanol and stirred until homogeneous. 7g of cholesterol, 40g of soybean lecithin and 3g of Tween-80 were added. The solvent was removed by rotary evaporation under reduced pressure to form a film. 100mL of purified water was added for hydration for 60min. The film was then sonicated at 100W for 20min to obtain Vitamin K2 / Vitamin D3 complex liposomes. S5. Preparation of fusion body: 150 mL of calcium-loaded exosomes and 100 mL of vitamin K2 / vitamin D3 complex liposomes were mixed, sonicated at 300 W for 1 min, and incubated at 38 °C for 0.5 h to obtain the fusion body; S6. Preparation of peptide calcium: 10g of collagen peptide was added to 200mL of water, 2g of calcium sulfate was added, and the mixture was stirred and chelated for 50min. The mixture was dialyzed for 24h using a dialysis bag with a pore size of 500Da. The unpermeated liquid was freeze-dried to obtain peptide calcium. S7. Preparation of liquid calcium preparation with high solubility and high absorption rate: 15g of peptide calcium was added to 100mL of fusion medium and subjected to ultra-high pressure homogenization treatment at a pressure of 120MPa and a flow rate of 200mL / min, once. After filtration, sterilization, and filling, a liquid calcium preparation with high solubility and high absorption rate was obtained.

[0027] Example 3 This embodiment describes a method for preparing a liquid calcium preparation with high solubility and high absorption rate, comprising the following steps: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant. The amount of anhydrous ethanol added is 10% of the mass of food-grade calcium citrate. Ultrafine grinding is carried out using a low-temperature impact mill at 2°C. After grinding, the mixture is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take 1g of calcium citrate that has been pulverized and sieved, add 0.15g of carrageenan, stir and mix in 170mL of purified water, and ultrasonically disperse at 70W for 50min to obtain calcium citrate dispersion emulsion. S3. Exosome loading: 2.5g of milk exosomes were added to 170g of calcium citrate dispersion emulsion and sonicated on ice with an ultrasonic power of 40W for 25min to obtain calcium-loaded exosomes; S4. Preparation of Vitamin K2 / Vitamin D3 Complex Liposomes: 0.5g of Vitamin K2 and 1.5g of Vitamin D3 were added to 100mL of ethanol and stirred until homogeneous. 6g of cholesterol, 30g of soybean lecithin and 2.5g of Tween-85 were added. The solvent was removed by rotary evaporation under reduced pressure to form a film. 100mL of purified water was added for hydration for 60min. The film was then sonicated at 100W for 20min to obtain Vitamin K2 / Vitamin D3 complex liposomes. S5. Preparation of fusion body: 120 mL of calcium-loaded exosomes and 100 mL of vitamin K2 / vitamin D3 complex liposomes were mixed, sonicated at 250 W for 2 min, and incubated at 37 °C for 1 h to obtain fusion body; S6. Preparation of peptide calcium: 10g of collagen peptide was added to 200mL of water, 1.5g of calcium nitrate was added, and the mixture was stirred and chelated for 40min. The mixture was dialyzed for 24h using a dialysis bag with a pore size of 500Da. The unpermeated liquid was freeze-dried to obtain peptide calcium. S7. Preparation of liquid calcium preparation with high solubility and high absorption rate: 12g of peptide calcium was added to 100mL of fusion medium and subjected to ultra-high pressure homogenization treatment at a pressure of 110MPa and a flow rate of 150mL / min for 2 times. After filtration, sterilization, and filling, a liquid calcium preparation with high solubility and high absorption rate was obtained.

[0028] Compared with Example 3, Comparative Examples 1-6 only performed steps S1 and S2. The differences between these two steps are shown in Table 1 below: Table 1

[0029] Test Example 1 The particle size distribution was determined using a Mastersizer 3000+ Ultra high-speed intelligent laser particle size analyzer, and the results were analyzed using Mastersizer 3000 software. Basic test parameters: stirring speed 1500 r / min, opacity 10%-20%, sample refractive index 1.69. The test samples were calcium citrate dispersion emulsions prepared in step S2 of Examples 1-3 and Comparative Examples 1-6.

[0030] The results are shown in Table 2.

[0031] Table 2: Particle size distribution results of Examples 1-3 and Comparative Examples 1-6 (unit: μm)

[0032] The experimental data in the table above show that the average particle size (D[4,3]) of the volumetric torque in Examples 1-3 is significantly lower than that in Comparative Examples 1-6, indicating the necessity of each treatment process formulation during the pulverization and ultrasonic treatment stages. Specifically, in the pulverization stage, the absence of anhydrous ethanol as a dispersant resulted in insufficient particle grinding and a larger overall particle size; a higher pulverization temperature also enhanced the intermolecular repolymerization effect. In the ultrasonic treatment stage, suboptimal control of each process condition led to intermolecular repolymerization, affecting the dispersibility of calcium citrate molecules.

[0033] Comparative Example 7 The difference from Example 3 is that steps S3 and S5 were not performed.

[0034] Includes the following steps: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant. The amount of anhydrous ethanol added is 10% of the mass of food-grade calcium citrate. Ultrafine grinding is carried out using a low-temperature impact mill at 2°C. After grinding, the mixture is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take 1g of calcium citrate that has been pulverized and sieved, add 0.15g of carrageenan, stir and mix in 170mL of purified water, and ultrasonically disperse to obtain calcium citrate dispersion emulsion; S3. Preparation of Vitamin K2 / Vitamin D3 Complex Liposomes: 0.5g of Vitamin K2 and 1.5g of Vitamin D3 were added to 100mL of ethanol and stirred until homogeneous. 6g of cholesterol, 30g of soybean lecithin and 2.5g of Tween-85 were added. The solvent was removed by rotary evaporation under reduced pressure to form a film. 100mL of purified water was added for hydration for 60min. The film was then sonicated at 100W for 20min to obtain Vitamin K2 / Vitamin D3 complex liposomes. S4. Preparation of the mixture: Mix 120 mL of calcium citrate dispersion emulsion and 100 mL of vitamin K2 / vitamin D3 complex liposomes evenly to obtain the mixture; S5. Preparation of peptide calcium: 10g of collagen peptide was added to 200mL of water, 1.5g of calcium nitrate was added, and the mixture was stirred and chelated for 40min. The mixture was dialyzed for 24h using a dialysis bag with a pore size of 500Da. The unpermeated liquid was freeze-dried to obtain peptide calcium. S6. Preparation of liquid calcium preparation with high solubility and high absorption rate: 12g of peptide calcium was added to 100mL of mixture and subjected to ultra-high pressure homogenization treatment at a pressure of 110MPa and a flow rate of 150mL / min for 2 times. After filtration, sterilization and filling, a liquid calcium preparation with high solubility and high absorption rate was obtained.

[0035] Comparative Example 8 The difference from Example 3 is that steps S4 and S5 were not performed.

[0036] Includes the following steps: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant. The amount of anhydrous ethanol added is 10% of the mass of food-grade calcium citrate. Ultrafine grinding is carried out using a low-temperature impact mill at 2°C. After grinding, the mixture is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take 1g of calcium citrate that has been pulverized and sieved, add 0.15g of carrageenan, stir and mix in 170mL of purified water, and ultrasonically disperse to obtain calcium citrate dispersion emulsion; S3. Exosome loading: 2.5g of milk exosomes were added to 170g of calcium citrate dispersion emulsion and sonicated on ice with an ultrasonic power of 40W for 25min to obtain calcium-loaded exosomes; S4. Preparation of peptide calcium: 10g of collagen peptide was added to 200mL of water, 1.5g of calcium nitrate was added, and the mixture was stirred and chelated for 40min. The mixture was dialyzed for 24h using a dialysis bag with a pore size of 500Da. The unpermeated liquid was freeze-dried to obtain peptide calcium. S5. Preparation of liquid calcium preparation with high solubility and high absorption rate: 12g of peptide calcium was added to 100mL of calcium-loaded exosomes and subjected to ultra-high pressure homogenization treatment at a pressure of 110MPa and a flow rate of 150mL / min for 2 times. After filtration, sterilization, and filling, a liquid calcium preparation with high solubility and high absorption rate was obtained.

[0037] Comparative Example 9 The difference from Example 3 is that steps S3 to S5 were not performed.

[0038] Includes the following steps: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant. The amount of anhydrous ethanol added is 10% of the mass of food-grade calcium citrate. Ultrafine grinding is carried out using a low-temperature impact mill at 2°C. After grinding, the mixture is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take 1g of calcium citrate that has been pulverized and sieved, add 0.15g of carrageenan, stir and mix in 170mL of purified water, and ultrasonically disperse to obtain calcium citrate dispersion emulsion; S3. Preparation of peptide calcium: 10g of collagen peptide was added to 200mL of water, 1.5g of calcium nitrate was added, and the mixture was stirred and chelated for 40min. The mixture was dialyzed for 24h using a dialysis bag with a pore size of 500Da. The unpermeated liquid was freeze-dried to obtain peptide calcium. S4. Preparation of liquid calcium preparation with high solubility and high absorption rate: 12g of peptide calcium was added to 100mL of calcium citrate dispersion emulsion, and subjected to ultra-high pressure homogenization treatment at a pressure of 110MPa and a flow rate of 150mL / min for 2 times. After filtration, sterilization, and filling, a liquid calcium preparation with high solubility and high absorption rate was obtained.

[0039] Comparative Example 10 The difference from Example 3 is that step S6 was not performed.

[0040] Includes the following steps: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant. The amount of anhydrous ethanol added is 10% of the mass of food-grade calcium citrate. Ultrafine grinding is carried out using a low-temperature impact mill at 2°C. After grinding, the mixture is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take 1g of calcium citrate that has been pulverized and sieved, add 0.15g of carrageenan, stir and mix in 170mL of purified water, and ultrasonically disperse to obtain calcium citrate dispersion emulsion; S3. Exosome loading: 2.5g of milk exosomes were added to 170g of calcium citrate dispersion emulsion and sonicated on ice with an ultrasonic power of 40W for 25min to obtain calcium-loaded exosomes; S4. Preparation of Vitamin K2 / Vitamin D3 Complex Liposomes: 0.5g of Vitamin K2 and 1.5g of Vitamin D3 were added to 100mL of ethanol and stirred until homogeneous. 6g of cholesterol, 30g of soybean lecithin and 2.5g of Tween-85 were added. The solvent was removed by rotary evaporation under reduced pressure to form a film. 100mL of purified water was added for hydration for 60min. The film was then sonicated at 100W for 20min to obtain Vitamin K2 / Vitamin D3 complex liposomes. S5. Preparation of fusion body: 120 mL of calcium-loaded exosomes and 100 mL of vitamin K2 / vitamin D3 complex liposomes were mixed, sonicated at 250 W for 2 min, and incubated at 37 °C for 1 h to obtain fusion body; S6. Preparation of liquid calcium preparation with high solubility and high absorption rate: 1.58g of calcium citrate that has been pulverized and sieved is added to 100mL of fusion medium and subjected to ultra-high pressure homogenization treatment at a pressure of 110MPa and a flow rate of 150mL / min for 2 times. After filtration, sterilization and filling, a liquid calcium preparation with high solubility and high absorption rate is obtained.

[0041] Test Example 2 The high-solubility and high-absorption liquid calcium preparations obtained in Examples 1-3 and Comparative Examples 7-10 were subjected to stability tests.

[0042] Referring to the accelerated stability verification methods for pharmaceuticals and food, the product was stored in a constant temperature and humidity chamber at 40℃±2℃ and 75%±5% relative humidity for 3 months. Based on the Arrhenius equation, this is equivalent to storing it at 25℃ for 720 days, simulating 24 months of stability.

[0043] The results are shown in Table 3.

[0044] Table 3: Stability verification results of Examples 1-3 and Comparative Examples 7-10

[0045] The experimental data in the table above show that the compositions in Examples 1-3 exhibited good stability after a 3-month accelerated stability test. However, Comparative Examples 7 and 9, due to the direct addition of calcium citrate dispersion emulsion, showed poor stability and stratification after the accelerated stability test.

[0046] Test Example 3: Verification of the effect of high-solubility, high-absorption-rate liquid calcium preparations on calcium absorption and utilization. (I) Experimental Methods Mice were fed samples prepared using the highly soluble and highly absorbable liquid calcium formulations obtained in Examples 1-3 and Comparative Examples 7-10, following the steps outlined below.

[0047] Mice were acclimatized for 6 days with free access to a basal maintenance diet and distilled water. They were then randomly divided into 9 groups of 10 mice each, based on their body weight. Except for the normal control group (with 0.4% calcium content in their diet), the other 8 groups were fed a low-calcium diet (0.1% calcium content in their diet). All groups were administered 0.1 mL / 10g BW via gavage. The normal control and low-calcium groups were administered an equal volume of ultrapure water via gavage to avoid calcium intake from drinking water. This gavage treatment continued for 4 weeks. Each cage was supplemented weekly with approximately 200g of feed and approximately 240 mL of ultrapure water. The composition of the low-calcium diet is shown in Table 4 below. Table 4: Composition Table of Low-Calcium Feed

[0048] The feeding groups and feeding conditions are shown in Table 5.

[0049] Table 5: Mouse grouping and feeding conditions

[0050] During the experiment, the weight of the mice was measured weekly. The mice were fasted for 12 hours before the measurement, and their coat color, activity status, and any adverse reactions before and after gavage were observed.

[0051] After 25 days of continuous gavage, the mice were transferred to individual metabolic cages for calcium metabolism testing. Daily food intake was accurately recorded for 3 consecutive days, and feces and urine were collected and stored at -20°C for later analysis.

[0052] Determination of calcium content in mouse feces and urine: Mouse feces and urine were transferred to 25 mL Erlenmeyer flasks, 5 mL of nitric acid and 2 mL of perchloric acid were added, and the mixture was placed on a hot plate to digest until it was colorless and transparent or slightly yellow. After removing the acid, the mixture was diluted with deionized water and then measured by ICP-OES.

[0053] Calculate absorbed calcium, retained calcium, apparent calcium absorption rate, and calcium retention rate using the following formulas: Calcium intake = calcium in feed + calcium administered via gavage Calcium absorption = calcium intake - fecal calcium Calcium retention = calcium intake - fecal calcium - urinary calcium Apparent calcium absorption rate (%) = (Absorbed calcium / Ingested calcium) × 100% Calcium retention rate (%) = Calcium retained / Calcium intake × 100% Determination of serum calcium content in mice: After 28 days of gavage, mice were fasted for 12 hours, and blood was collected by enucleation. The collected mouse blood was allowed to stand at 4℃ for 3 hours, then centrifuged. The supernatant was collected and stored in a -20℃ refrigerator. Serum calcium was measured using ICP-OES.

[0054] Mice were euthanized by cervical dislocation, and the femurs on both sides were quickly dissected. After removing the muscles and fascia, the femur samples were wrapped in gauze soaked in physiological saline and frozen for later use.

[0055] Femur length: The left femur of the mouse was thawed at room temperature and its length was accurately measured using digital vernier calipers.

[0056] Bone weight index: The left femur was placed in an oven at 105℃ and dried to constant weight. It was weighed on an analytical balance, and the bone weight was recorded and the bone weight index was calculated.

[0057] Bone density index (mg / g) = Left femoral bone weight / Body weight Determination of bone calcium content in mice: A certain mass of left femur bone, dried to constant weight, was ashed in a muffle furnace at high temperature. The bone ash was wet digested and then measured using ICP-OES.

[0058] The experimental results are as follows: The effects of high-solubility and high-absorption-rate liquid calcium preparations on calcium absorption and utilization are shown in Table 6 below.

[0059] Table 6: Effects of Examples 1-3 and Comparative Examples 7-10 on calcium absorption and utilization rate

[0060] Calcium is primarily absorbed through the small intestine and excreted in feces and urine, with a very small amount excreted through the skin via sweat. Calcium metabolism balance experiments are a classic method in nutritional research. Apparent calcium absorption rate and calcium retention rate indicate the absorption and utilization of calcium in the body; high apparent absorption rate and calcium retention rate indicate good absorption of ingested calcium. As shown in the table above, the calcium absorption and utilization data of Examples 1-3 are superior to those of Comparative Examples 7-10. Therefore, the high-solubility, high-absorption liquid calcium preparations obtained in Examples 1-3 of this invention are more conducive to calcium absorption and utilization.

[0061] The effects of liquid calcium preparations with high solubility and high absorption rate on bone development in mice are shown in Table 7 below.

[0062] Table 7: Effects of Examples 1-3 and Comparative Examples 7-10 on mouse skeletal development

[0063] The data in the table above show that the blood calcium content in each embodiment was not significantly different from that in the normal control group, but was significantly higher than that in the control group. However, the bone calcium content was significantly increased, indicating that more calcium was deposited in the bones. The bone dry weight index and bone calcium content in the experimental results also show that the promoting effect of each embodiment on mouse bone development was better than that in the control group. Because the experimental period was only 4 weeks, the growth time of the mice was short, resulting in no significant difference in tibia length among the control group, each embodiment group, and each control group.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a liquid calcium preparation with high solubility and high absorption rate, characterized in that, Food-grade calcium citrate was ultra-finely pulverized, ultrasonically treated, and loaded into milk exosomes. It was then mixed with liposomes loaded with vitamin K2 and vitamin D3, ultrasonically treated, and incubated to obtain a fusion. The fusion was then mixed with collagen peptides chelated with calcium, homogenized under ultra-high pressure, filtered, sterilized, and filled to obtain a liquid calcium preparation with high solubility and high absorption rate.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Ultrafine grinding treatment: Food-grade calcium citrate is added to food-grade anhydrous ethanol as a dispersant and ultrafine grinding is carried out under low temperature conditions. After grinding, it is passed through a 2000-mesh standard sieve. S2. Ultrasonic treatment: Take calcium citrate that has been crushed and sieved, add a certain amount of stabilizer, stir and mix it in purified water, and then ultrasonically disperse it to obtain a calcium citrate dispersion emulsion. S3. Exosome loading: Milk exosomes were added to a calcium citrate dispersion emulsion and sonicated on ice to obtain calcium-loaded exosomes; S4. Preparation of vitamin K2 / vitamin D3 complex liposomes: Vitamin K2 and vitamin D3 were added to ethanol and stirred until homogeneous. Cholesterol, soybean lecithin and Tween were added. The solvent was removed by rotary evaporation under reduced pressure to form a film. The film was hydrated with purified water and sonicated to obtain vitamin K2 / vitamin D3 complex liposomes. S5. Preparation of fusion body: Calcium-loaded exosomes and vitamin K2 / vitamin D3 complex liposomes were mixed, sonicated, and incubated to obtain the fusion body; S6. Preparation of peptide calcium: Collagen peptides are added to water, calcium is added, stirred and chelated, dialyzed, and freeze-dried to obtain peptide calcium; S7. Preparation of liquid calcium preparation with high solubility and high absorption rate: Add peptide calcium to the fusion body, perform ultra-high pressure homogenization, filter, sterilize, and fill to obtain liquid calcium preparation with high solubility and high absorption rate.

3. The preparation method according to claim 2, characterized in that, The amount of anhydrous ethanol added in step S1 is 8%-12% of the mass of food-grade calcium citrate. The temperature of the low-temperature ultrafine grinding is 0-5℃. The low-temperature ultrafine grinding adopts any one of the following: low-temperature airflow ultrafine grinding mill, low-temperature impact mill, or low-temperature vibration ultrafine grinding mill.

4. The preparation method according to claim 2, characterized in that, The stabilizer in step S2 includes one or more of xanthan gum, gellan gum, and carrageenan. The mass ratio of calcium citrate to stabilizer is 10:1-2. The mass ratio of calcium citrate to purified water is 1:150-200. The ultrasonic dispersion power is 60-80W and the time is 40-60min.

5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of milk exosomes to calcium citrate dispersion emulsion is 2-3:150-200, and the ultrasonic power for co-incubation on ice is 30-50W, with an incubation time of 15-35min.

6. The preparation method according to claim 2, characterized in that, In step S4, the mass ratio of vitamin K2, vitamin D3, cholesterol, soybean lecithin, and Tween is 0.4-0.7:1-2:5-7:20-40:2-3, and the Tween is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85.

7. The preparation method according to claim 2, characterized in that, In step S5, the volume ratio of calcium-loaded exosomes to vitamin K2 / vitamin D3 complex liposomes is 1-1.5:1, the ultrasonic treatment power is 200-300W, the time is 1-3min, and the incubation temperature is 36-38℃ for 0.5-1.5h.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of collagen peptides to calcium agent is 10:1-2, and the calcium agent is calcium chloride, calcium sulfate, or calcium nitrate. In step S7, the mass-to-volume ratio of peptide calcium to fusion body is 10-15:100g / mL. The pressure of the ultra-high pressure homogenization treatment is 100-120MPa, the flow rate is 100-200mL / min, and the treatment is performed 1-3 times.

9. A liquid calcium preparation with high solubility and high absorption rate prepared by the preparation method according to any one of claims 1-8.

10. The use of a liquid calcium preparation with high solubility and high absorption rate as described in claim 9 in the preparation of a medicament for the prevention and / or treatment of osteoporosis.

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