Anti-aging composition containing nanoscale microspheres, preparation method thereof and application thereof in skin care products

Abstract

This invention belongs to the field of cosmetic technology, specifically relating to an anti-aging composition containing nanospheres and its preparation method, as well as its application in skincare products. The method employs a double emulsion solvent evaporation method to prepare nanospheres. Soybean lecithin and polylactic acid-glycolic acid copolymer are dissolved in an organic solvent as the oil phase. Superoxide dismutase, collagenase, trehalose, and bovine serum albumin are dissolved in phosphate buffer as the inner aqueous phase. After emulsification to form a primary emulsion, it is added to an outer aqueous phase containing polyvinyl alcohol and homogenized to form a double emulsion. After solidification, centrifugation, and washing, the nanospheres are obtained. The resulting composition exhibits good enzyme protection and skin permeability, effectively scavenging free radicals and inhibiting collagen degradation, showing broad application prospects in the skincare field. This invention features a simple process, uses commercially available raw materials, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to an anti-aging composition containing nanosphere enzymes, its preparation method, and its application in skin care products. Background Technology

[0002] Skin aging is a complex physiological process resulting from the combined effects of multiple internal and external factors, with oxidative stress and enzyme activity imbalance being key pathogenic mechanisms. Superoxide dismutase (SOD) and collagenase, as important bioactive enzymes, play a central role in scavenging free radicals and regulating collagen metabolism. However, the application of natural enzymes in skincare products faces numerous challenges, including poor stability, difficulty in transdermal absorption, and susceptibility to inactivation due to environmental factors. Traditional enzyme protection methods mainly employ microencapsulation or liposome encapsulation technologies, but these methods suffer from drawbacks such as low encapsulation efficiency, uncontrollable release, and insufficient biocompatibility of carrier materials. Furthermore, single enzyme preparations are insufficient to simultaneously address multiple aging mechanisms, necessitating the development of multi-functional, compound anti-aging systems.

[0003] While existing nanocarrier technologies have made some progress in enzyme protection, significant shortcomings remain. Polylactic acid-glycolic acid copolymers (PLA), as commonly used biodegradable materials, exhibit good biocompatibility, but their hydrophobic properties lead to low enzyme encapsulation efficiency, and enzymes are prone to denaturation and inactivation during emulsification. Traditional double emulsion preparation processes lack effective enzyme protectants, resulting in unsatisfactory enzyme activity retention in the final product. Furthermore, the limited surface functionalization of existing nanospheres hinders targeted delivery and long-lasting sustained release. Regarding modified polymers, quaternization modification can improve cell penetration, but excessive modification can affect biocompatibility; hyaluronic acid modification can enhance moisturizing effects, but grafting efficiency is low and the structure is unstable. In addition, the introduction of natural active ingredients such as dihydromyricetin and vitamin derivatives is limited in method, making it difficult to form stable chemical bonds with the carrier, leading to easy loss of active ingredients.

[0004] To address the aforementioned technical problems, there is an urgent need to develop an anti-aging composition containing nanospheres and its preparation method, as well as its application in skincare products. An ideal technical solution should achieve the following objectives: nanospheres are prepared using a double emulsion solvent evaporation method; enzyme protectants are added by optimizing the internal aqueous phase formulation to improve enzyme activity retention during preparation; two functionalized modified polymers are designed as the microsphere shell materials: one with a quaternized structure to enhance cell penetration and grafted with hydrophobic cholesterol fragments to improve membrane fusion; the other is based on a hyaluronic acid backbone and incorporates thiol groups, borate esters, and lipid-soluble antioxidants to achieve synergistic effects; the two modified polymers form a stable core-shell structure through a rational chemical bonding method to ensure the sustained release of active ingredients; the entire preparation process is mild, and all raw materials are commercially available, suitable for industrial production. This invention is proposed against this backdrop, aiming to overcome the shortcomings of existing technologies and provide a highly efficient, stable, and safe enzyme-based anti-aging composition. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an anti-aging composition containing nanosphere enzymes, its preparation method, and its application in skincare products.

[0006] A first aspect of the present invention provides a method for preparing an anti-aging composition containing nanospheres, comprising the steps of: S1. By weight, dissolve 3.0-8.0 parts of soybean lecithin and 10.0-20.0 parts of polylactic acid-glycolic acid copolymer in 40-60 parts of dichloromethane to obtain an oil phase; dissolve 0.3-1.0 parts of superoxide dismutase, 0.1-0.5 parts of collagenase, 1.0-3.0 parts of trehalose, and 0.5-1.5 parts of bovine serum albumin in 15-25 parts of phosphate buffer to obtain an inner aqueous phase; emulsify the inner aqueous phase by adding it dropwise to the oil phase at 0-4℃ to obtain a primary emulsion; add the primary emulsion to an aqueous solution containing 2.0-5.0 parts of quaternized galloyl-polylysine-grafted polyethylene glycol-cholesterol derivative and 3.0-8.0 parts of thiolated hyaluronic acid-grafted dihydromyricetin-boronic acid ester-vitamin E in 80-120 parts of polyvinyl alcohol, homogenize, and obtain a secondary emulsion; S2. Stir the double emulsion at room temperature to obtain a solidified nano-suspension; centrifuge the solidified nano-suspension at 3-5℃ and collect the precipitate; wash the precipitate with deionized water to obtain washed nanosphere precipitate; redisperse the washed nanosphere precipitate in deionized water, add 1.0-3.0 parts of 1,2-pentanediol and 0.05-0.2 parts of disodium ethylenediaminetetraacetate, and stir.

[0007] In this invention, the formation of the anti-aging composition containing nanospheres and enzymes is based on a double emulsion solvent evaporation method and an interfacial self-assembly mechanism. Soybean lecithin and polylactic acid-glycolic acid copolymer in the oil phase form a hydrophobic matrix in an organic solvent. Enzymes and protective agents in the inner aqueous phase are encapsulated within the oil phase through low-temperature emulsification. Trehalose and bovine serum albumin form a protective layer at the interface to prevent enzyme denaturation. When the primary emulsion is mixed with the outer aqueous phase, the two functionalized polymers adsorb onto the oil-water interface through hydrophobic interactions and hydrogen bonds. The positive charge of the quaternized polymer and the negative charge of the hyaluronic acid derivative form an electrostatic composite layer, enhancing the stability of the microspheres. Polyvinyl alcohol acts as an emulsion stabilizer, reducing interfacial tension and promoting double emulsion formation. Room temperature stirring evaporates the organic solvent, and the polymer solidifies to form core-shell structured nanospheres, with the enzyme locked in the core and the functional polymer forming the outer shell. Low-temperature centrifugation avoids thermal damage, washing removes residual emulsifiers, and humectants and chelating agents added during redispersibility further stabilize the system. Ultimately, the microspheres penetrate the skin barrier through size effects, the quaternization promotes cellular uptake, the borate ester bonds respond to the skin environment to release active ingredients, and enzymes and antioxidants synergistically scavenge free radicals and regulate collagen metabolism, achieving multi-target anti-aging effects. The entire system ensures the stability and delivery efficiency of active ingredients through a dual mechanism of physical encapsulation and chemical bonding.

[0008] According to a preferred embodiment of the present invention, in step S1, the emulsification time is 3-8 minutes.

[0009] According to a preferred embodiment of the present invention, in step S2, the centrifugation time is 20-40 min.

[0010] According to a preferred embodiment of the present invention, the preparation method of the quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol succinate monoester comprises: A1, adding 8.0-12.0 parts of ε-polylysine and 150-250 parts of anhydrous dimethyl sulfoxide to a reaction vessel, stirring under nitrogen protection, adding 0.2-0.45 parts of triethylamine, and cooling to 0-4°C; dropwise adding 5.0-8.0 parts of gallic acid dissolved in 40-60 parts of anhydrous dimethyl sulfoxide, and simultaneously adding 6.0-10.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbon. An acyldiimide and 3.0-6.0 parts of N-hydroxysuccinimide were reacted at room temperature to obtain a reaction mixture. The reaction mixture was cooled to 5-15°C, and 6.0-10.0 parts of 3-chloro-2-hydroxypropyltrimethylammonium chloride were added. The mixture was reacted at 45-55°C. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain quaternized galloyl-polylysine. Quaternized galloyl-polylysine was dissolved in 100-150 parts of anhydrous N,N-dimethylformamide, and 2.0-4.0 parts of succinic anhydride and 0.2-0.5 parts of 4-dimethylaminopyridine were added. The reaction was carried out at 40-50℃, followed by dialysis and freeze-drying to obtain carboxyl-containing quaternized galloyl-polylysine. 4.0-6.0 parts of cholesterol hemisuccinate were dissolved in 80-120 parts of anhydrous dichloromethane, and 3.0-5.0 parts of amino-polyethylene glycol-hydroxyl were added, followed by 0.3-0.7 parts of 4-dimethylaminopyridine and 0.6-1.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The mixture was reacted at room temperature to obtain a reaction solution. The reaction solution was filtered to obtain a filtrate. The filtrate was concentrated, anhydrous diethyl ether was added, and the mixture was centrifuged and vacuum dried. Drying yields cholesterol-PEG-hydroxy linkage; A2, dissolve 2.0-4.0 parts of carboxyl-containing quaternized galloyl-polylysine and 1.0-2.0 parts of cholesterol-PEG-hydroxy linkage in 100-150 parts of phosphate buffer, add 0.5-1.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 0.3-0.6 parts of N-hydroxythiosuccinimide and 0.2-0.4 parts of 4-dimethylaminopyridine, stir and react at 20-30℃, after the reaction is completed, purify by gel filtration chromatography and freeze-dry.

[0011] In this invention, the preparation of the quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol succinate monoester involves a multi-step organic synthesis reaction. First, gallic acid is grafted onto the polylysine backbone via an amidation reaction. The carboxyl group of the gallic acid is activated using a carbodiimide condensing agent, forming a stable amide bond with the amino group of the polylysine, thus imparting antioxidant properties to the material. Subsequently, a quaternizing agent is introduced at low temperature, and a chlorinated quaternary ammonium salt is grafted onto the polylysine side chain via a nucleophilic substitution reaction, giving the material a positive surface charge and enhancing its electrostatic adsorption to skin cell membranes. Next, the remaining amino group is modified with succinic anhydride through carboxylation, introducing a carboxyl functional group that can be further reacted. On the other hand, a cholesterol derivative and a polyethylene glycol segment are linked via an esterification reaction to form an amphiphilic linker, where cholesterol provides membrane fusion capability and polyethylene glycol improves water solubility. Finally, carboxyl-containing quaternized polylysine and cholesterol-polyethylene glycol linkers are coupled via amide bonds in a buffer system. Carbodiimide and hydroxysuccinimide synergistically activate the carboxyl groups, promoting efficient grafting. The entire reaction process is purified by dialysis and freeze-drying to ensure the integrity of the product structure and the absence of residual reagents, ultimately yielding a multifunctional polymer with positive charge properties, antioxidant capacity, and membrane fusion function.

[0012] According to a preferred embodiment of the present invention, in step A1, the reaction time at 45-55°C is 10-14 hours.

[0013] According to a preferred embodiment of the present invention, in step A2, the stirring reaction time at 20-30°C is 40-56 hours.

[0014] According to a preferred embodiment of the present invention, the preparation method of the thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E comprises: B1, dissolving 4.0-6.0 parts of sodium hyaluronate in 200-300 parts of deionized water, adding 1.0-2.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.8-1.5 parts of N-hydroxythiosuccinimide, followed by adding 2.0-4.0 parts of cystamine dihydrochloride, stirring the reaction at room temperature, and after the reaction is completed, dialyzing with sodium chloride solution, reducing the reaction with dithiothreitol at pH=7.5-8.5, dialyzing, and freeze-drying to obtain thiolated hyaluronic acid; dissolving 1.5-2.5 parts of dihydromyricetin in 40-60 parts of anhydrous tetrahydrofuran, adding 0.4-0.8 parts of 4-carboxyphenylboronic acid and 0.5-1.0 parts of vitamin E. A1. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3-0.6 parts of 4-dimethylaminopyridine are reacted at 50-60℃. After the reaction, the mixture is rotary evaporated and recrystallized with a methanol-water mixture to obtain dihydromyricetin-phenylboronic acid ester. B2. 1.5-2.5 parts of thiolated hyaluronic acid are dissolved in 80-120 parts of phosphate buffer (pH 7.0-7.8), and 0.3-0.6 parts of dihydromyricetin-phenylboronic acid ester and 0.15-0.35 parts of vitamin E succinate are added. 0.2-0.5 parts of 4-dimethylaminopyridine are added, followed by 0.3-0.7 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.2-0.5 parts of N-hydroxythiosuccinimide. The mixture is reacted at 25-35℃ to obtain a reaction solution. The reaction solution is dialyzed and freeze-dried.

[0015] In this invention, the construction of the thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E is based on a dynamic covalent chemical and bio-coupling strategy. The hyaluronic acid backbone is first activated by carbodiimide to activate the carboxyl group, which then reacts with the amino group of cystamine to form an amide bond, introducing a disulfide bond structure. Subsequently, a reducing agent is used to cleave the disulfide bond, generating free thiol groups, enabling the material to form disulfide bonds with skin keratin and enhancing its retention effect. The phenolic hydroxyl group of dihydromyricetin and the carboxyphenylboronic acid form a borate ester bond under the action of a condensing agent. This bond has pH-responsive characteristics and can be reversibly cleaved in the slightly acidic environment of the skin, achieving intelligent release. Vitamin E is linked through a Michael addition reaction between the carboxyl group of its succinate and the thiol group of the thiolated hyaluronic acid, or through a condensing agent-mediated amidation reaction, forming a stable covalent bond. In the final product, hyaluronic acid provides a moisturizing matrix, the thiol groups enhance skin adhesion, the borate ester bond enables environmentally responsive release, and dihydromyricetin and vitamin E synergistically exert antioxidant and anti-inflammatory effects. The entire synthesis process removes small molecule byproducts through dialysis and maintains structural stability through freeze-drying, ensuring the integrity of the activity of each functional group.

[0016] According to a preferred embodiment of the present invention, in step B1, the reaction time at 50-60°C is 6-10 hours.

[0017] A second aspect of the present invention provides an anti-aging composition containing nanospheres prepared according to the method for preparing the aforementioned anti-aging composition containing nanospheres.

[0018] A third aspect of the present invention provides the application of the aforementioned anti-aging composition containing nanospheres in skincare products.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses a double emulsion solvent evaporation method to prepare nanospheres. By optimizing the formulation of the inner aqueous phase and adding enzyme protectants, the activity retention rate of the enzyme during the preparation process is significantly improved. Trehalose and bovine serum albumin added to the inner aqueous phase act as dual protectants, effectively maintaining the spatial conformation of the enzyme under low-temperature emulsification conditions and preventing the enzyme molecules from denaturing and becoming inactive under interfacial tension. Polyvinyl alcohol added to the outer aqueous phase acts as an emulsion stabilizer, forming a stable core-shell structure with the two functionalized modified polymers, giving the nanospheres a uniform particle size distribution and good dispersibility. The low-temperature centrifugation and washing process avoids thermal inactivation of the enzyme, and the final nanospheres have a high encapsulation efficiency and drug loading. The humectant and metal ion chelating agent added during the double dispersion process further improve the storage stability of the composition and extend the shelf life of the product. The entire preparation process is mild, simple to operate, and suitable for large-scale industrial production.

[0020] (2) The two functionalized modified polymers designed in this invention achieve multiple synergistic effects. In the quaternized galloyl polylysine grafted polyethylene glycol cholesterol derivative, the quaternization structure endows the material with positive charge properties, enhances the interaction with the negatively charged skin cell membrane, and improves cell penetration; the gallic acid fragment provides additional antioxidant activity; the polyethylene glycol segment improves the water solubility and biocompatibility of the material; the cholesterol fragment enhances the fusion with the cell membrane and promotes the release of contents. In the thiolized hyaluronic acid grafted dihydromyricetin borate vitamin derivative, the hyaluronic acid backbone provides excellent moisturizing properties; the thiol group can form disulfide bonds with skin keratin to enhance the retention effect; the borate bond forms a reversible bond with the vicinal diol structure of hyaluronic acid to achieve intelligent response release; dihydromyricetin and vitamin derivatives provide strong antioxidant and anti-inflammatory effects. The two modified polymers form a composite shell on the surface of the nanospheres, and the microsphere structure is stabilized synergistically through electrostatic interaction and hydrogen bonding.

[0021] (3) The anti-aging composition containing nanospheres obtained in this invention exhibits excellent technical effects in skincare applications. The synergistic effect of superoxide dismutase and collagenase can effectively scavenge free radicals and regulate collagen metabolism, thus delaying the skin aging process from multiple targets. The nanosphere carrier protects the active enzyme ingredients from external environmental factors while achieving transdermal delivery and improving bioavailability. The composition's multiple antioxidant system can neutralize different types of reactive oxygen species and reduce oxidative stress damage. The combined use of moisturizing and antioxidant ingredients maintains skin hydration while combating aging. All materials have good biocompatibility and safety, are non-irritating, and are suitable for long-term daily use. The overall formulation system is stable, does not easily separate or precipitate, and is easy to process. This invention provides a new technical solution for developing efficient, safe, and stable enzyme-based anti-aging skincare products, and has broad market application prospects. Detailed Implementation

[0022] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0023] Example 1 This embodiment provides a method for preparing an anti-aging composition containing nanospheres and enzymes, the steps of which include: S1. Dissolve 5.5g of soybean lecithin and 15.0g of polylactic acid-glycolic acid copolymer (molecular weight 10000 Da), with a lactic acid to glycolic acid molar ratio of 50:50, in 50g of dichloromethane. Stir at 500rpm for 30min to obtain the oil phase. Dissolve 0.65g of superoxide dismutase (3000U / mg), 0.3g of collagenase (125U / mg), 2.0g of trehalose, and 1.0g of bovine serum albumin in 20g of phosphate buffer (pH 7.4). Stir at 200rpm for 15min to obtain the inner aqueous phase. Under 2℃ ice-water bath conditions, add the inner aqueous phase dropwise to the oil phase at a rate of 2mL / min, while simultaneously homogenizing and emulsifying at 12000rpm for 5min to form the primary emulsion. Add 3.5g of quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol derivative and 5.5g of thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E to 100g of an aqueous solution containing 1.0% polyvinyl alcohol. Stir at 300rpm for 30min to dissolve and obtain the external aqueous phase. Under 2℃ ice-water bath conditions, rapidly pour the colostrum into the external aqueous phase and homogenize at 8000rpm for 8min to form a secondary emulsion.

[0024] S2. Transfer the double emulsion to a beaker and magnetically stir at 300 rpm for 4 hours at 25°C to evaporate and remove the organic solvent dichloromethane, thus solidifying the nanospheres and obtaining a nano-suspension. Centrifuge the nano-suspension at 10,000 rpm for 30 minutes at 4°C, discard the supernatant, and collect the precipitate. Resuspend the precipitate in deionized water, centrifuge at 10,000 rpm for 30 minutes at 4°C, discard the supernatant, and repeat the washing process three times to obtain the washed nanosphere precipitate. Redisperse the washed nanosphere precipitate in deionized water, add 2.0 g of 1,2-pentanediol and 0.12 g of disodium ethylenediaminetetraacetate, and stir at 500 rpm for 15 minutes to obtain an anti-aging composition containing nanosphere enzymes.

[0025] Preparation of quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol succinate monoester: A1. Add 10.0 g of ε-polylysine (average molecular weight 4000 Da) and 200 mL of anhydrous dimethyl sulfoxide to a reaction vessel. Dissolve under nitrogen protection by stirring at 300 rpm for 30 min. Add 0.35 g of triethylamine and cool to 2 °C in an ice-water bath. Dissolve 6.5 g of gallic acid in 50 mL of anhydrous dimethyl sulfoxide and slowly add it dropwise to the reaction system at a rate of 1 mL / min. Simultaneously add 8.0 g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 4.5 g of N-hydroxysuccinimide. Heat to 25 °C and stir for 24 h to obtain the reaction mixture. The reaction mixture was cooled to 10°C, and 8.0 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride was added. The mixture was heated to 50°C and stirred for 12 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in deionized water for 48 h, changing the dialysate every 6 h. After dialyzing, the solution was freeze-dried for 48 h to obtain quaternized galloyl-polylysine. Quaternized galloyl-polylysine was dissolved in 120 mL of anhydrous N,N-dimethylformamide, and 3.0 g of succinic anhydride and 0.35 g of 4-dimethylaminopyridine were added. The mixture was stirred at 45°C for 18 h. After the reaction was completed, the solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in deionized water for 48 h, changing the dialysate every 6 h. After dialyzing, the solution was freeze-dried for 48 h to obtain carboxyl-containing quaternized galloyl-polylysine. 5.0 g of cholesterol hemisuccinate was dissolved in 100 mL of anhydrous dichloromethane. 4.0 g of amino-PEG-hydroxyl compound with an average molecular weight of 2000 Da was added, followed by 0.5 g of 4-dimethylaminopyridine and 0.8 g of 1-ethyl-3-dimethylaminopropylcarbodiimide. The mixture was stirred at 25 °C for 24 h to obtain a reaction solution. The reaction solution was filtered through a 0.45 μm organic filter membrane to obtain a filtrate. The filtrate was concentrated to 1 / 3 of its original volume by rotary evaporation at 40 °C. 200 mL of anhydrous diethyl ether was slowly added, and the mixture was allowed to stand at 4 °C for 12 h. The precipitate was collected by centrifugation at 8000 rpm for 10 min, and the supernatant was discarded. The precipitate was then vacuum dried at 40 °C for 24 h to obtain the cholesterol-PEG-hydroxyl linkage.

[0026] A2. 3.0 g of carboxyl-containing quaternized galloyl-polylysine and 1.5 g of cholesterol-PEG-hydroxy linker were dissolved in 120 mL of pH 7.5 phosphate buffer. 0.8 g of 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.45 g of N-hydroxythiosuccinimide, and 0.3 g of 4-dimethylaminopyridine were added. The mixture was stirred at 200 rpm for 48 h at 25 °C. After the reaction, the reaction solution was loaded onto a Sephadex G-75 gel filtration chromatography column. Phosphate buffer at pH 7.5 was used as the eluent, and the flow rate was controlled at 0.5 mL / min. The elution peaks were collected, the target components were combined, and dialyzed in deionized water for 48 h, with the dialysate changed every 6 h. After dialyzing, the mixture was freeze-dried for 48 h to obtain the quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol derivative.

[0027] Preparation of thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E: B1. Dissolve 5.0 g of sodium hyaluronate (50 kDa) in 250 mL of deionized water and stir at 300 rpm for 1 h. Add 1.5 g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 1.2 g of N-hydroxythiosuccinimide and activate the carboxyl group at 25 °C for 30 min. Then add 3.0 g of cystamine dihydrochloride and stir at 25 °C for 24 h. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8000 Da. The hyaluronic acid was dialyzed in 0.1 mol / L sodium chloride solution for 3 days, with the dialysate changed every 12 hours. Then, it was dialyzed in deionized water for 24 hours. After dialyzing, the solution was transferred to a reaction vessel, and 0.1 mol / L dithiothreitol was added to adjust the pH to 8.0. The reduction reaction was carried out at 25°C for 4 hours. After the reaction was completed, the solution was transferred to a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed in deionized water for 48 hours, with the dialysate changed every 6 hours. After dialyzing, the solution was freeze-dried for 48 hours to obtain thiolated hyaluronic acid. 2.0 g of dihydromyricetin was dissolved in 50 mL of anhydrous tetrahydrofuran, and 0.6 g of 4-carboxyphenylboronic acid, 0.8 g of 1-ethyl-3-dimethylaminopropylcarbodiimide, and 0.45 g of 4-dimethylaminopyridine were added. The mixture was refluxed and stirred at 55 °C for 8 h. After the reaction was completed, the solvent was removed by rotary evaporation at 40 °C to obtain the crude product. The crude product was dissolved in a mixed solvent of methanol and water in a volume ratio of 3:1 at 60 °C, cooled to 4 °C, and recrystallized for 12 h. The crystals were collected by filtration and dried under vacuum at 40 °C for 24 h to obtain dihydromyricetin-phenylboronic acid ester.

[0028] B2. Dissolve 2.0 g of thiolated hyaluronic acid in 100 mL of pH 7.4 phosphate buffer and stir at 200 rpm for 30 min. Add 0.45 g of dihydromyricetin-phenylboronic acid ester and 0.25 g of vitamin E succinate, then add 0.35 g of 4-dimethylaminopyridine, followed by 0.5 g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 0.35 g of N-hydroxythiosuccinimide. Stir the reaction mixture at 30 °C for 36 h to obtain the reaction solution. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8000 Da and dialyze sequentially with 50% ethanol aqueous solution for 48 h and deionized water for 48 h, changing the dialysate every 6 h. After dialyzing, freeze-dry for 48 h to obtain thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E.

[0029] Example 1 The difference between this embodiment and Embodiment 1 is that this embodiment provides a method for preparing an anti-aging composition containing nanospheres, the steps of which include: S1. Dissolve 3.0g of soybean lecithin and 10.0g of polylactic acid-glycolic acid copolymer in 40g of dichloromethane to obtain the oil phase. Dissolve 0.3g of superoxide dismutase, 0.1g of collagenase, 1.0g of trehalose, and 0.5g of bovine serum albumin in 15g of phosphate buffer at pH 7.4 to obtain the inner aqueous phase. Add the inner aqueous phase dropwise to the oil phase at 0℃ and emulsify at 10000rpm for 3min to obtain the primary emulsion. Add the primary emulsion to 80g of an aqueous solution containing 0.5% polyvinyl alcohol, which contains 2.0g of quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol derivative and 3.0g of thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E, and homogenize at 6000rpm for 5min to obtain the secondary emulsion.

[0030] S2. The double emulsion was stirred at 25°C for 3 hours to obtain a solidified nano-suspension. The solidified nano-suspension was centrifuged at 8000 rpm for 20 minutes at 3°C, and the precipitate was collected. The precipitate was washed twice with deionized water to obtain washed nanosphere precipitate. The washed nanosphere precipitate was redispersed in deionized water, and 1.0 g of 1,2-pentanediol and 0.05 g of disodium ethylenediaminetetraacetate were added. The mixture was stirred evenly to obtain an anti-aging composition containing nanosphere enzymes.

[0031] Preparation of quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol succinate monoester: A1. Add 8.0 g of ε-polylysine and 150 mL of anhydrous dimethyl sulfoxide to a reaction vessel. Stir under nitrogen protection, add 0.20 g of triethylamine, and cool to 0 °C. Add dropwise 5.0 g of gallic acid dissolved in 40 mL of anhydrous dimethyl sulfoxide, simultaneously adding 6.0 g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 3.0 g of N-hydroxysuccinimide. Heat to 25 °C and react for 20 h to obtain the reaction mixture. Cool the reaction mixture to 5 °C, add 6.0 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride, and react at 45 °C for 10 h. After the reaction is complete, dialyze for 40 h, and freeze-dry to obtain quaternized galloyl-polylysine. Quaternized galloyl-polylysine was dissolved in 100 mL of anhydrous N,N-dimethylformamide, and 2.0 g of succinic anhydride and 0.20 g of 4-dimethylaminopyridine were added. The mixture was reacted at 40 °C for 12 h, dialyzed for 40 h, and freeze-dried to obtain carboxyl-containing quaternized galloyl-polylysine. 4.0 g of cholesterol hemisuccinate was dissolved in 80 mL of anhydrous dichloromethane, and 3.0 g of amino-polyethylene glycol-hydroxyl was added, followed by 0.3 g of 4-dimethylaminopyridine and 0.6 g of 1-ethyl-3-dimethylaminopropylcarbodiimide. The mixture was reacted at 25 °C for 20 h to obtain a reaction solution. The reaction solution was filtered to obtain a filtrate, which was concentrated and then added to anhydrous diethyl ether. The filtrate was centrifuged and vacuum-dried to obtain the cholesterol-PEG-hydroxyl linkage.

[0032] A2. 2.0 g of carboxyl-containing quaternized galloyl-polylysine and 1.0 g of cholesterol-PEG-hydroxy linker were dissolved in 100 mL of pH 7.5 phosphate buffer. 0.5 g of 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.3 g of N-hydroxythiosuccinimide and 0.2 g of 4-dimethylaminopyridine were added. The mixture was stirred at 20 °C for 40 h. After the reaction was completed, the mixture was purified by gel filtration chromatography and freeze-dried to obtain the quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol derivative.

[0033] Preparation of thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E: B1. Dissolve 4.0 g of sodium hyaluronate in 200 mL of deionized water, add 1.0 g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 0.8 g of N-hydroxythiosuccinimide, then add 2.0 g of cystamine dihydrochloride. Stir the reaction at 25 °C for 20 h. After the reaction is complete, dialyze with 0.1 mol / L sodium chloride solution for 2 days, reduce with 0.05 mol / L dithiothreitol at pH 7.5 for 3 h, dialyze for 40 h, and freeze dry to obtain thiolated hyaluronic acid. 1.5 g of dihydromyricetin was dissolved in 40 mL of anhydrous tetrahydrofuran, and 0.4 g of 4-carboxyphenylboronic acid, 0.5 g of 1-ethyl-3-dimethylaminopropylcarbodiimide, and 0.3 g of 4-dimethylaminopyridine were added. The mixture was reacted at 50 °C for 6 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the mixture was recrystallized with a 3:1 volume ratio of methanol to water to obtain dihydromyricetin-phenylboronic acid ester.

[0034] B2. Dissolve 1.5g of thiolated hyaluronic acid in 80mL of pH 7.0 phosphate buffer, add 0.3g of dihydromyricetin-phenylboronic acid ester and 0.15g of vitamin E succinate, add 0.2g of 4-dimethylaminopyridine, then add 0.3g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 0.2g of N-hydroxythiosuccinimide, react at 25℃ for 30h to obtain the reaction solution, dialyze the reaction solution for 60h, freeze dry to obtain thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E.

[0035] Example 2 The difference between this embodiment and Embodiment 1 is that this embodiment provides a method for preparing an anti-aging composition containing nanospheres, the steps of which include: S1. Dissolve 8.0g of soybean lecithin and 20.0g of polylactic acid-glycolic acid copolymer in 60g of dichloromethane to obtain the oil phase. Dissolve 1.0g of superoxide dismutase, 0.5g of collagenase, 3.0g of trehalose, and 1.5g of bovine serum albumin in 25g of phosphate buffer at pH 7.4 to obtain the inner aqueous phase. Add the inner aqueous phase dropwise to the oil phase at 4°C and emulsify at 15000rpm for 8min to obtain the primary emulsion. Add the primary emulsion to 120g of an aqueous solution containing 2.0% polyvinyl alcohol, which contains 5.0g of quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol derivative and 8.0g of thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E, and homogenize at 10000rpm for 10min to obtain the secondary emulsion.

[0036] S2. The double emulsion was stirred at 25℃ for 6 hours to obtain a solidified nano-suspension. The solidified nano-suspension was centrifuged at 12000 rpm for 40 minutes at 5℃, and the precipitate was collected. The precipitate was washed four times with deionized water to obtain washed nanosphere precipitate. The washed nanosphere precipitate was redispersed in deionized water, and 3.0 g of 1,2-pentanediol and 0.2 g of disodium ethylenediaminetetraacetate were added. The mixture was stirred evenly to obtain an anti-aging composition containing nanosphere enzymes.

[0037] Preparation of quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol succinate monoester: A1. Add 12.0 g of ε-polylysine and 250 mL of anhydrous dimethyl sulfoxide to a reaction vessel. Stir under nitrogen protection, add 0.45 g of triethylamine, and cool to 4 °C. Add dropwise 8.0 g of gallic acid dissolved in 60 mL of anhydrous dimethyl sulfoxide, simultaneously adding 10.0 g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 6.0 g of N-hydroxysuccinimide. Heat to 25 °C and react for 28 h to obtain the reaction mixture. Cool the reaction mixture to 15 °C, add 10.0 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride, and react at 55 °C for 14 h. After the reaction is complete, dialyze for 56 h, and freeze-dry to obtain quaternized galloyl-polylysine. Quaternized galloyl-polylysine was dissolved in 150 mL of anhydrous N,N-dimethylformamide, and 4.0 g of succinic anhydride and 0.5 g of 4-dimethylaminopyridine were added. The mixture was reacted at 50 °C for 24 h, dialyzed for 56 h, and freeze-dried to obtain carboxyl-containing quaternized galloyl-polylysine. 6.0 g of cholesterol hemisuccinate was dissolved in 120 mL of anhydrous dichloromethane, and 5.0 g of amino-polyethylene glycol-hydroxyl was added, followed by 0.7 g of 4-dimethylaminopyridine and 1.0 g of 1-ethyl-3-dimethylaminopropylcarbodiimide. The mixture was reacted at 25 °C for 28 h to obtain a reaction solution. The reaction solution was filtered to obtain a filtrate, which was concentrated and then added to anhydrous diethyl ether. The filtrate was centrifuged and vacuum-dried to obtain the cholesterol-PEG-hydroxyl linkage.

[0038] A2. 4.0 g of carboxyl-containing quaternized galloyl-polylysine and 2.0 g of cholesterol-PEG-hydroxy linker were dissolved in 150 mL of pH 7.5 phosphate buffer. 1.0 g of 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.6 g of N-hydroxythiosuccinimide and 0.4 g of 4-dimethylaminopyridine were added. The mixture was stirred at 30 °C for 56 h. After the reaction was completed, the mixture was purified by gel filtration chromatography and freeze-dried to obtain the quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol derivative.

[0039] Preparation of thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E: B1. Dissolve 6.0 g of sodium hyaluronate in 300 mL of deionized water, add 2.0 g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 1.5 g of N-hydroxythiosuccinimide, then add 4.0 g of cystamine dihydrochloride. Stir the mixture at 25 °C for 28 h. After the reaction is complete, dialyze with 0.1 mol / L sodium chloride solution for 4 days, reduce with 0.15 mol / L dithiothreitol at pH 8.5 for 5 h, dialyze for 56 h, and freeze dry to obtain thiolized hyaluronic acid. 2.5 g of dihydromyricetin was dissolved in 60 mL of anhydrous tetrahydrofuran, and 0.8 g of 4-carboxyphenylboronic acid, 1.0 g of 1-ethyl-3-dimethylaminopropylcarbodiimide, and 0.6 g of 4-dimethylaminopyridine were added. The mixture was reacted at 60 °C for 10 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the mixture was recrystallized with a 3:1 volume ratio of methanol to water to obtain dihydromyricetin-phenylboronic acid ester.

[0040] B2. Dissolve 2.5g of thiolated hyaluronic acid in 120mL of phosphate buffer at pH 7.8, add 0.6g of dihydromyricetin-phenylboronic acid ester and 0.35g of vitamin E succinate, add 0.5g of 4-dimethylaminopyridine, then add 0.7g of 1-ethyl-3-dimethylaminopropylcarbodiimide and 0.5g of N-hydroxythiosuccinimide, react at 35℃ for 42h to obtain the reaction solution, dialyze the reaction solution for 84h, freeze dry to obtain thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E.

[0041] Comparative Example 1 The difference between this comparative example and Example 1 is that, in step S1, quaternized galloyl-polylysine-grafted polyethylene glycol-cholesterol derivative was not added, but only 5.5g of thiolated hyaluronic acid-grafted dihydromyricetin-boronic acid ester-vitamin E was added, and the rest was the same as in Example 1.

[0042] Comparative Example 2 The difference between this comparative example and Example 1 is that in step S1, thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E was not added, but only 3.5g of quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol derivative was added, and the rest was the same as in Example 1.

[0043] Comparative Example 3 The difference between this comparative example and Example 1 is that the quaternized galloyl-polylysine-grafted polyethylene glycol-cholesterol derivative and thiolated hyaluronic acid-grafted dihydromyricetin-boronic acid ester-vitamin E were not added in step S1. The rest is the same as in Example 1.

[0044] According to relevant national and industry standards, the performance of the anti-aging compositions containing nanospheres provided in the above embodiments and comparative examples was tested, and the test methods are as follows: For the determination of nanosphere particle size and Zeta potential, 1.0 mL of the anti-aging composition containing nanospheres prepared in each example and comparative example was diluted with deionized water to 100 mL. After shaking well, 1.0 mL was injected into a polystyrene cuvette. The average particle size and polydispersity index of the nanospheres were determined using a dynamic light scattering particle size analyzer at 25℃. The laser wavelength was 633 nm and the scattering angle was 173°. Each sample was measured in triplicate, and the average value was taken. 1.0 mL of the diluted sample was injected into the Zeta potential sample cell. The Zeta potential was measured using electrophoretic light scattering on the same instrument. The electric field strength was 150 V / cm. Each sample was measured in triplicate, and the average value was taken.

[0045] Encapsulation efficiency was determined by taking 2.0 mL of the anti-aging compositions containing nanospheres prepared in each example and comparative example, placing them in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10000 Da, centrifuging at 8000 rpm for 30 min at 4 °C, collecting the filtrate, and determining the total protein content of free superoxide dismutase and collagenase in the filtrate using a BCA protein quantification kit. Specifically, 25 μL of the filtrate was added to 200 μL of BCA working solution, incubated at 37 °C for 30 min, and the absorbance was measured at 562 nm. The free protein content was calculated based on the bovine serum albumin standard curve. Another 2.0 mL of the composition was placed in a glass test tube and sonicated at 200 W for 10 min in an ice-water bath, with a 1 min interval after every 2 min of sonication to completely break down the nanospheres. The total protein content was determined using the same method. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency equals total protein content minus free protein content divided by total protein content multiplied by 100%. Each sample was measured in triplicate, and the average value was taken.

[0046] Superoxide dismutase (SOD) activity retention rate determination: 5.0 mL of the anti-aging compositions containing nanospheres prepared in each example and comparative example were stored in a 45℃ incubator in the dark for 90 days. Samples were taken at 0 and 90 days. SOD activity was determined using the xanthine oxidase method. Specifically, 50 μL of sample was added to a 2.5 mL reaction system containing 50 mmol / L phosphate buffer (pH 7.8), 0.1 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.1 mmol / L xanthine, and 0.05 mmol / L nitrotetrazolium chloride (NTC). 0.1 mL of xanthine oxidase was added to initiate the reaction. The reaction was carried out at 25℃ for 20 min, and the absorbance was measured at 560 nm. One unit of activity was defined as the amount of enzyme at which the inhibition rate of the reaction was 50%. The enzyme activity retention rate at 90 days was calculated with the enzyme activity at 0 days as 100%. Each sample was measured in triplicate, and the average value was taken.

[0047] To determine the retention rate of collagenase activity, 5.0 mL of the anti-aging compositions containing nanospheres prepared in each example and comparative example were stored in a 45℃ incubator in the dark for 90 days. Samples were taken at 0 and 90 days. Collagenase activity was determined using a colorimetric method with a collagenase activity assay kit. Specifically, 50 μL of sample was added to 1.0 mL of reaction buffer and 0.5 mL of chromogenic substrate solution, incubated at 37℃ for 30 min, and the reaction was terminated by adding a stop solution. The absorbance was measured at 450 nm, and the enzyme activity was calculated based on the p-nitroaniline standard curve. The enzyme activity retention rate at 90 days was calculated with the enzyme activity at 0 days as 100%. Each sample was measured in triplicate, and the average value was taken.

[0048] The cumulative permeation rate of dihydromyricetin was measured by removing subcutaneous fat tissue from isolated pig ear skin, washing it with physiological saline, and fixing it between the supply and receiving cells of a Franz diffusion cell. The effective diffusion area of ​​the skin was 2.0 cm², with the stratum corneum facing the supply cell and the dermis facing the receiving cell. 15 mL of pH 7.4 phosphate buffer was added to the receiving cell as the receiving medium, and the mixture was stirred at 300 rpm for 30 min in a 32°C water bath for equilibration. 1.0 g of the anti-aging composition containing nanospheres prepared in each example and comparative example was evenly applied to the surface of the stratum corneum. After 24 h, 1.0 mL of sample was taken from the receiving cell, and an isothermal and equal volume of receiving medium was added simultaneously. After filtration through a 0.22 μm filter membrane, the dihydromyricetin content was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were: a C18 column (4.6 mm × 250 mm, 5 μm); a mobile phase of methanol to water (60:40 v / v); a flow rate of 1.0 mL / min; a detection wavelength of 292 nm; a column temperature of 30 °C; and an injection volume of 20 μL. The concentration of dihydromyricetin in the receiving solution was calculated based on the standard curve. The cumulative permeation rate per unit area of ​​skin was calculated using the following formula: cumulative permeation rate equals the total amount of dihydromyricetin in the receiving cell divided by the effective diffusion area of ​​the skin. Each sample was measured in parallel six times, and the average value was taken.

[0049] To determine the DPPH free radical scavenging rate, take 1.0 mL of the anti-aging composition containing nanospheres prepared in each example and comparative example, add 2.0 mL of 0.1 mmol / L DPPH ethanol solution, mix well, and react in the dark at 25°C for 30 min. Measure the absorbance at 517 nm and record it as A1. Take 1.0 mL of deionized water instead of the sample and mix it with 2.0 mL of DPPH ethanol solution, and measure the absorbance using the same method, recording it as A0. Take 1.0 mL of the sample and mix it with 2.0 mL of anhydrous ethanol, and measure the absorbance using the same method, recording it as A2. Calculate the DPPH free radical scavenging rate using the following formula: Scavenging rate equals A0 minus A1 minus A2 divided by A0 multiplied by 100%. Each sample is measured in triplicate, and the average value is taken.

[0050] Human skin fibroblast collagen synthesis promotion assay: Human skin fibroblasts were resuscitated and passaged to passages 3-5. Cells were then collected after digestion with 0.25% trypsin and the cell density was adjusted to 1×10⁶ cells / mL using DMEM medium containing 10% fetal bovine serum. 5 Cells were seeded at a concentration of 100 μL / mL in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. The original culture medium was discarded, and 100 μL of fresh culture medium containing the anti-aging nanosphere enzyme composition prepared in each example and comparative example at a concentration of 0.1 mg / mL was added to each well. Culture was continued for 48 h. The cell culture supernatant was collected, and the type I collagen content in the supernatant was determined using enzyme-linked immunosorbent assay (ELISA). Specifically, 50 μL of the supernatant was added to an ELISA plate coated with type I collagen antibody and incubated at 37°C for 60 min. After washing, 50 μL of enzyme-labeled antibody was added, and the plate was incubated at 37°C for 30 min. After washing, 50 μL of chromogenic solution was added, and the plate was incubated in the dark for 15 min. 50 μL of stop solution was added, and the absorbance was measured at 450 nm. The type I collagen content was calculated based on the standard curve. Using normal cultured cells without samples as the control group, the relative collagen synthesis rate of each sample group and the control group was calculated. The relative collagen synthesis rate was equal to the collagen content of the sample group divided by the collagen content of the control group multiplied by 100%. Each sample was measured in parallel 6 times and the average value was taken.

[0051] To determine the inhibition rate of matrix metalloproteinase-1, human skin fibroblasts were resuscitated and passaged to passages 3-5. Cells were then digested with 0.25% trypsin and collected. The cell density was adjusted to 1×10⁶ cells / mL using DMEM medium containing 10% fetal bovine serum. 5Cells were seeded at a concentration of 100 μL / mL into 96-well plates and cultured at 37°C with 5% CO2 for 24 h. The original culture medium was discarded, and fresh culture medium containing 0.1 mg / mL of the anti-aging nanosphere enzyme composition prepared in each example and comparative example was added to each well at 100 μL. Simultaneously, 50 ng / mL of tumor necrosis factor-α was added to induce an inflammatory response, and the culture was continued for 48 h. The cell culture supernatant was collected, and the matrix metalloproteinase-1 (MMP-1) content in the supernatant was determined using enzyme-linked immunosorbent assay (ELISA). Specifically, 50 μL of the supernatant was added to an ELISA plate coated with MMP-1 antibody and incubated at 37°C for 60 min. After washing, 50 μL of the enzyme-labeled antibody was added, and the plate was incubated at 37°C for 30 min. After washing, 50 μL of the chromogenic solution was added, and the plate was developed in the dark for 15 min. 50 μL of the stop solution was added, and the absorbance was measured at 450 nm. The MMP-1 content was calculated based on the standard curve. Cells without the sample but with only tumor necrosis factor-α were used as the model control group, and normal cells without the sample and tumor necrosis factor-α were used as the blank control group. The matrix metalloproteinase-1 inhibition rate was calculated according to the following formula: the inhibition rate is equal to the matrix metalloproteinase-1 content of the model control group minus the matrix metalloproteinase-1 content of the sample group divided by the matrix metalloproteinase-1 content of the model control group minus the matrix metalloproteinase-1 content of the blank control group multiplied by 100%. Each sample was measured in parallel 6 times and the average value was taken.

[0052] The performance test data above are shown in Table 1.

[0053] Table 1 Performance Test Results

[0054] As can be seen from the above, Examples 1-3 show significant advantages over Comparative Examples 1-3 in several key performance indicators, and systematically solve the technical problems of unstable enzyme activity, low transdermal penetration efficiency, and single anti-aging effect in the prior art.

[0055] Regarding the physicochemical properties of the nanospheres, the average particle size of Examples 1-3 was 205.3-235.6 nm, and the polydispersity index was 0.118-0.148, both of which were superior to the 245.2-278.4 nm and 0.176-0.215 of Comparative Examples 1-3, indicating that the synergistic effect of the two modified compounds made the nanosphere particle size distribution more uniform and the system more stable. In terms of zeta potential, Examples 1-3 were -15.3 to -20.1 mV, which was significantly better than the -8.7 mV of Comparative Example 3, proving that the modified compound A, quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol derivative, enhanced the colloidal stability of the nanospheres through electrostatic interaction. Although the zeta potential of Comparative Example 2 reached -22.5 mV when only modified compound A was added, the encapsulation efficiency was lower than that of the Example group, indicating that although modified compound A alone can improve the surface charge, it cannot take into account the encapsulation efficiency.

[0056] Regarding the core issue of enzyme stability, the superoxide dismutase activity retention rates in Examples 1-3 were 91.5-95.6%, and the collagenase activity retention rates were 90.2-94.3%. In contrast, in Comparative Example 1 without the addition of modified compound A, the activity retention rates of the two enzymes decreased to 78.3% and 75.6%, respectively. In Comparative Example 2 with the addition of modified compound A but without the addition of modified compound B, the enzyme activity retention rates were 86.4% and 84.5%, respectively. In Comparative Example 3 without the addition of either modified compound, the enzyme activity retention rates were as low as 65.7% and 62.3%, respectively. This gradient difference directly proves that modified compound A significantly protects the native conformation of the enzyme through electrostatic interaction and hydrophobic anchoring, and is a key component in solving the problem of enzyme thermal inactivation. The protection of enzyme activity by modified compound B is mainly achieved indirectly by improving the encapsulation efficiency.

[0057] Regarding transdermal permeability, the cumulative permeation amount of dihydromyricetin in Examples 1-3 was 42.5-52.3 μg / cm² over 24 hours, significantly higher than that of Comparative Example 1 (35.2 μg / cm²), Comparative Example 2 (18.5 μg / cm²), and Comparative Example 3 (12.4 μg / cm²). The permeation amount of Comparative Example 2 with only modified compound A was much lower than that of the Example group. Although the permeation amount of Comparative Example 1 with only modified compound B was higher than that of Comparative Example 2, it was only about 70% of that of the Example group. This result indicates that the active targeting effect of hyaluronic acid to the CD44 receptor and the anchoring effect of thiol groups in modified compound B are the dominant factors in solving the transdermal permeability problem, while modified compound A plays a synergistic role by improving the lipid compatibility of nanospheres.

[0058] Regarding antioxidant efficacy, the DPPH free radical scavenging rates of Examples 1-3 were 82.3-89.2%, which were much higher than those of Comparative Example 1 (68.5%), Comparative Example 2 (72.4%), and Comparative Example 3 (45.6%). The lowest scavenging rate in Comparative Example 3 indicates that the antioxidant capacity of a single enzyme component is limited. The scavenging rate of Comparative Example 2 was slightly higher than that of Comparative Example 1, but both were significantly lower than those of the Example groups. This demonstrates that the synergistic antioxidant effect of gallic acid residues in modified compound A and dihydromyricetin residues in modified compound B is the key to solving the problem of poor efficacy of single antioxidants.

[0059] Regarding anti-aging efficacy at the cellular level, the relative collagen synthesis rate of Examples 1-3 was 176.8-192.3%, significantly higher than that of Comparative Example 1 (142.5%), Comparative Example 2 (151.2%), and Comparative Example 3 (112.3%). Simultaneously, the matrix metalloproteinase-1 inhibition rate of Examples 1-3 was 68.2-75.8%, significantly higher than that of Comparative Example 1 (52.3%), Comparative Example 2 (58.6%), and Comparative Example 3 (35.2%). Comparative Example 3, relying solely on enzyme components, had limited effects in promoting collagen synthesis and inhibiting collagen degradation. Although the effects of adding only a single modified compound in Comparative Examples 1 and 2 were improved, they were still far inferior to those in the Example Group. This difference fully demonstrates that the two modified compounds work synergistically through multiple targets. Modified compound A protects enzyme activity and exerts antioxidant effects, while modified compound B enables transdermal delivery and pH-responsive release of dihydromyricetin. At the same time, the active fragments in the two modified compounds jointly activate fibroblast function, systematically solving the technical problem of existing anti-aging products having a single target and being unable to simultaneously promote collagen synthesis and inhibit collagen degradation.

Claims

1. A method for preparing an anti-aging composition containing nanospheres and enzymes, characterized in that the steps include... include: S1. By weight, dissolve 3.0-8.0 parts of soybean lecithin and 10.0-20.0 parts of polylactic acid-glycolic acid copolymer in 40-60 parts of dichloromethane to obtain an oil phase; dissolve 0.3-1.0 parts of superoxide dismutase, 0.1-0.5 parts of collagenase, 1.0-3.0 parts of trehalose, and 0.5-1.5 parts of bovine serum albumin in 15-25 parts of phosphate buffer to obtain an inner aqueous phase; emulsify the inner aqueous phase by adding it dropwise to the oil phase at 0-4℃ to obtain a primary emulsion; add the primary emulsion to an aqueous solution containing 2.0-5.0 parts of quaternized galloyl-polylysine-grafted polyethylene glycol-cholesterol derivative and 3.0-8.0 parts of thiolated hyaluronic acid-grafted dihydromyricetin-boronic acid ester-vitamin E in 80-120 parts of polyvinyl alcohol, homogenize, and obtain a secondary emulsion; S2. Stir the double emulsion at room temperature to obtain a solidified nano suspension; centrifuge the solidified nano suspension at 3-5℃ and collect the precipitate; The precipitate was washed with deionized water to obtain the washed nanosphere precipitate. The washed nanosphere precipitate was redispersed in deionized water, and 1.0-3.0 parts of 1,2-pentanediol and 0.05-0.2 parts of disodium ethylenediaminetetraacetate were added and stirred.

2. The method for preparing the anti-aging composition containing nanospheres according to claim 1, characterized in that, In step S1, the emulsification time is 3-8 minutes.

3. The method for preparing the anti-aging composition containing nanospheres according to claim 1, characterized in that, In step S2, the centrifugation time is 20-40 minutes.

4. The method for preparing the anti-aging composition containing nanospheres according to claim 1, characterized in that, The preparation method of the quaternized galloyl-polylysine-grafted-polyethylene glycol-cholesterol succinate includes: A1. In a reaction vessel, add 8.0-12.0 parts of ε-polylysine and 150-250 parts of anhydrous dimethyl sulfoxide, stir under nitrogen protection, add 0.2-0.45 parts of triethylamine, and cool to 0-4℃; dropwise add 5.0-8.0 parts of gallic acid dissolved in 40-60 parts of anhydrous dimethyl sulfoxide, and simultaneously add 6.0-10.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 3.0-6... 0.0 parts of N-hydroxysuccinimide were heated to room temperature to obtain a reaction mixture; the reaction mixture was cooled to 5-15℃, and 6.0-10.0 parts of 3-chloro-2-hydroxypropyltrimethylammonium chloride were added, and the mixture was reacted at 45-55℃. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain quaternized galloyl-polylysine; quaternized galloyl-polylysine was dissolved in 100-150 parts of anhydrous N,N-dimethylformamide, and 2.0-4.0 parts of succinic anhydride and 0.2-0.5 parts of 4-dimethylaminopyridine were added, and the mixture was reacted at 40-50℃. The reaction was carried out at ℃, followed by dialysis and freeze-drying to obtain carboxyl-containing quaternized galloyl-polylysine. 4.0-6.0 parts of cholesterol hemisuccinate were dissolved in 80-120 parts of anhydrous dichloromethane, and 3.0-5.0 parts of amino-polyethylene glycol-hydroxyl were added, followed by 0.3-0.7 parts of 4-dimethylaminopyridine and 0.6-1.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The reaction was carried out at room temperature to obtain a reaction solution. The reaction solution was filtered to obtain a filtrate. The filtrate was concentrated, anhydrous diethyl ether was added, and the mixture was centrifuged and vacuum dried to obtain... To obtain cholesterol-PEG-hydroxy linker; A2, dissolve 2.0-4.0 parts of carboxyl-containing quaternized galloyl-polylysine and 1.0-2.0 parts of cholesterol-PEG-hydroxy linker in 100-150 parts of phosphate buffer, add 0.5-1.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 0.3-0.6 parts of N-hydroxythiosuccinimide and 0.2-0.4 parts of 4-dimethylaminopyridine, stir and react at 20-30℃, after the reaction is completed, purify by gel filtration chromatography and freeze dry.

5. The method for preparing the anti-aging composition containing nanospheres according to claim 4, characterized in that, In step A1, the reaction time is 10-14 hours at 45-55℃.

6. The method for preparing the anti-aging composition containing nanospheres according to claim 4, characterized in that, In step A2, the reaction is stirred at 20-30℃ for 40-56 hours.

7. The method for preparing the anti-aging composition containing nanospheres according to claim 1, characterized in that, The preparation method of the thiolated hyaluronic acid-grafted-dihydromyricetin-boronic acid ester-vitamin E includes: B1, dissolving 4.0-6.0 parts of sodium hyaluronate in 200-300 parts of deionized water, adding 1.0-2.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.8-1.5 parts of N-hydroxythiosuccinimide, followed by adding 2.0-4.0 parts of cystamine dihydrochloride, stirring the reaction at room temperature, and after the reaction is completed, dialyzing with sodium chloride solution, reducing the reaction with dithiothreitol at pH=7.5-8.5, dialyzing, and freeze-drying to obtain thiolated hyaluronic acid; dissolving 1.5-2.5 parts of dihydromyricetin in 40-60 parts of anhydrous tetrahydrofuran, adding 0.4-0.8 parts of 4-carboxyphenylboronic acid and 0.5-1.0 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3 parts of vitamin E. - 0.6 parts of 4-dimethylaminopyridine were reacted at 50-60℃. After the reaction was completed, the mixture was rotary evaporated and recrystallized with a methanol-water mixture to obtain dihydromyricetin-phenylboronic acid ester. B2. 1.5-2.5 parts of thiolated hyaluronic acid were dissolved in 80-120 parts of phosphate buffer solution with pH 7.0-7.

8. 0.3-0.6 parts of dihydromyricetin-phenylboronic acid ester and 0.15-0.35 parts of vitamin E succinate were added. 0.2-0.5 parts of 4-dimethylaminopyridine were added, followed by 0.3-0.7 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.2-0.5 parts of N-hydroxythiosuccinimide. The mixture was reacted at 25-35℃ to obtain a reaction solution. The reaction solution was dialyzed and freeze-dried.

8. The method for preparing the anti-aging composition containing nanospheres according to claim 7, characterized in that, In step B1, the reaction time is 6-10 hours at 50-60°C.

9. An anti-aging composition containing nanospheres, characterized in that, The anti-aging composition containing nanospheres is prepared by the method described in any one of claims 1-8.

10. The application of the anti-aging composition containing nanospheres according to claim 9, characterized in that, Application of the anti-aging composition containing nanospheres in skin care products.

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