Alpha-ketoglutaric acid composite liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and discloses an alpha-ketoglutaric acid composite liposome as well as a preparation method and application thereof. Alpha-ketoglutaric acid is preliminarily entrapped by lecithin and cholesterol to prepare a basic liposome, and then enzymolysis silkworm pupa protein is introduced to the surface of the liposome, so that the enzymolysis silkworm pupa protein and a lipid bilayer are subjected to hydrophobic and hydrogen-bond interaction to form a protein-lipid composite layer, and the stability of a membrane structure is enhanced; and the positively charged arginine modified starch is further deposited on the surface of the liposome to form a polysaccharide-protein-lipid composite layer, so that the electrostatic locking of the negatively charged alpha-ketoglutaric acid is realized, and the encapsulation efficiency and the controlled release performance are further improved. The alpha-ketoglutaric acid composite liposome provided by the invention has the advantages of being high in stability, safe to eat, high in encapsulation efficiency, good in gastric acid resistance stability and the like, has a good slow release characteristic, and can be applied to the fields of sports nutritional supplements, antioxidant products or anti-aging products and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically disclosing an α-ketoglutarate complex liposome, its preparation method, and its application. Background Technology

[0002] Alpha-ketoglutarate (α-KG) is a five-carbon dicarboxylic acid molecule. As a key metabolic intermediate in the tricarboxylic acid cycle (TCA cycle), it can generate succinyl-CoA through oxidative decarboxylation, effectively driving energy production and providing the necessary foundation for ATP synthesis, thus ensuring energy for cellular survival and function. With the deepening of metabolic research and inflammation biology, α-ketoglutarate has gradually demonstrated its central role in various biological functions. By participating in inflammation regulation, metabolic regulation, mitochondrial function maintenance, and epigenetic modification, α-ketoglutarate plays an important role in supporting cellular energy needs, maintaining cellular homeostasis, and coping with chronic diseases. Furthermore, increasing research indicates that α-KG, as a naturally occurring biological compound in the human body, has the potential for rapid clinical translation when provided as a dietary supplement.

[0003] α-Ketoglutarate is highly hydrophilic but unstable, easily degraded by the gastrointestinal environment after oral administration, and has a low absorption rate. Related studies have shown that oral administration of α-ketoglutarate can improve initial absorption efficiency to a limited extent, but approximately 80% is rapidly metabolized or excreted, limiting bioavailability. Furthermore, traditional liposomes have low encapsulation efficiency for α-KG and are prone to leakage during storage or application, failing to meet practical application requirements. Therefore, it is necessary to construct structurally stable composite liposomes to encapsulate α-ketoglutarate to improve encapsulation efficiency and system stability, while simultaneously achieving sustained release after oral administration, thereby improving its bioavailability. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention utilizes the synergistic effect of silkworm pupa protein and arginine-modified starch to construct a multi-layered, stable composite liposome. It provides an α-ketoglutarate composite liposome, its preparation method, and its application, thereby improving the encapsulation efficiency and stability of conventional α-ketoglutarate liposomes and thus enhancing their bioavailability.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an α-ketoglutarate complex liposome, comprising, from the inside out, a core layer, an intermediate layer and a composite protective layer; The core layer includes α-ketoglutaric acid and an osmotic pressure regulator; The intermediate layer comprises lecithin and cholesterol; The composite protective layer comprises enzymatically hydrolyzed silkworm pupa protein and arginine-modified starch.

[0006] This invention utilizes silkworm pupa protein and arginine-modified starch as functional wall materials for basic liposomes, synergistically constructing multi-layered composite liposomes to encapsulate α-ketoglutarate. This provides an α-ketoglutarate composite liposome with high encapsulation efficiency, good sustained-release performance, stability, and biocompatibility, thereby improving oral bioavailability. The invention initially prepares basic liposomes by encapsulating α-ketoglutarate with lecithin and cholesterol. Then, enzymatically hydrolyzed silkworm pupa protein is introduced onto the liposome surface, causing hydrophobic and hydrogen bonding interactions with the lipid bilayer to form a protein-lipid composite layer, enhancing membrane structural stability. Furthermore, positively charged arginine-modified starch is deposited on the liposome surface to form a polysaccharide-protein-lipid composite layer, achieving electrostatic locking of negatively charged α-ketoglutarate, further improving encapsulation efficiency and controlled-release performance.

[0007] This invention selects a food-derived macromolecule—silkworm pupa protein—because it contains a variety of amino acids, including hydrophobic amino acids and a certain proportion of charged amino acids, exhibiting good biocompatibility, membrane fusion, biodegradability, and non-toxicity. Silkworm pupa protein can interact with liposomes through hydrophobic and hydrogen bonding, thereby improving the stability of the liposomes. Partial enzymatic hydrolysis exposes more reactive groups, resulting in smaller peptides with better solubility and compatibility with liposome membranes. Starch itself lacks charged functional groups, while the guanidino group of arginine is positively charged. Arginine modification of the guanidino group in starch can interact electrostatically with the carboxyl group of α-ketoglutarate, improving encapsulation efficiency and stability.

[0008] Silkworm pupa protein is an amphoteric macromolecule. In the acidic environment of gastric acid, it can be protonated to form a positively charged barrier, competitively binding to pepsin molecules and resisting the damage of pepsin to liposome membranes. Arginine-modified starch introduces positively charged guanidin groups, which can electrostatically adsorb with the negatively charged gastrointestinal mucosa, prolonging the retention time. At the same time, the starch skeleton slowly swells in the acidic environment, further delaying drug release.

[0009] Preferably, the mass ratio of α-ketoglutaric acid to the osmotic pressure regulator is 1-2:5-10.

[0010] Preferably, the mass ratio of lecithin to cholesterol is 5:0.8-1.

[0011] Preferably, the mass ratio of the enzymatically hydrolyzed silkworm pupa protein to the arginine-modified starch is 0.8-1.2:1.

[0012] Preferably, the mass ratio of the α-ketoglutarate, cholesterol, and arginine-modified starch is 9-11:1.8-2.2:1.8-2.2.

[0013] Preferably, the osmotic pressure regulator includes at least one of glycerol, sucrose, or mannitol.

[0014] By way of example, the present invention is illustrated using glycerin as an example.

[0015] Preferably, the lecithin includes soybean lecithin.

[0016] Soy lecithin is a natural phospholipid with excellent biocompatibility. Its hydrophilic head and hydrophobic tail can self-assemble to form a bilayer, which is then bound to α-ketoglutarate through hydrogen bonds to achieve encapsulation. Cholesterol is embedded in the gaps between phospholipid molecules, which can reduce membrane fluidity and avoid drug leakage caused by brittleness or looseness due to excessive phospholipid arrangement, thus prolonging the release time.

[0017] Preferably, the degree of enzymatic hydrolysis of the silkworm pupa protein is 15%-20%.

[0018] Preferably, the preparation method of the arginine-modified starch includes the following steps: adding sodium trimetaphosphate and L-arginine to potato starch milk, and carrying out a grafting reaction at 55℃-62℃ to obtain the arginine-modified starch.

[0019] This invention introduces arginine groups into potato starch molecules through a composite modification technique involving sodium trimetaphosphate (STMP) crosslinking and arginine grafting. Sodium trimetaphosphate, as a food-grade crosslinking agent, can form phosphate ester bonds with starch hydroxyl groups under alkaline conditions, while simultaneously providing crosslinking sites for the guanidine groups of arginine, ultimately forming a stable three-dimensional network structure. This invention achieves simultaneous crosslinking and grafting in a one-step reaction, avoiding the complex operations of traditional multi-step methods.

[0020] Preferably, the concentration of potato starch milk is 200g / L-250g / L.

[0021] For example, potato starch and water are mixed in a mass-volume ratio and then dispersed by high-speed shearing to ensure that no particles agglomerate.

[0022] Preferably, the mass ratio of sodium trimetaphosphate, L-arginine and potato starch is 1.5-2.5:4.5-5.5:90-100.

[0023] Preferably, the preparation method of the arginine-modified starch specifically includes the following steps: adjusting the pH of potato starch milk to 8.5-9.0, activating it at 45℃-55℃ for 25min-40min, adding sodium trimetaphosphate and L-arginine, carrying out a grafting reaction at 55℃-62℃ for 1.5h-2h, adjusting the pH of the system to 6.5-7.0, separating the solid and liquid, and drying to obtain the arginine-modified starch.

[0024] Secondly, the present invention provides a method for preparing the above-mentioned α-ketoglutarate complex liposomes, the method comprising the following steps: S1. Dissolve lecithin and cholesterol in a chloroform-methanol mixture and rotary evaporate to form a lipid film; S2. Add α-ketoglutaric acid aqueous solution to the lipid membrane, hydrate for 25 min-35 min, and then perform intermittent ultrasonic refining treatment to obtain basic liposomes. S3. Add an enzymatically hydrolyzed silkworm pupa protein solution to the basic liposomes, sonicate, add an arginine-modified starch solution, adjust the pH to 4.5-4.7, freeze-dry, and obtain α-ketoglutarate complex liposomes.

[0025] The present invention provides a method for preparing α-ketoglutarate complex liposomes. After preparing basic liposomes via thin-film dispersion-ultrasound, enzymatic hydrolysis of silkworm pupa protein and arginine-modified starch are introduced to further enhance membrane structural stability. This preparation method utilizes biocompatible and synergistically functional raw materials, is based on mature technology, uses readily available and conventional equipment, and features seamless integration of each step, facilitating large-scale production.

[0026] Preferably, the volume ratio of chloroform to methanol in the chloroform-methanol mixture is 2-3.5:1.

[0027] Preferably, the α-ketoglutaric acid aqueous solution comprises 30 mg / mL-40 mg / mL of α-ketoglutaric acid and 150 mg / mL-200 mg / mL of glycerol.

[0028] Preferably, the concentration of the enzymatically hydrolyzed silkworm pupa protein solution is 2%-3% and the pH is 4.9-5.1.

[0029] Preferably, the concentration of the arginine-modified starch solution is 1.3%-2.0% and the pH is 4.75-4.85.

[0030] Preferably, in S2, the conditions for the intermittent ultrasonic refining process are: temperature 22℃-28℃, stirring speed 250rpm-300rpm, ultrasonic power 280W-320W, with an interval of 4s-6s between ultrasonic cycles of 2.5s-3.5s, and a total processing time of 8min-15min.

[0031] Preferably, in step S3, after adding the enzymatically hydrolyzed silkworm pupa protein solution, the mixture is kept under ultrasonic power of 200W-250W and temperature of 38℃-42℃ for 12min-20min, then arginine-modified starch solution is added, the pH is adjusted to 4.5-4.7, and the mixture is kept under stirring speed of 100rpm-150rpm for 25min-35min. The solid and liquid are separated and washed, and then freeze-dried to obtain α-ketoglutarate complex liposomes.

[0032] This step scientifically constructs a composite protective layer by enzymatically hydrolyzing silkworm pupa protein for coating, followed by a secondary coating with arginine-modified starch, enhancing the stability and functional synergy of liposomes. Specifically, after adding enzymatically hydrolyzed silkworm pupa protein solution to the base liposomes, ultrasonic vibration breaks down the hydration membrane on the liposome surface, increasing the collision frequency between partially positively charged small peptides in the enzymatically hydrolyzed silkworm pupa protein and the liposomes (whose surface is negatively charged due to phospholipid groups). This promotes efficient binding between the two through electrostatic attraction, achieving uniform protein coating and avoiding localized aggregation caused by simple stirring. First, enzymatically hydrolyzed silkworm pupa protein is coated to form the inner protective layer, then arginine-modified starch (a rigid polysaccharide) is added to form the outer layer. The two substances bind through hydrogen bonds (protein amide bonds and starch hydroxyl groups) and electrostatic interactions (arginine guanidine groups and protein carboxyl groups), constructing a composite protective layer that retains the protein's flexible buffering effect on the liposome membrane while reducing liposome aggregation through the steric hindrance of starch, synergistically improving system stability.

[0033] The reason for adjusting the pH to 4.5-4.7 is that this pH is close to the isoelectric point of enzymatic hydrolysis of silkworm pupa protein and the optimal adsorption pH of arginine-modified starch. This can reduce the electrostatic repulsion between protein molecules, promote their tight arrangement on the liposome surface, and enhance the interaction between arginine guanidine groups and protein / liposomes, making the protective layer denser.

[0034] Thirdly, the present invention provides the application of the α-ketoglutarate complex liposomes described in the first aspect or the α-ketoglutarate complex liposomes prepared by the preparation method described in the second aspect in the preparation of sports nutrition supplements, antioxidant products or anti-aging products.

[0035] Given that the α-ketoglutarate complex liposomes provided by this invention have advantages such as high stability, food safety, high encapsulation rate, and good gastric acid resistance, as well as good sustained-release properties, simple preparation process, and suitability for large-scale production, they have the potential for wide application in sports nutrition supplements, antioxidant products, or anti-aging products. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a scanning electron microscope image of the α-ketoglutarate complex liposomes prepared in Example 1 of this invention; Figure 2 The liquid chromatogram is used to determine the total amount of α-ketoglutarate complex liposomes prepared in Example 1 of this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] To better illustrate the embodiments provided by the present invention, further examples are given below.

[0040] The trypsin used in this invention was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., with an enzyme activity >250 U / mg. Silkworm pupa protein was purchased from Shaanxi Langde Biotechnology Co., Ltd.; α-ketoglutarate was produced by Jingjing Pharmaceutical Co., Ltd., with a content of 98%.

[0041] Example 1 This embodiment provides an α-ketoglutarate complex liposome, which includes a core layer, an intermediate layer and a composite protective layer from the inside out; The core layer comprises α-ketoglutaric acid and glycerol in a mass ratio of 1:5; The intermediate layer comprises soybean lecithin and cholesterol in a mass ratio of 5:1; The composite protective layer comprises enzymatically hydrolyzed silkworm pupa protein and arginine-modified starch in a mass ratio of 1:1.

[0042] This embodiment also provides a method for preparing the above-mentioned α-ketoglutarate complex liposomes, the preparation method comprising the following steps: S1. Dissolve 10g of soybean lecithin and 2g of cholesterol in 1L of chloroform-methanol mixture (chloroform to methanol volume ratio of 2:1), stir in a 50℃ water bath until dissolved, transfer to a rotary evaporator, evaporate under reduced pressure at 60rpm and 35℃ to remove organic solvent, and form a uniform lipid film on the inner wall of the flask. S2. Dissolve 10g of α-ketoglutaric acid and 50g of glycerol in 250mL of ultrapure water, stir magnetically at 30℃ for 30min, filter through a 0.22μm filter membrane to obtain an aqueous solution of α-ketoglutaric acid, and store at 4℃ for later use. An aqueous solution of α-ketoglutaric acid was added to the lipid membrane, and the mixture was hydrated by stirring in a water bath at 37°C for 30 min. The membrane was then subjected to ultrasonic refinement at 25°C, 250 rpm, 300 W ultrasonic power, and a working time of 3 s followed by a 5 s interval for 10 min to obtain the basic liposomes. S3、(1) Dissolve 10g of silkworm pupa protein powder in deionized water to obtain a silkworm pupa protein solution with a protein concentration of 5% (w / v). Adjust the pH to 8.0, add 0.3g of trypsin, and maintain the solution in a 37℃ water bath with magnetic stirring at 150rpm for 2.5h. Determine the degree of hydrolysis (DH) by pH-stat method to obtain a silkworm pupa protein solution with a degree of hydrolysis of 15.5%. The above enzymatically hydrolyzed silkworm pupa protein solution was placed in a 95°C water bath and heated for 10 minutes. The high temperature denatured and inactivated the enzyme, thus terminating the hydrolysis. After cooling to room temperature, it was freeze-dried to obtain the enzymatically hydrolyzed silkworm pupa protein. Add 6.25g of enzymatically hydrolyzed silkworm pupa protein to 200mL of deionized water, stir in a 50℃ water bath until dissolved, adjust the pH to 5.0 with 1M sodium hydroxide / hydrochloric acid solution, and bring the volume to 250mL to obtain a 2.5% enzymatically hydrolyzed silkworm pupa protein solution. (2) Take 250g of potato starch, add deionized water and bring the volume to 1L, stir evenly, and disperse at 1000rpm for 30min using a high-speed shear machine to ensure no particle agglomeration, and obtain potato starch milk with a concentration of 250g / L. Add 1M sodium hydroxide solution to 500mL of potato starch milk, adjust the pH to 8.5, and stir in a 50℃ water bath for 30min to activate the starch granules and allow them to swell initially. Add 2.5g sodium trimetaphosphate as a crosslinking agent and 6.25g L-arginine as a functional group donor to the pre-swollen starch milk. Carry out the grafting reaction at 60℃ and 200rpm for 1.5h. Adjust the pH of the reaction solution to 6.8 with 1.5M hydrochloric acid to terminate the reaction. After the grafting reaction was terminated, the starch was centrifuged at 3500 rpm for 15 min. The precipitate was washed three times with deionized water to obtain wet starch, which was then vacuum dried at 45℃ until the moisture content was <10%. The starch was then pulverized and passed through a 100-mesh sieve to obtain arginine-modified starch for later use. Add 5g of arginine-modified starch to 200mL of deionized water, stir the solution, adjust the pH to 4.8, and make up the volume to 250mL to obtain a 2.0% arginine-modified starch solution. (3) Add 80 mL of enzymatically hydrolyzed silkworm pupa protein solution to the basic liposomes, sonicate at 200 W and 40 °C for 15 min, add 100 mL of arginine-modified starch solution, adjust the pH to 4.6, keep stirring at 100 rpm for 30 min, centrifuge and wash with water 3 times, freeze dry to obtain α-ketoglutarate complex liposomes.

[0043] The scanning electron microscope image of the α-ketoglutarate complex liposomes prepared in this embodiment is shown below. Figure 1 As shown.

[0044] Example 2 This embodiment provides an α-ketoglutarate complex liposome, which includes a core layer, an intermediate layer and a composite protective layer from the inside out; The core layer comprises α-ketoglutaric acid and glycerol in a mass ratio of 1:5; The intermediate layer comprises soybean lecithin and cholesterol in a mass ratio of 6.25:1; The composite protective layer comprises enzymatically hydrolyzed silkworm pupa protein and arginine-modified starch in a mass ratio of 1.17:1.

[0045] This embodiment also provides a method for preparing the above-mentioned α-ketoglutarate complex liposomes, the preparation method comprising the following steps: S1. Dissolve 12.5g of soybean lecithin and 2g of cholesterol in 1L of chloroform-methanol mixture (chloroform to methanol volume ratio of 3:1), stir in a 50℃ water bath until dissolved, transfer to a rotary evaporator, evaporate under reduced pressure at 60rpm and 37℃ to remove organic solvent, and form a uniform lipid film on the inner wall of the flask. S2. Dissolve 10g of α-ketoglutaric acid and 60g of glycerol in 250mL of ultrapure water, stir magnetically at 30℃ for 30min, filter through a 0.22μm filter membrane to obtain an aqueous solution of α-ketoglutaric acid, and store at 4℃ for later use. An aqueous solution of α-ketoglutaric acid was added to the lipid membrane, and the mixture was hydrated by stirring in a water bath at 37°C for 35 min. The membrane was then subjected to ultrasonic refinement at 28°C, 300 rpm, 280 W ultrasonic power, and a working time of 2.5 s followed by a 4 s interval for 15 min to obtain the basic liposomes. S3, (1) Prepare enzymatically hydrolyzed silkworm pupa protein according to the method described in Example 1; Add 7.5g of enzymatically hydrolyzed silkworm pupa protein to 200mL of deionized water, stir in a 50℃ water bath until dissolved, adjust the pH to 5.0, and bring the volume to 250mL to obtain a 3% enzymatically hydrolyzed silkworm pupa protein solution. (2) Arginine-modified starch was prepared according to the method described in Example 1; Take 3.75g of arginine-modified starch and add it to 200mL of deionized water. Stir the solution, adjust the pH to 4.85, and make up the volume to 250mL to obtain a 1.5% arginine-modified starch solution. (3) Add 70 mL of enzymatically hydrolyzed silkworm pupa protein solution to the basic liposomes, sonicate at 250 W and 38 °C for 20 min, add 120 mL of arginine-modified starch solution, adjust the pH to 4.55, keep stirring at 150 rpm for 25 min, centrifuge and wash with water 3 times, freeze dry to obtain α-ketoglutarate complex liposomes.

[0046] Example 3 This embodiment provides an α-ketoglutarate complex liposome, which includes a core layer, an intermediate layer and a composite protective layer from the inside out; The core layer comprises α-ketoglutaric acid and glycerol in a mass ratio of 1:5; The intermediate layer comprises soybean lecithin and cholesterol in a mass ratio of 5:1; The composite protective layer comprises enzymatically hydrolyzed silkworm pupa protein and arginine-modified starch in a mass ratio of 0.82:1.

[0047] This embodiment also provides a method for preparing the above-mentioned α-ketoglutarate complex liposomes, the preparation method comprising the following steps: S1. Dissolve 10g of soybean lecithin and 2g of cholesterol in 1L of chloroform-methanol mixture (chloroform to methanol volume ratio of 3:1), stir in a 50℃ water bath until dissolved, transfer to a rotary evaporator, evaporate under reduced pressure at 60rpm and 37℃ to remove organic solvent, and form a uniform lipid film on the inner wall of the flask. S2. Dissolve 10g of α-ketoglutaric acid and 50g of glycerol in 250mL of ultrapure water, stir magnetically at 30℃ for 30min, filter through a 0.22μm filter membrane to obtain an aqueous solution of α-ketoglutaric acid, and store at 4℃ for later use. An aqueous solution of α-ketoglutaric acid was added to the lipid membrane, and the membrane was hydrated by stirring in a water bath at 38°C for 25 min. Then, it was ultrasonically refined for 8 min at 22°C, 250 rpm, 320 W ultrasonic power, and 3.5 s working time followed by 6 s intermittent time to obtain the basic liposomes. S3, (1) Prepare enzymatically hydrolyzed silkworm pupa protein according to the method described in Example 1; Add 5g of enzymatically hydrolyzed silkworm pupa protein to 200mL of deionized water, stir in a 50℃ water bath until dissolved, adjust the pH to 4.9, and bring the volume to 250mL to obtain a 2% enzymatically hydrolyzed silkworm pupa protein solution. (2) Arginine-modified starch was prepared according to the method described in Example 1; Take 5g of arginine-modified starch and add it to 200mL of deionized water. Stir the solution, adjust the pH to 4.75, and make up the volume to 250mL to obtain a 2% arginine-modified starch solution. (3) Add 90 mL of enzymatically hydrolyzed silkworm pupa protein solution to the basic liposomes, sonicate at 200 W and 42 °C for 12 min, add 110 mL of arginine-modified starch solution, adjust the pH to 4.65, keep stirring at 100 rpm for 35 min, centrifuge and wash with water 3 times, freeze dry to obtain α-ketoglutarate complex liposomes.

[0048] Example 4 This embodiment provides an α-ketoglutarate complex liposome and its preparation method. The complex liposome includes a core layer, an intermediate layer and a composite protective layer from the inside to the outside. The core layer comprises α-ketoglutaric acid and glycerol in a mass ratio of 1:5; The intermediate layer comprises soybean lecithin and cholesterol in a mass ratio of 5:1; The composite protective layer comprises enzymatically hydrolyzed silkworm pupa protein and arginine-modified starch in a mass ratio of 4:1.

[0049] The preparation method of this composite liposome is basically the same as that in Example 1, except that in S3, when preparing the arginine-modified starch solution, 20g of arginine-modified starch is taken and an 8.0% arginine-modified starch solution is prepared under the same conditions; the rest of the preparation methods and parameters are the same as those in Example 1.

[0050] Comparative Example 1 This comparative example provides an α-ketoglutarate liposome and its preparation method, the liposome comprising a core layer and a coating layer; The core layer comprises α-ketoglutaric acid and glycerol in a mass ratio of 1:5; The coating layer comprises soybean lecithin and cholesterol in a mass ratio of 5:1.

[0051] The specific method for preparing these liposomes is as follows: S1. Dissolve 10g of soybean lecithin and 2g of cholesterol in 1L of chloroform-methanol mixture (chloroform to methanol volume ratio of 2:1), stir in a 50℃ water bath until dissolved, transfer to a rotary evaporator, evaporate under reduced pressure at 60rpm and 35℃ to remove organic solvent, and form a uniform lipid film on the inner wall of the flask. S2. Dissolve 10g of α-ketoglutaric acid and 50g of glycerol in 250mL of ultrapure water, stir magnetically at 30℃ for 30min, filter through a 0.22μm filter membrane to obtain an aqueous solution of α-ketoglutaric acid, and store at 4℃ for later use. An aqueous solution of α-ketoglutaric acid was added to the lipid membrane, and the membrane was hydrated by stirring in a water bath at 37°C for 30 min. The membrane was then subjected to ultrasonic refinement at 25°C, 250 rpm, 300 W ultrasonic power, and a working time of 3 s followed by a 5 s interval for 10 min to obtain basic liposomes. The liposomes were then separated by centrifugation, washed three times with water, and freeze-dried to obtain α-ketoglutaric acid liposomes.

[0052] Example of effect 1 The present invention characterized the average particle size, particle size distribution (PDI, polydispersity index) and zeta potential of the α-ketoglutarate complex liposomes prepared in Examples 1-4 and Comparative Example 1. Each sample was measured in parallel three times, and the results are shown in Table 1.

[0053] Table 1

[0054] As shown in Table 1, due to the positive charge of arginine-modified starch, the Zeta potentials in Examples 1-4 were all positive. In Comparative Example 1, the liposomes, lacking positively charged components like arginine-modified starch, exhibited negative Zeta potentials. In Example 4, the Zeta potential was higher due to the greater amount of arginine-modified starch compared to the examples, but the PDI value was significantly higher than in Examples 1-3, indicating that the composite liposomes in Examples 1-3 had a more uniform particle size distribution than those in Example 4.

[0055] Example 2 This invention measures the encapsulation efficiency of liposomes prepared in Examples 1-4 and Comparative Example 1. Details are as follows: Take 1g of each liposome prepared in different examples or comparative examples, slowly add 10mL of 0.9% physiological saline, let stand in a 37℃ water bath for 10min to allow the powder to initially absorb moisture and swell; then stir magnetically at 100rpm for 15min to obtain the corresponding reconstituted suspension. Determination of total drug content: Take 1 mL of the reconstituted suspension, add 1 mL of demulsifier containing 1% Triton X-100, vortex for 10 min, let stand at 4℃ for 5 min, filter through a 0.22 μm filter membrane, and determine the α-ketoglutarate content by HPLC.

[0056] Determination of free drug content: Take 1 mL of the reconstituted suspension, centrifuge at 15000 rpm for 25 min at 4℃, take the supernatant after centrifugation, filter through a 0.22 μm filter membrane, and determine the content of α-ketoglutarate by HPLC under the same conditions.

[0057] Encapsulation rate (%) = [(total drug amount - free drug amount) / total drug amount] × 100%, and the average value is taken after three parallel determinations.

[0058] The liquid chromatogram for determining the total amount of α-ketoglutaric acid complex liposomes prepared in Example 1 in this invention is shown below. Figure 2 As shown in Table 2, the encapsulation efficiency of different liposomes was determined.

[0059] Table 2

[0060] As shown in Table 2, the encapsulation efficiency of the composite liposomes prepared in Examples 1-4 of this invention is significantly improved compared to Comparative Example 1. The liposomes provided in Comparative Example 1 lack a composite protective layer. Since α-ketoglutarate is only soluble in the aqueous core of the liposome, the simple bilayer has limited encapsulation capacity for water-soluble small molecules and cannot embed into the membrane structure like lipophilic drugs. After hydration and sonication, some incompletely encapsulated drugs remain free in the solution. During washing and freeze-drying, without a protective layer, the liposomes easily fuse, potentially leading to drug leakage from the aqueous core, resulting in a lower encapsulation efficiency. Although the composite liposomes in Example 4 have a protective layer, the amount of arginine-modified starch is significantly increased. The starch is too rigid, and excessive starch may disrupt the uniformity of the protective layer and the structural stability of the liposomes, causing the liposome membrane to deform under pressure, increasing drug leakage, and resulting in a lower encapsulation efficiency than in the examples.

[0061] Example 3 This invention aims to investigate the stability of α-ketoglutarate composite liposomes prepared by different preparation processes in the gastrointestinal environment. Therefore, the physiological environment of the human gastrointestinal tract (gastric acid pH 1.2, intestinal pH 6.8) was simulated. The stability of the liposomes prepared in Example 1, Example 4, and Comparative Example 1 was evaluated by detecting the α-ketoglutarate leakage rate. The specific details are as follows: Simulated gastric acid: Prepared according to USP standards, containing pepsin (1 mg / mL, Sigma), and adjusted to pH 1.2 with 1M hydrochloric acid; Simulated intestinal fluid: Prepared according to USP standards, containing pancreatic enzymes (0.1 mg / mL, Sigma) and ox bile salts (5 mg / mL, Sigma), and adjusted to pH 6.8 with 0.2 M phosphate buffer.

[0062] Take 100 mg (accurate to 0.1 mg) of each of the three liposome samples prepared in Example 1, Example 4 and Comparative Example 1, and add them to 10 mL centrifuge tubes respectively. Take 3 portions of each sample.

[0063] Gastric acid resistance test: Add 10 mL of simulated gastric acid solution (pH 1.2) to each centrifuge tube and vortex to mix; place in a 37℃ constant temperature water bath shaker and incubate at 120 rpm; take 1 mL of the mixture at 0 h (initial), 2 h and 4 h respectively, centrifuge at 3000 rpm for 10 min, take the supernatant and filter through a 0.22 μm filter membrane, load onto liquid phase and measure; Intestinal stability test: Take 5 mL of the above mixture after 4 h of gastric acid incubation and transfer it to a new centrifuge tube. Add 5 mL of simulated intestinal fluid to each tube and adjust the pH of the mixture to 6.8±0.1. Vortex to mix. Continue incubation at 37℃ and 120 rpm. After 4 h of incubation, take 1 mL of the mixture, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane. Publish the solution in liquid chromatography and perform the determination.

[0064] Leakage rate (%) = (Actual content of α-ketoglutarate in supernatant / Total content of α-ketoglutarate in liposomes) × 100%.

[0065] The method for determining the total α-ketoglutarate content in liposomes is as follows: take 100 mg of liposomes, break the membrane with 5 mL of methanol, sonicate for 10 min, and then adjust the volume to 10 mL to determine the total α-ketoglutarate content.

[0066] The measurement results are shown in Table 3.

[0067] Table 3

[0068] As shown in Table 3, the α-ketoglutarate composite liposomes provided in Example 1 of this invention contain a composite protective layer of enzymatically hydrolyzed silkworm pupa protein and arginine-modified starch. The enzymatically hydrolyzed silkworm pupa protein carries a positive charge under acidic conditions, and the arginine-modified starch is protonated at pH 1.2. The two can form a double electrostatic barrier, inhibiting the damage of gastric acid to the liposome membrane. In contrast, the liposomes prepared in Comparative Example 1 are coated with soybean lecithin and cholesterol. Under low pH conditions, the phospholipids hydrolyze, leading to a large amount of α-ketoglutarate leakage. The arginine-modified starch content in the composite protective layer of the α-ketoglutarate composite liposomes provided in Example 4 is significantly increased, which can enhance the thickness and charge density of the protective layer and further improve acid resistance. However, excessive starch leads to an increase in liposome particle size, reduced dispersibility, and a slower enzymatic hydrolysis rate in the intestine, affecting the release of the target substance in the intestine.

[0069] Given the advantages of the α-ketoglutarate complex liposomes provided by this invention in terms of particle size uniformity, encapsulation efficiency, gastric acid resistance, and intestinal stability, they can be used to prepare sports nutrition supplements, antioxidant products, or anti-aging products.

[0070] 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 or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An α-ketoglutarate complex liposome, characterized in that, From the inside out, it includes a core layer, an intermediate layer, and a composite protective layer; The core layer includes α-ketoglutaric acid and an osmotic pressure regulator; The intermediate layer comprises lecithin and cholesterol; The composite protective layer comprises enzymatically hydrolyzed silkworm pupa protein and arginine-modified starch.

2. The α-ketoglutarate complex liposome as described in claim 1, characterized in that, The mass ratio of α-ketoglutaric acid to the osmotic pressure regulator is 1-2:5-10; and / or The mass ratio of lecithin to cholesterol is 5:0.8-1; and / or The mass ratio of enzymatically hydrolyzed silkworm pupa protein to arginine-modified starch is 0.8-1.2:1; and / or The mass ratio of α-ketoglutarate, cholesterol, and arginine-modified starch is 9-11:1.8-2.2:1.8-2.

2.

3. The α-ketoglutarate complex liposome as described in claim 1 or 2, characterized in that, The osmotic pressure regulator includes at least one of glycerol, sucrose, or mannitol; and / or The lecithin includes soybean lecithin; and / or The degree of enzymatic hydrolysis of the silkworm pupa protein is 15%-20%.

4. The α-ketoglutarate complex liposome as described in claim 1, characterized in that, The preparation method of the arginine-modified starch includes the following steps: adding sodium trimetaphosphate and L-arginine to potato starch milk, and carrying out a grafting reaction at 55℃-62℃ to obtain the arginine-modified starch.

5. The α-ketoglutarate complex liposome as described in claim 4, characterized in that, The concentration of the potato starch milk is 200 g / L-250 g / L; and / or The mass ratio of sodium trimetaphosphate, L-arginine and potato starch is 1.5-2.5:4.5-5.5:90-100.

6. The α-ketoglutarate complex liposome according to claim 4, characterized in that, The preparation method of the arginine-modified starch specifically includes the following steps: adjusting the pH of potato starch milk to 8.5-9.0, activating it at 45℃-55℃ for 25min-40min, adding sodium trimetaphosphate and L-arginine, and carrying out a grafting reaction at 55℃-62℃ for 1.5h-2h. Adjusting the pH of the system to 6.5-7.0, separating the solid and liquid, and drying to obtain the arginine-modified starch.

7. The method for preparing α-ketoglutaric acid complex liposomes according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: S1. Dissolve lecithin and cholesterol in a chloroform-methanol mixture and rotary evaporate to form a lipid film; S2. Add α-ketoglutaric acid aqueous solution to the lipid membrane, hydrate for 25 min-35 min, and then perform intermittent ultrasonic refining treatment to obtain basic liposomes. S3. Add an enzymatically hydrolyzed silkworm pupa protein solution to the basic liposomes, sonicate, add an arginine-modified starch solution, adjust the pH to 4.5-4.7, freeze-dry, and obtain α-ketoglutarate complex liposomes.

8. The method for preparing α-ketoglutarate complex liposomes as described in claim 7, characterized in that, The volume ratio of chloroform to methanol in the chloroform-methanol mixture is 2-3.5:1; and / or The α-ketoglutaric acid aqueous solution comprises 30 mg / mL-40 mg / mL α-ketoglutaric acid and 150 mg / mL-200 mg / mL glycerol; and / or The concentration of the enzymatically hydrolyzed silkworm pupa protein solution is 2%-3%, and the pH is 4.9-5.1; and / or The concentration of the arginine-modified starch solution is 1.3%-2.0%, and the pH is 4.75-4.

85.

9. The method for preparing α-ketoglutarate complex liposomes as described in claim 7, characterized in that, In S2, the conditions for the intermittent ultrasonic refinement process are: ultrasonic power 280W-320W, 2.5s-3.5s interval between ultrasonic waves, 4s-6s interval between ultrasonic waves, and a total processing time of 8min-15min; and / or In step S3, after adding the enzymatically hydrolyzed silkworm pupa protein solution, the mixture is kept under ultrasonic power of 200W-250W and temperature of 38℃-42℃ for 12min-20min. Then, arginine-modified starch solution is added, the pH is adjusted to 4.5-4.7, and the mixture is kept under stirring speed of 100rpm-150rpm for 25min-35min. The solid and liquid are separated and washed, and then freeze-dried to obtain α-ketoglutarate complex liposomes.

10. The use of the α-ketoglutarate complex liposomes according to any one of claims 1-6 or the α-ketoglutarate complex liposomes prepared by the preparation method according to any one of claims 7-9 in the preparation of sports nutrition supplements, antioxidant products or anti-aging products.

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