A composition based on 2-oxoglutaric acid and a method of preparation

Abstract

The present application relates to a kind of compositions based on 2-oxoglutaric acid and its preparation method, the composition includes copolymer acid 2-oxoglutaric acid, nicotinamide, gamma-aminobutyric acid etc..Copolymer acid is prepared by double emulsification-solvent evaporation method, and the copolymer acid used is polylactic acid-glycolic acid copolymer acid with molecular weight of 15~20 kDa, and the average particle size of copolymer acid 2-oxoglutaric acid is 100~500 nm.Preparation method includes solution preparation, emulsification, ultrasonic, stirring, centrifugation and other steps, to ensure that the average particle size of nanoparticle is uniform and encapsulation efficiency.The composition promotes collagen production, and improves physiological adaptability by osmotic pressure regulator and pH buffer, realizes the stable encapsulation and slow-release performance of 2-oxoglutaric acid, improves antioxidant property and biocompatibility, and is widely used in tissue repair and anti-aging field.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical preparations, and particularly relates to a 2-oxoglutaric acid-based composition and a preparation method. BACKGROUND

[0002] In the field of modern medicine and biological engineering, the demand for chronic disease prevention, treatment, tissue repair and anti-aging is increasing. The significant role of 2-oxoglutaric acid in promoting collagen production in fibroblasts and skin epithelial cells shows a wide application potential. However, the main challenges in its practical application are easy oxidative degradation, high difficulty in release control and uneven distribution in vivo, which significantly limit its effect in collagen production. Therefore, it is crucial to develop a delivery material that can stably encapsulate and release 2-oxoglutaric acid. The ideal delivery system needs to have the following performances: first, the carrier material must have high encapsulation ability and protection performance to avoid the oxidative degradation of 2-oxoglutaric acid in the processing and physiological environment, so as to ensure its support for collagen production; second, the average particle size of the nanoparticles should be controlled in the nanometer range to ensure the targeted delivery and uniform distribution of the drug in the cell microenvironment, and to improve the efficiency of collagen production promotion; third, the material should have excellent biocompatibility and environmental stability to avoid immune rejection and ensure its safety and activity in fibroblasts and skin epithelial cells; fourth, the material system should be able to accurately control the release rate of the drug to achieve the effect of sustained release and continuously promote the production of collagen for a long time. Breaking through the above performances will significantly improve the application effect of 2-oxoglutaric acid in collagen production, enhance the patient compliance, and promote its wide application in the fields of chronic disease treatment, tissue repair, beauty and skin care, and anti-aging.

[0003] In the prior art, although some progress has been made in the study of 2-oxoglutaric acid, there are still significant limitations in its application in promoting collagen production. For example, although the scheme disclosed in CN103476274A proposes a combination of adding alpha-ketoglutaric acid, it does not use coating and antioxidant protection, resulting in unstable drug release rate and easy oxidation degradation of activity, which cannot continuously promote the generation of collagen. In addition, this scheme lacks comprehensive regulation of material biocompatibility and osmotic pressure, making it difficult to adapt to complex physiological environments, thereby limiting its practical application in tissue repair and beauty fields. Further analysis shows that these problems mainly come from the following reasons: first, no coating or loading treatment is used, making it difficult to accurately control the drug release rate and continuously support collagen production; second, no antioxidant protection system is introduced, making 2-oxoglutaric acid lose activity due to oxidation degradation during processing, storage and use; third, the material system lacks optimal design of biocompatibility and osmotic pressure, resulting in low adaptability in cell environment. Therefore, it is urgent to develop an efficient delivery system to achieve the comprehensive performance of continuous and stable drug release, effective antioxidant protection and promotion of collagen production. SUMMARY

[0004] (1) Technical problems solved

[0005] The purpose of the present application is to provide a 2-oxoglutaric acid-based composition and a preparation method to solve the problems of current 2-oxoglutaric acid.

[0006] (2) Technical solutions

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] A 2-oxoglutaric acid-based composition, comprising the following raw materials by weight: 10-20 parts of copolymer acid-coated 2-oxoglutaric acid, 0.1-1.5 parts of nicotinamide, 0.2-1.0 parts of gamma-aminobutyric acid, 0.1-0.5 parts of superoxide dismutase, 2-6 parts of polyethylene glycol 400, 2.5-4.5 parts of pH buffer, 3.5-8.0 parts of biological repair promoter, 0.5-4.5 parts of osmotic pressure regulator, 2.0-6.0 parts of beta-cyclodextrin, 50-70 parts of deionized water, 0.02-0.1 parts of pyrroloquinoline quinone disodium salt, 0.2-0.8 parts of catechin, 0.5-2.0 parts of L-carnitine, 0.5-2.5 parts of 1, 6-fructose disodium phosphate, and 0.002-0.02 parts of vitamin B12;

[0009] The copolymer acid-coated 2-oxoglutaric acid is a polylactic acid-glycolic acid copolymer acid-coated 2-oxoglutaric acid and vitamin composition prepared by double emulsification-solvent evaporation method;

[0010] The polylactic acid-glycolic acid copolymer acid is obtained by polymerization of glycolic acid and lactic acid.

[0011] Further, the preparation method of the copolymer acid encapsulating 2-oxoglutaric acid is as follows: first, 0.5-1.5 parts of Tween 80 is dissolved in 20-40 parts of 2 wt% citric acid aqueous solution, then 1.25-4.5 parts of vitamin composition is added, followed by 45-65 parts of 2.0 wt% polyvinyl alcohol aqueous solution, and the mixture is uniformly obtained as A solution, then 2.5-4.5 parts of 2-oxoglutaric acid is uniformly dissolved in 60-85 parts of glycerol to obtain B solution; then 10-20 parts of polylactic acid-glycolic acid copolymer acid is uniformly dissolved in 100-200 parts of ethyl acetate to obtain an organic solution; the A solution and the B solution are uniformly mixed at a mass ratio of 1:3, and then added dropwise into the organic solution to form a primary emulsion, then the primary emulsion is added into a vortex instrument container for emulsification treatment, the vortex time is 30-60 s, the vortex instrument rotation speed is 2000-3000 rpm, then ultrasonic treatment is performed using an ultrasonic processor, the ultrasonic conditions are 10 s on / off cycle each time, the ultrasonic amplitude is 60-70%, the frequency is 20-24 kHz, the ultrasonic treatment cycle number is set to 3-5 times, after ultrasonic treatment, 50 parts of 4 wt% PVA aqueous solution is added into 100 parts of the ultrasonic-treated primary emulsion, and vortex and ultrasonic treatment are continued to form a double emulsion, the vortex time is 5-10 min, the ultrasonic treatment cycle number is 2-4 times, the treated double emulsion is added into a magnetic stirrer, the stirring speed is set to 500-1000 rpm, the stirring temperature is 30-40℃, and the stirring time is 240-320 min, and then the solid nanoparticles are obtained; then 10-18 parts of the solid nanoparticles and 100 parts of deionized water are uniformly mixed, and then centrifuged at 10000-15000 rpm for 180-360 s, then the upper layer of the centrifuged suspension is dried at room temperature under vacuum, the vacuum condition is 40-100 Pa, the drying time is 6-12 h, and the copolymer acid encapsulating 2-oxoglutaric acid is obtained after complete drying.

[0012] Further, the mass ratio of the vitamin composition and 2-oxoglutaric acid is (0.5-1.0):1.

[0013] Further, the vitamin composition is prepared by mixing vitamin D, vitamin B1 and vitamin B2 at a mass ratio of (2.0-4.5):1:1.

[0014] Further, the preparation method of the polylactic acid-glycolic acid copolymer acid is as follows: 3.0-6.0 parts of glycolic acid, 12.9-25.8 parts of lactic acid and 3.0-5.5 parts of deionized water are stirred at 100-120 °C for 90-140 min, then the temperature is lowered to 60-70 °C, 0.10-0.25 parts of zinc lactate is added and stirred uniformly, then the temperature is raised to 160-170 °C, and stirred under vacuum for 14-18 h, during the reaction, the by-products are continuously removed through a Dean-Stark device, after the reaction, the temperature is lowered to 80-90 °C, then the product is dried under vacuum for 12-20 h to obtain the copolymer crude product, then the copolymer crude product is dissolved in 60-80 parts of ethanol to form a mixed solution, then the mixed solution is added dropwise into a mixed solution of 200 parts of methanol and water in a drop-by-drop manner, wherein the volume ratio of methanol to water is 1:3, after the dropwise addition is completed, the mixed solution is separated by decantation and centrifugation to remove the residual solvent and retain the precipitate, then the obtained precipitate is dried under vacuum at 60-70 °C for 18-24 h, then freeze-dried at -20 °C for 36-48 h, and finally the polylactic acid-glycolic acid copolymer is obtained.

[0015] The purpose of the present scheme is to realize the protection of functional ingredients, the improvement of stability, the controllable release and the functional synergies by the technical design of the copolymer acid wrapping 2-oxoglutaric acid combined with the synergies of vitamin composition. Specifically, the use of copolymer acid can effectively wrap 2-oxoglutaric acid, form a protective barrier, prevent its degradation in the external environment (such as light, heat, oxidation, etc.), and improve its stability; at the same time, the slow-release characteristics of the copolymer acid can realize the precise release of 2-oxoglutaric acid in specific application scenarios, prolong its action time, improve the bioavailability, and reduce the dosage and frequency of use. In addition, by introducing the vitamin composition (vitamin D, vitamin B1 and vitamin B2), using its multifunctional properties (such as the immune regulation and calcium and phosphorus metabolism of vitamin D, the metabolic support function of vitamin B1, and the antioxidant effect of vitamin B2), and forming synergies with 2-oxoglutaric acid, the biological functionality of the product is further enhanced. The mass ratio of the vitamin composition to 2-oxoglutaric acid is designed as (0.5~1.0):1, and the ratio of each component (vitamin D: vitamin B1: vitamin B2) in the vitamin composition is (2.0~4.5):1:1, which ensures the scientific collocation and functional balance of the two, avoiding the functional weakening or potential adverse effects caused by the imbalance of the ratio. Through the action of emulsifiers and stabilizers such as Tween 80 and polyvinyl alcohol, combined with processes such as vortex treatment, ultrasonic treatment and vacuum drying, uniform distribution and stable nanoparticles are formed, and key process parameters such as emulsification conditions, ultrasonic intensity and stirring rate are refined and optimized to ensure the stability and repeatability of the preparation process, thereby obtaining high-purity and stable solid nanoparticles. Through this design, the present scheme can be widely applied in the fields of drug delivery, functional food, health products and biological materials, providing efficient solutions for metabolic regulation, antioxidant and nutritional supplementation, and improving the functionality and market value of the product.

[0016] Further, the molecular weight of the polylactic acid-glycolic acid copolymer acid is 15~20 kDa.

[0017] Further, the average diameter of the copolymer acid wrapping 2-oxoglutaric acid is 100~500nm.

[0018] Further, the mass ratio of glycolic acid and lactic acid is 1:4.3.

[0019] Further, the biological repair promoter is hyaluronic acid or chitosan;

[0020] Further, the osmotic pressure regulator is mannitol or sodium chloride;

[0021] Further, the pH buffer is a phosphate buffer or a citrate buffer or a mixture of the two;

[0022] Further, the pH value of the 2-oxoglutaric acid-based composition is 6.8-7.2, and the osmotic pressure is 280-320 mOsm / L.

[0023] The design of the copolymer acid wrapping 2-oxoglutaric acid is mainly used to enhance the stability, slow-release performance and biocompatibility of 2-oxoglutaric acid. The molecular weight of the polylactic acid-glycolic acid copolymer acid is 15-20 kDa, and the mass ratio of glycolic acid to lactic acid is 1:4.3, so that it has a suitable degradation rate and mechanical properties, and gradually degrades in the body to achieve precise slow release. The average diameter of the nanoparticles formed by wrapping is 100-500 nm, and the average particle size control optimizes the dispersibility, stability and in vivo absorption efficiency of the drug. The pH value is adjusted to 6.8-7.2 and the osmotic pressure is adjusted to 280-320 mOsm / L, which further enhances the adaptability of the composition in the physiological environment and the protection of drug activity. Hyaluronic acid or chitosan as a biological repair promoter, by promoting tissue repair and providing antibacterial protection, improves the biological functionality of the composition; mannitol or sodium chloride as an osmotic pressure regulator ensures the stability in the physiological environment, avoids the adverse effects of osmotic pressure fluctuations on drug release, pyrroloquinoline quinone disodium salt as an active ingredient with antioxidant and cell protection functions can effectively reduce the damage of oxidative stress to cells; catechin further enhances the free radical scavenging capacity through its strong antioxidant property, and also has the effect of protecting cardiovascular health; L-carnitine is mainly involved in fat metabolism, promotes energy generation and improves cell activity; 1,6-fructose diphosphate trisodium salt as an important intermediate in sugar metabolism can improve the energy metabolism efficiency of cells; vitamin B12 is involved in DNA synthesis and cell metabolism, which helps to maintain the normal function of cells. The synergistic effect of each component significantly improves the antioxidant stability, slow-release performance and environmental adaptability of the drug, providing innovative technical support for efficient drug delivery.

[0024] The application also provides a preparation method of the 2-oxoglutaric acid-based composition, which specifically comprises the following steps: according to the weight fraction, the copolymer acid wrapping 2-oxoglutaric acid, polyethylene glycol 400 and β-cyclodextrin are weighed and added into deionized water, and stirred at a speed of 400-600 rpm at 20-30 DEG C for 30-60 min; then the pH buffer solution is added, and the stirring is continued for 10-20 min; then the biological repair promoter and the osmotic pressure regulator are added, and stirred uniformly at a speed of 500-800 rpm at 25-35 DEG C for 30-60 min; then the mixture is centrifuged at 3000-5000 rpm for 15-30 min, and the supernatant is taken, filtered through a 0.22 mu m filter membrane, and the obtained filtrate is the 2-oxoglutaric acid-based composition.

[0025] (3)Beneficial technical effects

[0026] The present application solves the problems of poor drug stability, uneven release and insufficient biocompatibility in the prior art by the innovative design of the copolymer acid wrapping 2-oxoglutaric acid, significantly improving the overall performance of the drug delivery system. The molecular weight and component ratio of the polylactic acid-glycolic acid copolymer acid are optimized to have suitable degradation rate and mechanical properties, gradually degrade in the body and achieve precise release, effectively protecting 2-oxoglutaric acid from oxidative degradation. The nanoparticles prepared by the double-emulsion-solvent evaporation method have an average particle size controlled within 100-500 nm, further enhancing the dispersibility and absorption efficiency of the drug, and through the regulation of pH value 6.8-7.2 and osmotic pressure 280-320 mOsm / L, the stability and activity of the drug in the complex physiological environment are ensured. Hyaluronic acid or chitosan as a biological repair promoter not only promotes collagen generation, but also provides tissue repair and antibacterial protection functions; mannitol or sodium chloride as an osmotic pressure regulator ensures a smooth release process, and the synergistic effect of the components significantly improves the antioxidant property, slow-release performance and biocompatibility of the system; pyrroloquinoline quinone disodium salt as an active ingredient with antioxidant and cell protection functions can effectively reduce the damage of oxidative stress to cells; catechin further enhances the free radical scavenging capacity through its strong antioxidant property, and also has the effect of protecting cardiovascular health; L-carnitine is mainly involved in fat metabolism, promotes energy generation and improves cell activity; 1,6-fructose diphosphate trisodium salt as an important sugar metabolism intermediate can improve the energy metabolism efficiency of cells; vitamin B12 participates in DNA synthesis and cell metabolism, and helps to maintain the normal function of cells. The present application effectively promotes collagen generation, significantly improves the tissue repair capacity, and has broad application prospects in the fields of cosmetic care, anti-aging and chronic disease treatment, providing important support for the upgrading of the industry. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The infrared Fourier spectrum of the polylactic acid-glycolic acid copolymer prepared in Example 1;

[0028] Figure 2 The morphology diagram of the copolymer acid-wrapped 2-oxoglutaric acid prepared in Example 1;

[0029] Figure 3 The MTT experiment results of the 3T3 cells stimulated by the 2-oxoglutaric acid-based composition in Example 1.

[0030] Figure 4 The MTT experiment results of the HaCaT cells stimulated by the 2-oxoglutaric acid-based composition in Example 1.

[0031] Figure 5WB imaging and gray scale analysis results of the effect of the 2-oxoglutaric acid-based composition in Example 2 on the amount of type I collagen produced in 3T3 cells.

[0032] Figure 6 WB imaging and gray scale analysis results of the effect of the 2-oxoglutaric acid-based composition in Example 2 on the amount of type III collagen produced in 3T3 cells.

[0033] Figure 7 WB imaging and gray scale analysis results of the effect of the 2-oxoglutaric acid-based composition in Example 2 on the amount of type I collagen produced in HaCaT cells.

[0034] Figure 8 WB imaging and gray scale analysis results of the effect of the 2-oxoglutaric acid-based composition in Example 2 on the amount of type XVII collagen produced in HaCaT cells.

[0035] Figure 9 WB imaging of the effect of the 2-oxoglutaric acid-based composition in Example 2 on the amount of type XVII collagen produced in 3T3 cells.

[0036] Figure 10 WB imaging of the effect of the 2-oxoglutaric acid-based composition in Example 2 on the amount of type III collagen produced in HaCaT cells.

[0037] Figure 11 MTT experiment results of the construction of a senescent cell model using D-galactose in Example 3.

[0038] Figure 12 β-galactosidase staining results of the construction of a senescent cell model using D-galactose in Example 3.

[0039] Figure 13 WB imaging and gray scale analysis results of the effect of D-galactose on the amount of TERT protein produced in 3T3 cells in Example 3.

[0040] Figure 14 MTT experiment results of the activation of senescent cells by the 2-oxoglutaric acid-based composition in Example 4.

[0041] Figure 15 β-galactosidase staining results of the control group, the senescent cell group, and the senescent cell + AKG group in Example 4.

[0042] Figure 16 WB imaging and gray scale analysis results of the effect of the 2-oxoglutaric acid-based composition in Example 4 on the amount of TERT protein produced in senescent cells. DETAILED DESCRIPTION

[0043] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Embodiment 1

[0044] A 2-oxoglutaric acid-based composition includes the following raw materials in parts by weight: 10 parts of 2-oxoglutaric acid coated with copolymer acid, 0.1 part of nicotinamide, 0.2 part of gamma-aminobutyric acid, 0.1 part of superoxide dismutase, 2 parts of polyethylene glycol 400, 2.5 parts of pH buffer, 3.5 parts of bioremediation promoter, 0.5 part of osmotic pressure regulator, 2.0 parts of β-cyclodextrin, 50 parts of deionized water, 0.02 parts of pyrroloquinoline quinone disodium salt, 0.2 parts of catechin, 0.5 parts of L-carnitine, 0.5 parts of 1, 6-fructose diphosphate trisodium salt, and 0.002 parts of vitamin B12; the 2-oxoglutaric acid coated with copolymer acid is a polylactic acid-glycolic acid copolymer acid coated with 2-oxoglutaric acid and vitamin composition V prepared by double-emulsification-solvent evaporation method; the polylactic acid-glycolic acid copolymer acid is obtained by polymerization reaction of glycolic acid and lactic acid;

[0045] The preparation method of the copolymer acid-encapsulated 2-oxoglutaric acid in this embodiment is as follows: first, 0.5 parts of Tween 80 is dissolved in 20 parts of 2 wt% citric acid aqueous solution, then 1.25 parts of the vitamin composition is added, followed by 45-65 parts of 2.0 wt% polyvinyl alcohol aqueous solution, and the mixture is uniformly mixed to obtain solution A, then 2.5 parts of 2-oxoglutaric acid is uniformly dissolved in 60 parts of glycerol to obtain solution B; then 10 parts of polylactic acid-glycolic acid copolymer acid is uniformly dissolved in 100 parts of ethyl acetate to obtain an organic solution; solution A and solution B are uniformly mixed in a mass ratio of 1:3, and then added dropwise into the organic solution in a drop-by-drop manner to form a primary emulsion, then the primary emulsion is added into a vortex instrument container for emulsification treatment, the vortex time is 30 s, the vortex instrument rotation speed is 2000 rpm, then ultrasonic treatment is performed using an ultrasonic processor, the ultrasonic conditions are 10 s on / off cycle each time, the ultrasonic amplitude is 60%, the frequency is 20 kHz, the ultrasonic treatment cycle number is set to 3 times, after ultrasonic treatment, 50 parts of 4 wt% PVA aqueous solution is added into 100 parts of the ultrasonic-treated primary emulsion, and vortex and ultrasonic treatment are continued to form a double emulsion, the vortex time is 5 min, the ultrasonic treatment cycle number is 2 times, the treated double emulsion is added into a magnetic stirrer, the stirring speed is set to 500 rpm, the stirring temperature is 30°C, the stirring time is 240 min, and the solid nanoparticles are obtained after stirring; then 10 parts of the solid nanoparticles and 100 parts of deionized water are uniformly mixed, centrifuged at 10000 rpm for 180 s, and then the upper layer of the centrifuged suspension is dried at room temperature under vacuum, the vacuum condition is 40 Pa, the drying time is 6 h, and the copolymer acid-encapsulated 2-oxoglutaric acid is obtained after complete drying.

[0046] The vitamin composition in this embodiment is prepared by mixing vitamin D, vitamin B1 and vitamin B2 in a mass ratio of 2.0:1:1.

[0047] The preparation method of the polylactic acid-glycolic acid copolymer acid of the present embodiment is as follows: 3.0 parts of glycolic acid, 12.9 parts of lactic acid and 3.0 parts of deionized water are stirred at 100°C for 90 min, then the temperature is lowered to 60°C, 0.10 parts of zinc lactate is added and stirred uniformly, then the temperature is raised to 160°C, and stirred under vacuum for 14 h. During the reaction, the by-products are continuously removed through a Dean-Stark device. After the reaction is completed, the temperature is lowered to 80°C, and then dried under vacuum for 12 h to obtain the copolymer crude product. Then the copolymer crude product is dissolved in 60 parts of ethanol to form a mixed solution, which is then added dropwise into a mixed solution of 200 parts of methanol and water in a volume ratio of 1:3. After the addition is completed, the mixed solution is separated by decantation and centrifugation to remove the residual solvent and retain the precipitate. Then the obtained precipitate is dried under vacuum at 60°C for 18 h, and then freeze-dried at -20°C for 36 h, to finally obtain the polylactic acid-glycolic acid copolymer.

[0048] The molecular weight of the polylactic acid-glycolic acid copolymer acid of the present embodiment is 15 kDa; and the average diameter of the 2-oxoglutaric acid wrapped by the copolymer acid is 100 nm.

[0049] The biorepairing promoter of the present embodiment is hyaluronic acid; the osmotic pressure regulator is mannitol; and the pH buffer is citrate buffer, and the pH value of the 2-oxoglutaric acid composition is 6.8, and the osmotic pressure is 280 mOsm / L.

[0050] The preparation method of the 2-oxoglutaric acid-based composition of the present embodiment specifically comprises the following steps: according to the weight fraction, the copolymer acid wrapped 2-oxoglutaric acid, polyethylene glycol 400 and β-cyclodextrin are weighed and added into deionized water, and stirred at 400 rpm at 20°C for 30 min; then the pH buffer is added and stirred for 10 min; then the biorepairing promoter and the osmotic pressure regulator are added and stirred uniformly at 500 rpm at 25°C for 30 min; then the mixture is centrifuged at 3000 rpm for 15 min, and the supernatant is taken and filtered through a 0.22 μm filter membrane to obtain the filtrate as the 2-oxoglutaric acid-based composition.

[0051] Figure 1 The infrared Fourier spectrum of the polylactic acid-glycolic acid copolymer prepared in Example 1 is shown, in which the absorption bands of CH bond stretching vibration are observed at 2949, 2887, 2824 cm -1 , which correspond to the characteristic of asymmetric group νCH2and δCH2. In addition, the absorption peaks attributed to the bidentate asymmetric and symmetric modes of COO-are recorded at 1622 and 1408 cm -1 , while the absorption peak of the carbonyl group is recorded at 1747 cm -1the most important band of the spectrum, belonging to the ester carbonyl group (vC=0 stretching vibration) in the copolyester. At the same time, two strong absorption bands belonging to the C-O-C ester group are recorded at 1180 and 1086 cm -1 , while the vCH3 and vCH2 absorption bands of the lactic and glycolic acid units are recorded at 2997 and 2949 cm -1 , respectively. In addition, the asymmetric and symmetric deformation vibrations δasCH3 and δsCH3 are present at 1452 and 1383 cm -1 , respectively, while the stretching vibration δCH is recorded at 1267 cm -1 . Figure 2 The morphology of the 2-oxoglutaric acid-coated nanoparticles of the copolymer prepared in Example 1 can be seen as nanoparticles with a uniform average particle size.

[0052] To evaluate the effect of the 2-oxoglutaric acid-based composition on cell viability, an MTT experiment was performed on the 2-oxoglutaric acid-based composition of the present example, according to the following method and results. First, BALB / 3T3 cells (mouse embryonic fibroblasts) and HaCaT cells (human epithelial keratinocytes) were selected as experimental cells and cultured at 37°C, 5% CO2, and the cell density and state were observed daily by inverted microscope. When the cells grew to 80-90% of the culture bottle, the cells were subcultured or inoculated. Subsequently, the cells were seeded in a 96-well plate at a density of 3x10 3 cells / well using complete culture medium, four replicate wells were set, and cultured at 37°C, 5% CO2 for 24 hours, and then replaced with maintenance medium for starvation treatment for 24 hours to eliminate the influence of growth factors in the culture medium on the experimental results. Then, the maintenance medium after starvation treatment was discarded, and the maintenance medium containing different concentrations of the 2-oxoglutaric acid-based composition was added, with a final concentration of 0.01, 0.1, 1, 5, 10, 20, 50, 100, 200, 500, 1000 μM, respectively. The blank control group was only added with the maintenance medium without 2-oxoglutaric acid. Then, the cells were further cultured at 37°C, 5% CO2 for 48 hours. To detect cell viability, 10 μL of MTT solution was added to each well after 48 hours of culture, and the cells were further cultured under the same conditions for 4 hours. Then, the supernatant (including the maintenance medium and MTT solution) in the wells was carefully aspirated, 100 μL of DMSO was added to each well, and the mixture was thoroughly mixed by blowing. The absorbance (OD value) was detected at 570 nm wavelength using a microplate reader to reflect the proliferation activity of the cells. The experimental results showed (as shown in Figure 1) that the 2-oxoglutaric acid-based composition of the present example had no significant effect on the cell viability of BALB / 3T3 cells and HaCaT cells, and the OD values of the experimental groups were similar to those of the blank control group, indicating that the 2-oxoglutaric acid-based composition of the present example had no significant effect on the cell viability of BALB / 3T3 cells and HaCaT cells. Figures 3-4As shown in the figure, the 2-oxoglutaric acid-based composition had no significant effect on the viability of fibroblasts (BALB / 3T3) and skin epithelial cells (HaCaT) at low to moderate concentrations (≤500 μM), and only at high concentrations (500 μM and 1000 μM) did the cell viability decrease. This indicates that the 2-oxoglutaric acid-based composition has good biological safety for cells and skin tissues at a regular use dose (less than 500 μM), and its safety and stability are embodied in Example 1. Therefore, the composition is suitable for application as an active ingredient in tissue repair products or skin care products. Example 2

[0053] A 2-oxoglutaric acid-based composition, comprising the following raw materials in parts by weight: 2-oxoglutaric acid coated with copolymer acid 13 parts, nicotinamide 0.5 parts, γ-aminobutyric acid 0.4 parts, superoxide dismutase 0.2 parts, polyethylene glycol 400 3 parts, pH buffer 3.1 parts, biological repair promoter 4.9 parts, osmotic pressure regulator 1.7 parts, β-cyclodextrin 3.2 parts, deionized water 56 parts, pyrroloquinoline quinone disodium salt 0.04 parts, catechin 0.4 parts, L-carnitine 0.8 parts, 1, 6-fructose diphosphate trisodium salt 1.1 parts, vitamin B12 0.008 parts; 2-oxoglutaric acid coated with copolymer acid is a polylactic acid-glycolic acid copolymer acid coated with 2-oxoglutaric acid and vitamin composition prepared by double emulsification-solvent evaporation method; the polylactic acid-glycolic acid copolymer acid is obtained by polymerization reaction of glycolic acid and lactic acid;

[0054] The preparation method of the copolymer acid-encapsulated 2-oxoglutaric acid in the embodiment is as follows: 0.8 parts of Tween 80 is first dissolved in 25 parts of a 2 wt% citric acid aqueous solution, 2 parts of a vitamin composition is then added, 50 parts of a 2.0 wt% polyvinyl alcohol aqueous solution is then added, and the mixture is uniformly mixed to obtain an A solution; 3.0 parts of 2-oxoglutaric acid is then uniformly dissolved in 68 parts of glycerol to obtain a B solution; 13 parts of polylactic acid-glycolic acid copolymer acid is then uniformly dissolved in 130 parts of ethyl acetate to obtain an organic solution; the A solution and the B solution are uniformly mixed at a mass ratio of 1:3, and the mixture is then added dropwise into the organic solution in a drop-by-drop manner to form a primary emulsion; the primary emulsion is then added into a vortex instrument container for emulsification treatment of the primary emulsion, the vortex time is 39 s, and the vortex instrument rotation speed is 2300 rpm; an ultrasonic processor is then used for ultrasonic treatment, the ultrasonic conditions are 10 s on / off cycles each time, the ultrasonic amplitude is 63%, the frequency is 21 kHz, the ultrasonic treatment cycle number is set to 4 times, and after the ultrasonic treatment, 50 parts of a 4 wt% PVA aqueous solution is added into 100 parts of the primary emulsion after the ultrasonic treatment, and the mixture is subjected to vortex and ultrasonic treatment to form a double emulsion, the vortex time is 7 min, the ultrasonic treatment cycle number is 3 times, the processed double emulsion is added into a magnetic stirrer, the stirring speed is set to 650 rpm, the stirring temperature is 33℃, the stirring time is 264 min, and after the stirring, solid nanoparticles are obtained; 12 parts of the solid nanoparticles and 100 parts of deionized water are then uniformly mixed, and the mixture is centrifuged at 11500 rpm for 234 s, the upper layer of the centrifuged suspension is then dried at room temperature under vacuum, the vacuum condition is 58 Pa, the drying time is 8 h, and after the drying, copolymer acid-encapsulated 2-oxoglutaric acid is obtained.

[0055] The vitamin composition is prepared by mixing vitamin D, vitamin B1 and vitamin B2 at a mass ratio of 3:1:1.

[0056] The preparation method of the polylactic acid-glycolic acid copolymer acid of the embodiment is as follows: 3.9 parts of glycolic acid, 16.8 parts of lactic acid and 3.8 parts of deionized water are stirred at 106°C for 105 min, then the temperature is lowered to 63°C, 0.15 parts of zinc lactate is added and stirred uniformly, then the temperature is raised to 163°C, and stirring is carried out under vacuum for 15 h, during which the by-products are continuously removed through a Dean-Stark device, after the reaction is completed, the temperature is lowered to 83°C, and then the mixture is dried under vacuum for 14 h to obtain a copolymer crude product, then the copolymer crude product is dissolved in 66 parts of ethanol to form a mixed solution, then the mixed solution is added dropwise into a mixed solution of 200 parts of methanol and water in a drop-by-drop manner, wherein the volume ratio of methanol to water is 1:3, after the dropwise addition is completed, the mixed solution is separated by decantation and centrifugation to remove residual solvents and retain the precipitate, then the obtained precipitate is dried under vacuum at 63°C for 20 h, and then freeze-dried at -20°C for 40 h, and finally the polylactic acid-glycolic acid copolymer is obtained.

[0057] The molecular weight of the polylactic acid-glycolic acid copolymer acid of the embodiment is 17 kDa; and the average diameter of the 2-oxoglutaric acid wrapped by the copolymer acid is 220 nm.

[0058] The bioremediation promoter of the embodiment is hyaluronic acid or chitosan; the osmotic pressure regulator is mannitol or sodium chloride; the pH buffer is a mixture of phosphate buffer and citrate buffer in a mass ratio of 1:1, the pH value of the 2-oxoglutaric acid-based composition is 6.9, and the osmotic pressure is 292 mOsm / L.

[0059] The preparation method of the 2-oxoglutaric acid-based composition of the embodiment specifically comprises the following steps: according to the weight fraction, the copolymer acid wrapped 2-oxoglutaric acid, polyethylene glycol 400 and β-cyclodextrin are weighed and added into deionized water, and stirred at a speed of 460 rpm for 39 min at 23°C; then the pH buffer is added and stirred for 13 min; then the bioremediation promoter and the osmotic pressure regulator are added and stirred uniformly at a speed of 590 rpm for 39 min at 28°C; then the mixture is centrifuged at 3600 rpm for 20 min, and the supernatant is taken and filtered through a 0.22 μm filter membrane to obtain the filtrate as the 2-oxoglutaric acid-based composition.

[0060] In order to explore the effect of the 2-oxoglutaric acid-based composition on collagen production in different cells, a WB experiment was conducted, and BALB / 3T3 cells (mouse embryonic fibroblasts) and HaCaT cells (human epithelial keratinocytes) were selected as research objects. In the experiment, the cells were cultured in a 37°C, 5% CO2 cell incubator, and when the cell density reached 80%-90%, the cells were passaged or inoculated, and then the cells were inoculated at a concentration of 3×10 5Cells were seeded at a density of cells / well in 6-well plates and cultured for 24 hours. After discarding the culture medium, maintenance medium containing different concentrations (0.01, 0.1, 1, 10, 50, 100, 200 μM) of the 2-oxoglutaric acid-based composition was added. The blank control group only received maintenance medium. After culturing for another 48 hours, total protein was extracted from the cells, and protein concentration was detected by the BCA method. Western blotting was then performed. After electrophoresis, the cells were transferred to membranes, blocked with 5% skim milk, and incubated with rabbit polyclonal antibodies (1:1000 dilution) targeting type I, III, and XVII collagen, and HRP-labeled goat anti-rabbit IgG secondary antibody (1:1000 dilution). The primary and secondary antibody information used in this example is shown in Table 1 below. The internal control protein was β-actin (42 kDa). After development, grayscale analysis was performed using ImageJ software. The experimental results show that the 2-oxoglutaric acid-based composition can significantly promote the production of various collagens in different cells. Specific results are as follows: Figure 4 The composition based on 2-oxoglutarate at concentrations of 1 μM and above significantly increased the production of type I collagen (COL1A1) in 3T3 cells, reaching the highest levels at concentrations of 10 μM and 50 μM, and then slightly decreased with further increases in concentration but still significantly higher than the control group. Figure 5 The images shown are Western blotting images and grayscale analysis results illustrating the effect of AKG on type I collagen production in HaCaT cells in Example 2. Figure 6 As stated, in 3T3 cells, concentrations of 1 μM and above of the 2-oxoglutaric acid-based composition significantly increased the production of type III collagen (COL3A1), showing a dose-dependent trend consistent with type I collagen. Figure 7 As stated, in HaCaT cells, concentrations of 1 μM and above of the 2-oxoglutaric acid-based composition significantly promoted the production of type I collagen, peaking at 10 μM and then slightly decreasing at higher concentrations; Figure 7 The composition based on 2-oxoglutarate significantly promoted the production of type XVII collagen in HaCaT cells, with the highest production levels observed at 10 μM and 50 μM, further demonstrating its promoting effect on specific collagen in epithelial cells; Figure 9 As stated, type XVII collagen expression was not detected in 3T3 cells, which is consistent with the characteristic that type XVII collagen is mainly expressed in epithelial cells; Figure 10The expression of collagen type III was not detected in HaCaT cells, which is consistent with the characteristic that collagen type III is mainly expressed in connective tissue. Based on the above results, the 2-oxoglutaric acid-based composition can significantly promote the production of collagen type I and III in fibroblasts, and significantly promote the production of collagen type I and XVII in epithelial cells, showing a significant collagen production-promoting potential, verifying its role in regulating collagen synthesis in extracellular matrix, and providing an important theoretical basis for its application in the field of anti-aging and tissue repair.

[0061] Table 1 Information parameters of primary antibody and secondary antibody

[0062] Example 3

[0063] A 2-oxoglutaric acid-based composition includes the following raw materials in parts by weight: 16 parts of 2-oxoglutaric acid coated with copolymer acid, 1 part of nicotinamide, 0.8 parts of gamma-aminobutyric acid, 0.4 parts of superoxide dismutase, 4.4 parts of polyethylene glycol 400, 3.7 parts of pH buffer, 6.2 parts of biological repair promoter, 2.9 parts of osmotic pressure regulator, 4.4 parts of beta-cyclodextrin, 62 parts of deionized water, 0.06 parts of pyrroloquinoline quinone disodium salt, 0.5 parts of catechin, 1.2 parts of L-carnitine, 1.5 parts of 1, 6-fructose diphosphate trisodium salt, and 0.012 parts of vitamin B12; the 2-oxoglutaric acid coated with copolymer acid is a polylactic acid-glycolic acid copolymer acid coated with 2-oxoglutaric acid and vitamin composition prepared by double emulsification-solvent evaporation method; the polylactic acid-glycolic acid copolymer acid is obtained by polymerization reaction of glycolic acid and lactic acid;

[0064] The preparation method of the copolymer acid-encapsulated 2-oxoglutaric acid in this embodiment is as follows: first, 1.2 parts of Tween 80 is dissolved in 30 parts of 2wt% citric acid aqueous solution, then 3.5 parts of vitamin composition is added, followed by 45-65 parts of 2.0wt% polyvinyl alcohol aqueous solution, and then A solution is obtained after mixing uniformly. Then, 4.3 parts of 2-oxoglutaric acid is uniformly dissolved in 75 parts of glycerol to obtain B solution. Then, 16 parts of polylactic acid-glycolic acid copolymer acid is uniformly dissolved in 160 parts of ethyl acetate to obtain an organic solution. After mixing A solution and B solution uniformly at a mass ratio of 1:3, the primary emulsion is formed by adding the mixture into the organic solution drop by drop. Then, the primary emulsion is added into a vortex instrument container for emulsification treatment, the vortex time is 48s, and the vortex instrument rotation speed is 2600 rpm. Then, the ultrasonic processor is used for ultrasonic treatment, the ultrasonic conditions are 10s on / off cycle each time, the ultrasonic amplitude is 66%, the frequency is 22.4 kHz, the ultrasonic treatment cycle number is set to 4 times, and after the ultrasonic treatment, 50 parts of 4wt% PVA aqueous solution is added into 100 parts of the primary emulsion after ultrasonic treatment, and the double emulsion is formed by further vortexing and ultrasonic treatment, the vortex time is 8min, and the ultrasonic treatment cycle number is 3 times. The treated double emulsion is added into a magnetic stirrer, the stirring speed is set to 800 rpm, the stirring temperature is 36℃, the stirring time is 288min, and then the solid nanoparticles are obtained after stirring. Then, 15 parts of the solid nanoparticles and 100 parts of deionized water are mixed uniformly, and then centrifuged at 13000 rpm for 288s, and then the upper layer of the suspension after centrifugation is dried at room temperature under vacuum, the vacuum condition is 76 Pa, the drying time is 9.6h, and then the copolymer acid-encapsulated 2-oxoglutaric acid is obtained after drying completely.

[0065] The vitamin composition in this embodiment is prepared by mixing vitamin D, vitamin B1 and vitamin B2 at a mass ratio of 4:1:1.

[0066] The preparation method of the polylactic acid-glycolic acid copolymer acid of the embodiment is as follows: 4.8 parts of glycolic acid, 20.6 parts of lactic acid and 4.5 parts of deionized water are stirred at 112°C for 120 min, then the temperature is lowered to 66°C, 0.19 parts of zinc lactate is added and stirred uniformly, then the temperature is raised to 166°C, and stirring is carried out under vacuum for 16.4 h, during which the by-products are continuously removed through a Dean-Stark device; after the reaction is completed, the temperature is lowered to 86°C, and then the mixture is dried under vacuum for 17 h to obtain a copolymer crude product; then the copolymer crude product is dissolved in 72 parts of ethanol to form a mixed solution, which is then added dropwise into a mixed solution of 200 parts of methanol and water in a volume ratio of 1:3; after the addition is completed, the mixed solution is separated by decantation and centrifugation to remove residual solvents and retain the precipitate; then the obtained precipitate is dried under vacuum at 66°C for 21.6 h, and then freeze-dried at -20°C for 43.2 h, and finally the polylactic acid-glycolic acid copolymer is obtained.

[0067] The molecular weight of the polylactic acid-glycolic acid copolymer acid of the embodiment is 18 kDa; and the average diameter of the 2-oxoglutaric acid wrapped by the copolymer acid is 340 nm.

[0068] The bioremediation promoter of the embodiment is hyaluronic acid or chitosan; the osmotic pressure regulator is mannitol or sodium chloride; the pH buffer is a mixture of phosphate buffer and citrate buffer in a mass ratio of 3:1, and the pH value of the 2-oxoglutaric acid-based composition is 7.0, and the osmotic pressure is 304 mOsm / L.

[0069] The preparation method of the 2-oxoglutaric acid-based composition of the embodiment specifically comprises the following steps: according to the weight fraction, the copolymer acid wrapped 2-oxoglutaric acid, polyethylene glycol 400 and β-cyclodextrin are weighed and added into deionized water, and stirred at a speed of 520 rpm at 26°C for 48 min; then the pH buffer is added and stirred for 16 min; then the bioremediation promoter and the osmotic pressure regulator are added and stirred uniformly at a speed of 680 rpm at 31°C for 48 min; then the mixture is centrifuged at 4200 rpm for 24 min, and the supernatant is taken and filtered through a 0.22 μm filter membrane to obtain the filtrate as the 2-oxoglutaric acid-based composition.

[0070] D-galactose is a classic reagent for inducing cells to produce a large number of free radicals and causing subacute aging, through which the antioxidant capacity can be weakened and the lipid level can be increased, and it is widely used in the construction of a model for studying aging. This embodiment uses D-galactose to construct a model of aging of BALB / 3T3 cells (mouse embryonic fibroblasts). First, the cells are cultured at 37°C, 5% CO2, and the density and state of the cells are observed daily, and when the cells grow to 80-90% of the culture bottle, they are passaged or inoculated. Subsequently, the cell viability is detected by an MTT experiment, 3x10 3 cells / well are inoculated in a 96-well plate, and after 24 hours of culture, D-galactose is added at a final concentration of 10, 20, 30 mg / mL, respectively, and a control group is set up by adding only a maintenance medium, and after 24 hours of continued culture, the cell viability is detected according to the standard method. Next, the aging state of the cells is further verified by a β-galactosidase staining experiment, 3x10 5 cells / well are inoculated in a 6-well plate, and after 24 hours of culture with the same concentration of D-galactose, the cells are stained using a cell aging β-galactosidase staining kit, and the staining results are observed under a general optical microscope. In addition, the protein expression changes of telomerase reverse transcriptase (TERT) and the internal reference actin (β-actin) are detected by a WB experiment, 5x10 5 cells / well are inoculated in a 6-well plate, and after 24 hours of stimulation with D-galactose, the proteins are extracted, and rabbit polyclonal antibodies (TERT) and HRP-goat anti-rabbit IgG secondary antibodies are used for detection, and Table 2 shows the information of the primary and secondary antibodies used in Example 3. The experimental results show that, as shown in Figure 11 , D-galactose significantly reduces the cell viability after 24 hours of stimulation, which is consistent with the characteristics of aging cells, and a D-galactose-induced cell aging model is successfully constructed.

[0071] Table 2 Information of primary and secondary antibodies used in Example 3

[0072]

[0073] One of the classic markers of aging cells is the expression of β-galactosidase, and its activity can be detected by a β-galactosidase staining experiment. In the detection process, the substrate X-Gal in the kit is hydrolyzed to form an insoluble deep blue product under the catalysis of β-galactosidase, so that the cells showing blue staining are galactosidase-positive cells, which are usually considered to be aging cells. The experimental results show that, as shown in Figure 12As shown, with the gradual increase of D-galactose concentration, the number of blue-stained cells increased significantly, indicating that D-galactose can effectively induce cells into the aging state. Another classic marker of senescent cells is the decrease in the expression level of telomerase reverse transcriptase (TERT). As the catalytic subunit of telomerase, the expression level of TERT directly determines the activity of telomerase, and the decrease in TERT expression will lead to the loss of telomerase activity, thereby failing to maintain telomere length. With cell division, the telomere gradually shortens, and when the telomere shortens to a certain extent, the cell enters the aging stage. As shown in FIG. 4, with the increase of D-galactose concentration, the expression level of TERT protein gradually decreased, further verifying the mechanism of D-galactose-induced cell senescence. Figure 13 As shown in FIG. 4, with the increase of D-galactose concentration, the expression level of TERT protein gradually decreased, further verifying the mechanism of D-galactose-induced cell senescence. Example 4

[0074] A 2-oxoglutaric acid-based composition includes the following raw materials in parts by weight: 20 parts of 2-oxoglutaric acid coated with a copolymer acid, 1.5 parts of nicotinamide, 1.0 parts of gamma-aminobutyric acid, 0.5 parts of superoxide dismutase, 6 parts of polyethylene glycol 400, 4.5 parts of pH buffer, 8.0 parts of biological repair promoter, 4.5 parts of osmotic pressure regulator, 6.0 parts of beta-cyclodextrin, 70 parts of deionized water, 0.1 parts of pyrroloquinoline quinone disodium salt, 0.8 parts of catechin, 2.0 parts of L-carnitine, 2.5 parts of 1, 6-fructose diphosphate trisodium salt, and 0.02 parts of vitamin B12; the 2-oxoglutaric acid coated with a copolymer acid is a polylactic acid-glycolic acid copolymer acid coated with 2-oxoglutaric acid and a vitamin composition prepared by double-emulsification-solvent evaporation method; the polylactic acid-glycolic acid copolymer acid is obtained by polymerization reaction of glycolic acid and lactic acid;

[0075] The preparation method of the copolymer acid-encapsulated 2-oxoglutaric acid in the embodiment is as follows: first, 1.5 parts of Tween 80 is dissolved in 40 parts of 2 wt% citric acid aqueous solution, then 4.5 parts of the vitamin composition is added, followed by 45-65 parts of 2.0 wt% polyvinyl alcohol aqueous solution, and then A solution is obtained after mixing uniformly; then 4.5 parts of 2-oxoglutaric acid is uniformly dissolved in 85 parts of glycerol to obtain B solution; then 20 parts of polylactic acid-glycolic acid copolymer acid is uniformly dissolved in 200 parts of ethyl acetate to obtain an organic solution; A solution and B solution are mixed uniformly at a mass ratio of 1:3, and then added dropwise into the organic solution in a drop-by-drop manner to form a primary emulsion; then the primary emulsion is added into a vortex instrument container for emulsification treatment of the primary emulsion, the vortex time is 60 s, and the vortex instrument rotation speed is 3000 rpm; then ultrasonic treatment is performed using an ultrasonic processor, the ultrasonic conditions are 10 s on / off cycle each time, the ultrasonic amplitude is 70%, the frequency is 24 kHz, the ultrasonic treatment cycle number is set to 5 times, and after the ultrasonic treatment, 50 parts of 4 wt% PVA aqueous solution is added into 100 parts of the primary emulsion after ultrasonic treatment, and vortex and ultrasonic treatment are continued to form a double emulsion, the vortex time is 10 min, the ultrasonic treatment cycle number is 4 times, the treated double emulsion is added into a magnetic stirrer, the stirring speed is set to 1000 rpm, the stirring temperature is 40℃, the stirring time is 320 min, and then solid nanoparticles are obtained after stirring; then 18 parts of the solid nanoparticles and 100 parts of deionized water are mixed uniformly, centrifuged at 15000 rpm for 360 s, and then the upper layer of the suspension after centrifugation is dried at room temperature under vacuum, the vacuum condition is 100 Pa, the drying time is 12 h, and then copolymer acid-encapsulated 2-oxoglutaric acid is obtained after complete drying.

[0076] The vitamin composition is prepared by mixing vitamin D, vitamin B1 and vitamin B2 at a mass ratio of 4.5:1:1.

[0077] The preparation method of the polylactic acid-glycolic acid copolymer acid of the embodiment is as follows: 6.0 parts of glycolic acid, 25.8 parts of lactic acid and 5.5 parts of deionized water are stirred at 120℃ for 140 min, then the temperature is lowered to 70℃, 0.25 parts of zinc lactate is added and stirred uniformly, then the temperature is raised to 170℃, and stirring is carried out under vacuum for 18 h. During the reaction, the by-products are continuously removed through a Dean-Stark device. After the reaction is completed, the temperature is lowered to 90℃, and then the mixture is dried under vacuum for 20 h to obtain a copolymer crude product. Then the copolymer crude product is dissolved in 80 parts of ethanol to form a mixed solution. The mixed solution is added dropwise into a mixed solution of 200 parts of methanol and water in a drop-by-drop manner, wherein the volume ratio of methanol to water is 1:3. After the dropwise addition is completed, the mixed solution is separated by decantation and centrifugation to remove residual solvents and retain the precipitate. Then the obtained precipitate is dried under vacuum at 70℃ for 24 h, and then freeze-dried at -20℃ for 48 h. Finally, the polylactic acid-glycolic acid copolymer is obtained.

[0078] The molecular weight of the polylactic acid-glycolic acid copolymer acid of the embodiment is 20 kDa; and the average diameter of the 2-oxoglutaric acid wrapped by the copolymer acid is 500 nm.

[0079] The bioremediation promoter of the embodiment is hyaluronic acid; the osmotic pressure regulator is mannitol; the pH buffer is phosphate buffer, and the pH value of the 2-oxoglutaric acid-based composition is 7.2, and the osmotic pressure is 320 mOsm / L.

[0080] The preparation method of the 2-oxoglutaric acid-based composition of the embodiment specifically comprises the following steps: according to the weight fraction, the copolymer acid wrapped 2-oxoglutaric acid, polyethylene glycol 400 and β-cyclodextrin are weighed and added into deionized water, and stirred at 30℃ at a speed of 600 rpm for 60 min; then the pH buffer is added and stirred for 20 min; then the bioremediation promoter and the osmotic pressure regulator are added, and stirred uniformly at 35℃ at a speed of 800 rpm for 60 min; then the mixture is centrifuged at 5000 rpm for 30 min, and the supernatant is taken and filtered through a 0.22μm filter membrane to obtain the filtrate as the 2-oxoglutaric acid-based composition.

[0081] The anti-aging activity of alpha-ketoglutaric acid (AKG) was verified by using the D-galactose-induced cell aging model constructed in Example 3. First, the BALB / 3T3 cells were subcultured to 80-90% confluence and inoculated in 96-well plates or 6-well plates according to the method in Example 3, and treated with 30 mg / mL D-galactose for 24 hours to construct the aging cell model, and the control group was only added with the maintenance medium. Subsequently, the culture solution was discarded, and different concentrations (1, 2, 3, and 4 mM) of AKG were added for stimulation, and the control group was still only added with the maintenance medium, and after 24 hours of continuous culture, MTT experiment, β-galactosidase staining experiment, and WB experiment were performed to detect the related indexes. The experimental results showed that, as shown in Figure 14 , D-galactose treatment led to a significant decrease in cell viability, while AKG could significantly improve the viability of aging cells, and the effect was optimal at a concentration of 2 mM, and the viability improvement was no longer significant beyond this concentration. Therefore, 2 mM AKG was used for subsequent staining experiments and WB experiments. The β-galactosidase staining results Figure 15 showed that the number of blue-stained cells increased significantly after D-galactose treatment, while the number of blue-stained cells decreased significantly after 2 mM AKG stimulation, and there was no significant difference compared with the control group, indicating that AKG could effectively reduce the number of aging cells. The WB experiment results Figure 16 further verified the anti-aging effect of AKG, and D-galactose treatment led to a significant decrease in TERT protein level, while 2 mM AKG stimulation led to a significant up-regulation of TERT protein level. In summary, AKG exhibited significant anti-aging activity by improving cell viability, reducing the number of aging cells, and restoring TERT protein level.

[0082] Comparative Example 1

[0083] The same as Example 1, except that the 2-oxoglutaric acid was not subjected to coating treatment, but the 2-oxoglutaric acid, polylactic acid-glycolic acid copolymer acid, and vitamin composition were added in the form of separate addition.

[0084] In this comparative example, compared with Example 1, the 2-oxoglutaric acid was not subjected to coating treatment, but was added in the form of separate addition with the polylactic acid-glycolic acid copolymer acid and the vitamin composition in the composition. Due to the lack of coating protection, the stability of 2-oxoglutaric acid in solution was significantly reduced, and it was easy to be oxidized or degraded, leading to a decrease in biological effectiveness. At the same time, the uncoated form cannot achieve sustained release, and the peak of drug efficacy decays rapidly, making it difficult to continuously exert the effect. In addition, the advantages of synergistic protection and stability provided by the coating technology are weakened, and the overall effect is reduced. The experimental results showed that, compared with Example 1, the collagen production in this comparative example was significantly reduced, and the effect was unstable.

[0085] Comparative Example 2

[0086] The same as Example 1, except that no chitosan was added to the copolymer acid-encapsulated 2-oxoglutaric acid.

[0087] The present comparative example is substantially the same as Example 1, but no chitosan was added to the copolymer acid-encapsulated 2-oxoglutaric acid nanoparticles. The stability and functionality of the copolymer acid-encapsulated 2-oxoglutaric acid nanoparticles were significantly reduced. Specifically, the particle size of the nanoparticles increased, the dispersibility deteriorated, and both the encapsulation efficiency and drug release performance were reduced. This indicates that chitosan plays an important role in the formation and stabilization of the nanoparticles, and its addition can effectively improve the structure and performance of the nanoparticles. Specifically, the promotion of collagen synthesis and secretion was significantly lower than that of Example 1. The absence of chitosan can lead to a decrease in the structural stability of the nanoparticles, thereby affecting the release efficiency of 2-oxoglutaric acid and its regulatory effect on the collagen metabolism-related pathway. This indicates that chitosan not only plays a key role in the stability of the nanoparticles, but also makes an important contribution to their biological activity, especially the enhancement of collagen production.

[0088] Comparative Example 3

[0089] The same as Example 1, except that no bio-repair promoter was added to the composition.

[0090] In the present comparative example, no bio-repair promoter (such as hyaluronic acid or chitosan) was added to the composition compared to Example 1. Bio-repair promoters play an important role in promoting extracellular matrix repair, maintaining cell activity, and providing a suitable microenvironment, and their absence can significantly weaken the collagen production-promoting ability and tissue repair effect of the composition. In particular, the moisturizing effect of hyaluronic acid helps to maintain cell activity, while chitosan can support the reconstruction of the extracellular matrix. The experimental results show that the amount of collagen produced in the present comparative example was significantly reduced compared to Example 1, and the cell activity and tissue repair ability also decreased.

[0091] Comparative Example 4

[0092] The same as Example 1, except that no osmotic pressure regulator was added to the composition.

[0093] The main difference between the present comparative example and Example 1 is that no osmotic pressure regulator (such as mannitol or sodium chloride) is added to the composition. The osmotic pressure regulator is used to maintain the osmotic pressure balance of the composition, so that it is close to the physiological range (280-320 mOsm / L). Without the addition of the osmotic pressure regulator, the osmotic pressure of the composition may deviate from the physiological range, causing the cells to swell or shrink, disrupting the normal growth environment of the cells, and thus affecting the absorption efficiency of the active ingredients by the cells. The experimental results show that the collagen production of the present comparative example is significantly lower than that of Example 1, and the cell activity and tissue repair function are significantly weakened.

[0094] Comparative Example 5

[0095] The composition is substantially the same as in Example 1, except that no pH buffer is added to the composition.

[0096] In the present comparative example, compared with Example 1, no pH buffer (such as phosphate buffer or citrate buffer) is added to the composition. The role of the pH buffer is to maintain the pH value of the composition in the physiological range (6.8-7.2) to ensure the stability of the 2-oxoglutaric acid and vitamin composition. Without the addition of the pH buffer, the pH value of the composition may fluctuate, causing the degradation of the active ingredients to accelerate, and at the same time affecting the acid-base balance of the cell growth environment. The experimental results show that the collagen production in the present comparative example is lower than that in Example 1, and the collagen-promoting effect is significantly weakened, especially during long-term use.

[0097] In summary, through comparative analysis of each comparative example and Example 1, it can be clearly seen that the synergistic effect of the coating treatment of 2-oxoglutaric acid, chitosan, the biological repair promoter, the osmotic pressure regulator, and the pH buffer is the key to achieving the high efficiency and stability of the composition. The coating treatment significantly improves the stability and slow-release effect of 2-oxoglutaric acid, chitosan plays an important role in antioxidant protection and collagen synthesis promotion, the biological repair promoter effectively promotes extracellular matrix repair and cell activity, and the osmotic pressure regulator and the pH buffer ensure the environmental adaptability and ingredient stability of the composition. The absence of any one of the ingredients will significantly reduce the collagen production and tissue repair capacity, verifying the scientificity and necessity of the formula design in Example 1, and providing a solid theoretical basis and experimental support for subsequent optimization and application.

[0098] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application and not to limit them, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that any equivalent structural transformation made within the concept of the present application, using the contents of the present application specification and drawings, should be covered within the protection scope of the claims of the present application.

Claims

1. A 2-oxoglutaric acid-based composition, characterized in that, The raw materials include the following weight parts: 10-20 parts of copolymer acid-encapsulated 2-oxoglutaric acid, 0.1-1.5 parts of nicotinamide, 0.2-1.0 parts of gamma-aminobutyric acid, 0.1-0.5 parts of superoxide dismutase, 2-6 parts of polyethylene glycol 400, 2.5-4.5 parts of pH buffer, 3.5-8.0 parts of bioremediation promoter, 0.5-4.5 parts of osmotic pressure regulator, 2.0-6.0 parts of beta-cyclodextrin, 50-70 parts of deionized water, 0.02-0.1 parts of pyrroloquinoline quinone disodium salt, 0.2-0.8 parts of catechin, 0.5-2.0 parts of L-carnitine, 0.5-2.5 parts of 1, 6-fructose disodium phosphate, and 0.002-0.02 parts of vitamin B12; The copolymer acid-encapsulated 2-oxoglutaric acid is a polylactic acid-glycolic acid copolymer acid-encapsulated 2-oxoglutaric acid and vitamin composition prepared by double emulsification-solvent evaporation method; The polylactic acid-glycolic acid copolymer acid is obtained by polymerization reaction of glycolic acid and lactic acid; The preparation method of the copolymer acid-wrapped 2-oxoglutaric acid is as follows: first, 0.5-1.5 parts of Tween 80 is dissolved in 20-40 parts of a 2 wt% citric acid aqueous solution, then 1.25-4.5 parts of a vitamin composition is added, followed by the addition of 45-65 parts of a 2.0 wt% polyvinyl alcohol aqueous solution, and the mixture is uniformly mixed to obtain an A solution; then 2.5-4.5 parts of 2-oxoglutaric acid is uniformly dissolved in 60-85 parts of glycerol to obtain a B solution; then 10-20 parts of a polylactic acid-glycolic acid copolymer acid is uniformly dissolved in 100-200 parts of ethyl acetate to obtain an organic solution; the A solution and the B solution are uniformly mixed at a mass ratio of 1:3, and then added dropwise into the organic solution in a drop-by-drop manner to form a primary emulsion; then the primary emulsion is added into a vortex instrument container for emulsification treatment of the primary emulsion, the vortex time is 30-60 s, the vortex instrument rotation speed is 2000-3000 rpm; then an ultrasonic processor is used for ultrasonic treatment, the ultrasonic conditions are as follows: 10 s on / off cycle each time, the ultrasonic amplitude is 60-70%, the frequency is 20-24 kHz, the ultrasonic treatment cycle number is set to 3-5 times; after the ultrasonic treatment, 50 parts of a 4 wt% PVA aqueous solution is added into 100 parts of the ultrasonically treated primary emulsion, and vortexing and ultrasonic treatment are continued to form a double emulsion, the vortexing time is 5-10 min, the ultrasonic treatment cycle number is 2-4 times; the treated double emulsion is added into a magnetic stirrer, the stirring speed is set to 500-1000 rpm, the stirring temperature is 30-40°C, and the stirring time is 240-320 min; after the stirring is completed, solid nanoparticles are obtained; then 10-18 parts of the solid nanoparticles and 100 parts of deionized water are uniformly mixed, and then centrifuged at 10000-15000 rpm for 180-360 s; then the upper layer of the centrifuged suspension is dried at room temperature under vacuum, the vacuum condition is 40-100 Pa, and the drying time is 6-12 h; after the drying is completed, the copolymer acid-wrapped 2-oxoglutaric acid is obtained. The mass ratio of the vitamin composition to 2-oxoglutaric acid is (0.5-1.0):

1. The vitamin composition is a mixture of vitamin D, vitamin B1 and vitamin B2 in a mass ratio of (2.0-4.5):1:

1. The biological repair promoter is hyaluronic acid or chitosan. The osmotic pressure regulator is mannitol or sodium chloride. The pH buffer is a phosphate buffer, a citrate buffer or a mixture of the two.

2. A 2-oxoglutaric acid-based composition according to claim 1, wherein, The preparation method of the polylactic acid-glycolic acid copolymer acid comprises the following steps: 3.0-6.0 parts of glycolic acid, 12.9-25.8 parts of lactic acid and 3.0-5.5 parts of deionized water are stirred at 100-120 ℃ for 90-140 min, then the temperature is lowered to 60-70 ℃, 0.10-0.25 parts of zinc lactate is added and stirred uniformly, then the temperature is raised to 160-170 ℃, and stirring is carried out under vacuum for 14-18 h; during the reaction, the by-products are continuously removed through a Dean-Stark device; after the reaction is completed, the temperature is lowered to 80-90 ℃, and then the copolymer crude product is obtained by drying under vacuum for 12-20 h; then the copolymer crude product is dissolved in 60-80 parts of ethanol to form a mixed solution, and then the mixed solution is added dropwise into a mixed solution of 200 parts of methanol and water in a drop-by-drop manner, wherein the volume ratio of methanol to water is 1:3; after the dropwise addition is completed, the mixed solution is separated by decantation and centrifugation to remove residual solvents and retain the precipitate; then the obtained precipitate is dried under vacuum at 60-70 ℃ for 18-24 h, and then freeze-dried at-20 ℃ for 36-48 h, and finally the polylactic acid-glycolic acid copolymer is obtained.

3. A 2-oxoglutaric acid-based composition according to claim 1, wherein, The molecular weight of the polylactic acid-glycolic acid copolymer acid is 15-20 kDa.

4. A 2-oxoglutaric acid-based composition according to claim 1, wherein, Further, the average diameter of the 2-oxoglutaric acid wrapped by the copolymer acid is 100-500 nm.

5. A 2-oxoglutaric acid-based composition according to claim 1, wherein, The mass ratio of the glycolic acid to the lactic acid is 1:4.

3.

6. A 2-oxoglutaric acid-based composition according to claim 1, wherein, The pH value of the composition is 6.8-7.2, and the osmotic pressure is 280-320 mOsm / L.

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