Preparation method and application of alpha-ketoglutaric acid liposome

Abstract

The invention relates to a preparation method of alpha-ketoglutaric acid liposome, which comprises the following steps: (1) adding purified water into AKG for stirring and dispersing, and then adding a coating material for shearing emulsification and high-pressure homogenization; and (2) performing spray drying after high-pressure homogenization is completed. The liposome disclosed by the invention does not use an organic solvent, obviously improves the stability, bioavailability and targeting property of AKG, and is suitable for the fields of aging resistance, metabolic regulation, cancer rehabilitation, sports nutrition and the like. The average particle size of the liposome is 100-400 nm, the PDI can reach 0.523, the particle size is uniform and concentrated in distribution, the encapsulation efficiency is high, and the stability is high.

Description

Technical Field

[0001] The present invention relates to the technical field of food and health products, and in particular to a method for preparing alpha-ketoglutarate liposomes. Background Art

[0002] α-Ketoglutarate (AKG) is a key intermediate in the tricarboxylic acid cycle, regulating energy metabolism, delaying aging, enhancing immunity, and inhibiting tumor growth. However, AKG suffers from issues such as high water solubility, a short half-life in vivo, and susceptibility to enzymatic degradation, resulting in low bioavailability and difficulty in targeted delivery to lesions.

[0003] A liposome is an artificial membrane. When the hydrophilic heads of phospholipid molecules are inserted into the water, while the hydrophobic tails of the liposomes extend into the air, agitation results in a spherical bilayer of lipid molecules with diameters ranging from 25 to 1000 nm. Liposomes can be used for transgenics or pharmaceutical preparations, leveraging their ability to fuse with cell membranes to deliver drugs into cells. Biological definition: When amphiphilic molecules such as phospholipids and sphingolipids are dispersed in water, the hydrophobic tails of the molecules tend to aggregate, avoiding the aqueous phase, while the hydrophilic heads are exposed, forming a closed bilayer vesicle called a liposome. Pharmaceutical definition: A liposome is a microscopic vesicle formed by encapsulating a drug within a lipid bilayer.

[0004] While traditional liposome preparation techniques can encapsulate some active ingredients, they suffer from low encapsulation efficiency and poor stability. The production process for liposome products is challenging due to its complexity, difficulty in scale-up, large batch-to-batch variability, and high quality control challenges. Common liposome preparation techniques include thin film hydration, reverse evaporation, and ethanol injection, all of which involve the use of organic solvents.

[0005] There are many disadvantages of organic solvents: Residual toxicity risk: Commonly used organic solvents such as chloroform and methanol are toxic to a certain extent and need to be purified later. If they cannot be completely removed, they will cause damage to the liver and kidneys after entering the human body.

[0006] Operational safety issues: Most organic solvents are flammable and volatile, so the workshop needs to be explosion-proof. The volatilization of organic solvents may form a flammable and explosive gas mixture. Exposure to open flames or static electricity may cause fire or explosion accidents, threatening the safety of personnel and equipment.

[0007] Environmental pollution issues: The large-scale use and discharge of organic solvents will pollute the environment and affect the ecological balance and environmental quality.

[0008] Increased costs: In order to remove organic solvents, additional purification steps and equipment are required, such as evaporation equipment, ultrafiltration equipment, etc., which will increase costs and process complexity. Summary of the Invention

[0009] The object of the present invention is to provide a method for preparing α-ketoglutaric acid liposomes. The liposomes of the present invention have an average particle size of 100-400 nm, a PDI of up to 0.523, uniform and concentrated particle size distribution, high encapsulation efficiency and high stability.

[0010] To achieve the above object, the present invention provides the following technical solutions: A method for preparing α-ketoglutarate liposomes comprises the following steps: (1) Add purified water to AKG in batches and stir to disperse. Then add the coating material and shear emulsify at 10,000-20,000 rpm. Emulsify until the material becomes creamy and does not separate after standing for 10 minutes. Homogenize under high pressure at 10kg-60kg for 4-5 times. (2) After high-pressure homogenization is completed, spray drying is carried out. The slurry is pressed into the atomizer at a pressure of 70 to 200 atmospheres using a high-pressure pump, so that the slurry is dispersed into mist-like particles, which directly contact with hot air and complete the drying.

[0011] The particle size of AKG is 100-200 mesh.

[0012] The mass ratio of AKG to purified water is 1-2:1.

[0013] The coating material consists of soybean lecithin and cholesterol, with the mass ratio of soybean lecithin to cholesterol being 15-20:1; or consists of palm oil and sunflower oil, with the mass ratio of palm oil to sunflower oil being 5-10:1.

[0014] In step (2), the slurry feed rate is 15-30 ml / min, the air inlet temperature is 120-140°C, and the air outlet temperature is 70-110°C.

[0015] The present invention also provides the use of the liposome in anti-aging, metabolic regulation, cancer rehabilitation and sports nutrition preparations.

[0016] The beneficial effects produced by adopting the above technical solution are:

[0017] The liposomes of the present invention do not use organic solvents, significantly improving the stability, bioavailability and targeting of AKG, and are suitable for anti-aging, metabolic regulation, cancer rehabilitation and sports nutrition. The present invention adopts a solvent-free liposome preparation process to prepare AKG liposomes. Phospholipids and cholesterol or palm oil and sunflower oil are directly added to a nearly saturated AKG aqueous solution, high shear dispersion is performed, and then high-pressure homogenization is used to reduce the particle size and improve the distribution. Finally, the AKG liposome solid powder is dried. The liposomes of the present invention have an average particle size of 100-400nm, a PDI of up to 0.523, uniform and concentrated particle size, high encapsulation efficiency, and high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is an electron microscope photograph of the liposomes prepared in Example 1 of the present invention at 40 times magnification.

[0019] Figure 2 This is an electron microscope photograph of the liposomes prepared in Example 1 of the present invention, magnified 200 times. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example 1

[0021] Use a hammer mill to crush AKG to below 200 mesh, take 450g of the crushed AKG, add 260ml of purified water twice, 130ml each time, stirring while adding, and after stirring evenly, add 100g of soybean lecithin and 6g of cholesterol respectively. Use 316 stainless steel equipment for shear emulsification, 15,000 rpm for 15min, the emulsification effect is good, 15kg high-pressure homogenization 4 times, and finally spray drying, controlling the feed rate at 15ml / min, the inlet air temperature at 120℃, and the outlet air temperature at 85℃. Example 2

[0022] AKG was crushed to a size of less than 200 mesh using a hammer mill. 450g of the crushed AKG was added to 450ml of purified water in three 150ml portions, stirring each time. After uniform mixing, 100g of soybean lecithin and 6g of cholesterol were added. Shear emulsification was performed at 20,000 rpm for 15 minutes. However, the emulsification effect was poor and the material separated. Repeated emulsification resulted in separation. Five passes of 15kg high-pressure homogenization were performed with poor results. Finally, spray drying was performed with a feed rate of 15ml / min, an inlet air temperature of 140°C, and an outlet air temperature of 100°C. Example 3

[0023] AKG was crushed to less than 200 mesh using a hammer mill. 450g of the crushed AKG was added to 200ml of purified water in two 100ml portions, stirring while adding. After uniform mixing, 100g of soybean lecithin and 6g of cholesterol were added, respectively. Shear emulsification was performed at 20,000 rpm for 15 minutes, but no emulsification occurred, indicating poor emulsification. A 20kg high-pressure homogenizer was used five times with poor results, and the mixture was spray-dried at a feed rate of 20ml / min, an inlet air temperature of 130°C, and an outlet air temperature of 90°C. Example 4

[0024] Use a hammer mill to crush AKG to below 200 mesh, take 450g of the crushed AKG, add 260ml of purified water twice, 130ml each time, stirring while adding, and after stirring evenly, add 95g of soybean lecithin and 11g of cholesterol respectively, and perform shear emulsification at 15,000 rpm for 15min. The emulsification effect is good. Homogenize with 20kg high pressure for 4 times, and finally spray dry. Control the feed rate at 30ml / min, the inlet air temperature at 120℃, and the outlet air temperature at 75℃. Example 5

[0025] Use a hammer mill to crush AKG to below 200 mesh, take 450g of the crushed AKG, add 260ml of purified water twice, 130ml each time, stirring while adding, and after stirring evenly, add 102.5g of soybean lecithin and 3.5g of cholesterol respectively, and perform shear emulsification at 18,000 rpm for 15min. The emulsification effect is good. Homogenize under 18kg high pressure for 5 times, and finally spray dry. Control the feed rate at 25ml / min, the inlet air temperature at 140℃, and the outlet air temperature at 100℃. Example 6

[0026] AKG was crushed to a size of less than 200 mesh using a hammer mill. 450g of the crushed AKG was added to 260ml of purified water in two 130ml portions, stirring while adding. After uniform mixing, 90g of palm oil and 10g of sunflower oil were added, respectively. Shear emulsification was performed at 15,000 rpm for 15 minutes, with good emulsification. Four passes of high-pressure homogenization at 20kg kg yielded poor results, and the mixture was spray-dried at a feed rate of 25ml / min, an inlet air temperature of 100°C, and an outlet air temperature of 70°C. Example 7

[0027] AKG was crushed to a size of less than 200 mesh using a hammer mill. 450g of the crushed AKG was added to 450ml of purified water in three 150ml portions, stirring continuously. After uniform mixing, 90g of palm oil and 10g of sunflower oil were added, respectively. Shear emulsification was performed at 20,000 rpm for 15 minutes. Emulsification was poor, and the material separated. High-pressure homogenization at 15kg was performed five times, with poor results. Finally, spray drying was performed, with a feed rate of 15ml / min, an inlet air temperature of 120°C, and an outlet air temperature of 85°C. Example 8

[0028] AKG was crushed to a size of less than 200 mesh using a hammer mill. 450g of the crushed AKG was added to 200ml of purified water in two 100ml portions, stirring while adding. After uniform mixing, 90g of palm oil and 10g of sunflower oil were added, respectively. Shear emulsification was performed at 15,000 rpm for 15 minutes, but no emulsification occurred, indicating poor emulsification. Five passes of 20kg high-pressure homogenization also failed, with poor results. Finally, spray drying was performed, with a feed rate of 20ml / min, an inlet air temperature of 140°C, and an outlet air temperature of 100°C. Example 9

[0029] AKG was crushed to a size of less than 200 mesh using a hammer mill. 450g of the crushed AKG was added to 260ml of purified water in two 130ml portions, stirring while adding. After uniform mixing, 82g of palm oil and 18g of sunflower oil were added, respectively. Shear emulsification was performed at 15,000 rpm for 15 minutes, achieving good emulsification. A 15kg high-pressure homogenizer was used three times, achieving poor results. Finally, spray drying was performed, controlling the feed rate at 15-30ml / min, the inlet air temperature at 130°C, and the outlet air temperature at 90°C. Example 10

[0030] AKG was crushed to a size of less than 200 mesh using a hammer mill. 450g of the crushed AKG was added to 260ml of purified water in two 130ml portions, stirring while adding. After uniform mixing, 95g of palm oil and 5g of sunflower oil were added, respectively. Shear emulsification was performed at 20,000 rpm for 15 minutes, with good emulsification. High-pressure homogenization at 20kg was performed five times, with poor results. Finally, spray drying was performed, with a feed rate of 25ml / min, an inlet air temperature of 110°C, and an outlet air temperature of 80°C.

[0031] Table 1 Test results

[0032] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing α-ketoglutarate liposomes, characterized in that: The following steps are involved: (1) Add purified water to AKG in batches and stir to disperse. Then add the coating material and shear emulsify at 10,000-20,000 rpm. Emulsify until the material becomes creamy and does not separate after standing for 10 minutes. Homogenize under high pressure at 10kg-60kg for 4-5 times. (2) After high-pressure homogenization is completed, spray drying is carried out. The slurry is pressed into the atomizer at a pressure of 70 to 200 atmospheres using a high-pressure pump, so that the slurry is dispersed into mist-like particles, which directly contact with hot air and complete the drying.

2. The method for preparing α-ketoglutarate liposomes according to claim 1, wherein: The particle size of AKG is 100-200 mesh.

3. The method for preparing α-ketoglutarate liposomes according to claim 1, wherein: The mass ratio of AKG to purified water is 1-2:

1.

4. The method for preparing α-ketoglutarate liposomes according to claim 1, wherein: The coating material consists of soy lecithin and cholesterol, or of palm oil and sunflower oil.

5. The method for preparing α-ketoglutarate liposomes according to claim 4, characterized in that: The mass ratio of soybean lecithin to cholesterol is 15-20:

1.

6. The method for preparing α-ketoglutarate liposomes according to claim 4, characterized in that: The mass ratio of palm oil to sunflower oil is 5-10:

1.

7. The method for preparing α-ketoglutarate liposomes according to claim 1, wherein: In step (2), the slurry feed rate is 15-30 ml / min, the air inlet temperature is 120-140°C, and the air outlet temperature is 70-110°C.

8. Use of the liposomes prepared by the preparation method according to any one of claims 1 to 7 in anti-aging, metabolic regulation, cancer rehabilitation and sports nutrition preparations.

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