Application of chondroitin sulfate A / resveratrol micelle and antioxidant repair essence

Abstract

The invention relates to the technical field of cosmetics, and discloses application of chondroitin sulfate A / resveratrol micelles in preparation of antioxidant products or cell oxidative damage repair products. In addition, the invention further provides the antioxidant repair essence. The chondroitin sulfate A / resveratrol micelle has better antioxidant effect and oxidation repair effect, and is high in stability, safe and non-toxic.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to the application of chondroitin sulfate A / resveratrol micelles. Furthermore, this invention also relates to an antioxidant repair serum. Background Technology

[0002] Oxidative stress is a state of imbalance between the production and clearance of reactive oxygen species (ROS) in the body. Excessive ROS can attack biomolecules, leading to cell damage, tissue inflammation, and aging. Therefore, there is a huge market demand for exogenous antioxidants in the pharmaceutical, cosmetic, and functional food industries.

[0003] Currently, the mainstream antioxidants on the market are mainly divided into two categories: synthetic antioxidants and traditional natural antioxidants. Synthetic antioxidants include butylated hydroxytoluene (BHA) and butylated hydroxyanisole (BHT). While these antioxidants have strong antioxidant effects, long-term use poses potential cytotoxicity and safety risks. Traditional natural antioxidants include vitamin C (VC) and vitamin E (VE). Although these antioxidants have relatively high safety, they suffer from poor stability (VC is easily oxidized and inactivated, and is unstable when exposed to light and heat), low bioavailability (some fat-soluble components are difficult to absorb), and limited specific functions. Summary of the Invention

[0004] The purpose of this invention is to overcome the safety risks of synthetic antioxidants and the problems of poor stability, low bioavailability, and limited function of traditional natural antioxidants in the existing technology. It provides an application of chondroitin sulfate A / resveratrol micelles and an antioxidant repair essence. The chondroitin sulfate A / resveratrol micelles have good antioxidant and antioxidant repair effects, and are highly stable, safe and non-toxic.

[0005] To achieve the above objectives, the first aspect of the present invention provides the application of chondroitin sulfate A / resveratrol micelles in the preparation of antioxidant products.

[0006] Preferably, the antioxidant product is an antioxidant product that scavenge DPPH free radicals and / or superoxide anion free radicals.

[0007] Preferably, the preparation steps of the chondroitin sulfate A / resveratrol micelles include: mixing a solution containing resveratrol and a solution containing chondroitin sulfate A under ultrasonic conditions.

[0008] Preferably, the ultrasonic conditions include: power of 150-250W, frequency of 30-50kHz, and duration of 20-40min.

[0009] Preferably, the mixing step includes: adding a solution containing resveratrol dropwise to the solution containing chondroitin sulfate A.

[0010] More preferably, the concentration of resveratrol in the solution containing resveratrol is 2-4 mg / mL, and the concentration of chondroitin sulfate A in the solution containing chondroitin A is 3-8 mg / mL.

[0011] Preferably, the preparation step of the chondroitin sulfate A / resveratrol micelles further includes: dialysis treatment of the mixture obtained by mixing, wherein the dialysis bag used for dialysis has a molecular weight cutoff of 3500-4000 Da.

[0012] Preferably, the amount of resveratrol used is 3-8 mg relative to 100 mg of chondroitin sulfate A.

[0013] Preferably, the product is selected from at least one of topical skin care products, health products, and pharmaceuticals.

[0014] A second aspect of this invention provides the application of chondroitin sulfate A / resveratrol micelles in the preparation of products for repairing cellular oxidative damage.

[0015] Preferably, the preparation steps of the chondroitin sulfate A / resveratrol micelles include: mixing a solution containing resveratrol and a solution containing chondroitin sulfate A under ultrasonic conditions.

[0016] Preferably, the ultrasonic conditions include: power of 150-250W, frequency of 30-50kHz, and duration of 20-40min.

[0017] Preferably, the mixing step includes: adding a solution containing resveratrol dropwise to the solution containing chondroitin sulfate A.

[0018] More preferably, the concentration of resveratrol in the solution containing resveratrol is 2-4 mg / mL, and the concentration of chondroitin sulfate A in the solution containing chondroitin A is 3-8 mg / mL.

[0019] Preferably, the preparation step of the chondroitin sulfate A / resveratrol micelles further includes: dialysis treatment of the mixture obtained by mixing, wherein the dialysis bag used for dialysis has a molecular weight cutoff of 3500-4000 Da.

[0020] Preferably, the amount of resveratrol used is 3-8 mg relative to 100 mg of chondroitin sulfate A.

[0021] Preferably, the product is selected from at least one of topical skin care products, health products, and pharmaceuticals.

[0022] A third aspect of the present invention provides an antioxidant repair serum containing chondroitin sulfate A / resveratrol micelles.

[0023] Preferably, based on the total mass of the antioxidant repair essence as 100%, the content of chondroitin sulfate A / resveratrol micelles is 1-3%.

[0024] Preferably, the preparation steps of the chondroitin sulfate A / resveratrol micelles include: mixing a solution containing resveratrol and a solution containing chondroitin sulfate A under ultrasonic conditions.

[0025] Preferably, the ultrasonic conditions include: power of 150-250W, frequency of 30-50kHz, and duration of 20-40min.

[0026] Preferably, the mixing step includes: adding a solution containing resveratrol dropwise to the solution containing chondroitin sulfate A.

[0027] More preferably, the concentration of resveratrol in the solution containing resveratrol is 2-4 mg / mL, and the concentration of chondroitin sulfate A in the solution containing chondroitin A is 3-8 mg / mL.

[0028] Preferably, the preparation step of the chondroitin sulfate A / resveratrol micelles further includes: dialysis treatment of the mixture obtained by mixing, wherein the dialysis bag used for dialysis has a molecular weight cutoff of 3500-4000 Da.

[0029] Preferably, the amount of resveratrol used is 3-8 mg relative to 100 mg of chondroitin sulfate.

[0030] Preferably, the antioxidant repair serum also contains sodium hyaluronate, butylene glycol, vitamin B5, and water; Preferably, based on the total mass of the antioxidant repair essence as 100%, the content of sodium hyaluronate is 0.1-0.3%, the content of butylene glycol is 3-5%, the content of vitamin B5 is 0.8-1.2%, and the balance is water.

[0031] Through the above technical solution, chondroitin sulfate A in chondroitin sulfate A / resveratrol micelles can interact with resveratrol through a specific sulfation structure and its amphiphilic properties, thereby improving its free radical scavenging effect. At the same time, it can also enhance the activity of intracellular antioxidant enzymes and improve its repair effect on cellular oxidative damage. It can be used to prepare antioxidant products and cellular oxidative damage repair products, and has the advantages of high stability, safety and non-toxicity. Attached Figure Description

[0032] Figure 1This is the micelle solution prepared in Example 1. Detailed Implementation

[0033] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0034] As previously stated, the first aspect of this invention provides the application of chondroitin sulfate A / resveratrol micelles in the preparation of antioxidant products.

[0035] During their research, the inventors discovered that chondroitin sulfate A in chondroitin sulfate A / resveratrol micelles can interact with resveratrol through a specific sulfation structure and its amphiphilic properties, thereby enhancing its free radical scavenging effect. It can also increase the activity of intracellular antioxidant enzymes and improve its repair effect on cellular oxidative damage. It can be used to prepare antioxidant products and cellular oxidative damage repair products, and has the advantages of high stability, safety and non-toxicity.

[0036] Preferably, the antioxidant product is an antioxidant product that scavenges DPPH free radicals and / or superoxide anion free radicals. Chondroitin sulfate A / resveratrol micelles have a better scavenging effect on DPPH free radicals and superoxide anion free radicals, and therefore have better applications in the preparation of antioxidant products that scavenge DPPH free radicals and / or superoxide anion free radicals.

[0037] Preferably, the preparation steps of the chondroitin sulfate A / resveratrol micelles include: mixing a solution containing resveratrol and a solution containing chondroitin sulfate A under ultrasonic conditions. The chondroitin sulfate A / resveratrol micelles prepared by the above method exhibit better antioxidant properties, cell oxidative damage repair effects, and structural stability.

[0038] Preferably, the ultrasonic conditions include: a power of 150-250W, which can be 150W, 170W, 190W, 210W, 230W, 250W, or any value within any two of the above ranges; a frequency of 30-50kHz, which can be 30kHz, 35kHz, 40kHz, 45kHz, 50kHz, or any value within any two of the above ranges; and a time of 20-40min, which can be 20min, 25min, 30min, 35min, 40min, or any value within any two of the above ranges. Controlling the ultrasonic conditions within the above ranges can further improve the yield and structural stability of chondroitin sulfate A / resveratrol micelles.

[0039] According to the present invention, preferably, the mixing step includes: adding a solution containing resveratrol dropwise to the solution containing chondroitin sulfate A. This can further improve the reaction effect of resveratrol and chondroitin sulfate A, thereby further improving the yield and structural stability of chondroitin sulfate A / resveratrol micelles.

[0040] From the perspective of further improving the yield of chondroitin sulfate A / resveratrol micelles, preferably, the concentration of resveratrol in the solution containing resveratrol is 2-4 mg / mL, which can be 2 mg / mL, 3 mg / mL, 4 mg / mL, or any value within any two of the above ranges; the concentration of chondroitin sulfate A in the solution containing chondroitin sulfate is 3-8 mg / mL, which can be 3 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, or any value within any two of the above ranges.

[0041] Preferably, the preparation step of the chondroitin sulfate A / resveratrol micelles further includes: dialysis of the obtained mixture, wherein the dialysis bag used for dialysis has a molecular weight cutoff of 3500-4000 Da, which can be 3500 Da, 3600 Da, 3700 Da, 3800 Da, 3900 Da, 4000 Da, or any value within any two of the above ranges. Controlling the molecular weight of the chondroitin sulfate A / resveratrol micelles within the above range can further improve the antioxidant effect and cell oxidative damage repair effect of the chondroitin sulfate A / resveratrol micelles.

[0042] In order to further improve the antioxidant effect and cell oxidative damage repair effect of the prepared chondroitin sulfate A / resveratrol micelles, preferably, the amount of resveratrol used is 3-8 mg relative to 100 mg of chondroitin sulfate A, which can be 3 mg, 4 mg, 6 mg, 8 mg, or any value within any two of the above ranges.

[0043] Preferably, the product is selected from at least one of topical skin care products, health products, and pharmaceuticals. More preferably, it is a topical skin care product, such as a skin cream, skin lotion, skin lotion, skin serum, or ointment.

[0044] According to the present invention, chondroitin A sulfate is commercially available or can be prepared. As a specific embodiment of the present invention, chondroitin A sulfate is obtained by enzymatic extraction from bovine tracheal cartilage. Resveratrol is commercially available.

[0045] A second aspect of this invention provides the application of chondroitin sulfate A / resveratrol micelles in the preparation of products for repairing cellular oxidative damage.

[0046] Studies have found that chondroitin sulfate A in chondroitin sulfate A / resveratrol micelles can interact with resveratrol through a specific sulfation structure and its amphiphilic properties, thereby enhancing its free radical scavenging effect. It can also increase the activity of intracellular antioxidant enzymes and improve its repair effect on cellular oxidative damage. It can be used to prepare antioxidant products and cellular oxidative damage repair products, and has the advantages of high stability, safety and non-toxicity.

[0047] Preferably, the preparation steps of the chondroitin sulfate A / resveratrol micelles include: mixing a solution containing resveratrol and a solution containing chondroitin sulfate A under ultrasonic conditions. The chondroitin sulfate A / resveratrol micelles prepared by the above method exhibit better antioxidant properties, cell oxidative damage repair performance, and structural stability.

[0048] Preferably, the ultrasonic conditions include: a power of 150-250W, which can be 150W, 170W, 190W, 210W, 230W, 250W, or any value within any two of the above ranges; a frequency of 30-50kHz, which can be 30kHz, 35kHz, 40kHz, 45kHz, 50kHz, or any value within any two of the above ranges; and a time of 20-40min, which can be 20min, 25min, 30min, 35min, 40min, or any value within any two of the above ranges. Controlling the ultrasonic conditions within the above ranges can further improve the yield and structural stability of chondroitin sulfate A / resveratrol micelles.

[0049] According to the present invention, preferably, the mixing step includes: adding a solution containing resveratrol dropwise to the solution containing chondroitin sulfate A. This can further improve the reaction effect of resveratrol and chondroitin sulfate A, thereby further improving the yield and structural stability of chondroitin sulfate A / resveratrol micelles.

[0050] From the perspective of further improving the yield of chondroitin sulfate A / resveratrol micelles, preferably, the concentration of resveratrol in the solution containing resveratrol is 2-4 mg / mL, which can be 2 mg / mL, 3 mg / mL, 4 mg / mL, or any value within any two of the above ranges; the concentration of chondroitin sulfate A in the solution containing chondroitin sulfate is 3-8 mg / mL, which can be 3 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, or any value within any two of the above ranges.

[0051] Preferably, the preparation step of the chondroitin sulfate A / resveratrol micelles further includes: dialysis of the obtained mixture, wherein the dialysis bag used for dialysis has a molecular weight cutoff of 3500-4000 Da, which can be 3500 Da, 3600 Da, 3700 Da, 3800 Da, 3900 Da, 4000 Da, or any value within any two of the above ranges. Controlling the molecular weight of the chondroitin sulfate A / resveratrol micelles within the above range can further improve the antioxidant effect and cell oxidative damage repair effect of the chondroitin sulfate A / resveratrol micelles.

[0052] In order to further improve the antioxidant effect and cell oxidative damage repair effect of the prepared chondroitin sulfate A / resveratrol micelles, preferably, the amount of resveratrol used is 3-8 mg relative to 100 mg of chondroitin sulfate A, which can be 3 mg, 4 mg, 6 mg, 8 mg, or any value within any two of the above ranges.

[0053] Preferably, the product is selected from at least one of topical skin care products, health products, and pharmaceuticals. More preferably, it is a topical skin care product, such as a skin cream, skin lotion, skin lotion, skin serum, or ointment.

[0054] A third aspect of the present invention provides an antioxidant repair serum containing chondroitin sulfate A / resveratrol micelles.

[0055] Research has found that the chondroitin A sulfate / resveratrol micelles in this antioxidant repair serum have good antioxidant and cell damage repair effects, and can be used as an effective ingredient in antioxidant repair serums.

[0056] Preferably, based on the total mass of the antioxidant repair serum (100%), the content of chondroitin sulfate A / resveratrol micelles is 1-3%, which can be 1%, 1.5%, 2%, 2.5%, 3%, or any value within any two of the above ranges. Controlling the content of chondroitin sulfate A / resveratrol micelles within the above range ensures its antioxidant and cell-damage repair effects while reducing its usage, thereby lowering the cost of the antioxidant repair serum.

[0057] Preferably, the preparation steps of the chondroitin sulfate A / resveratrol micelles include: mixing a solution containing resveratrol and a solution containing chondroitin sulfate A under ultrasonic conditions. The chondroitin sulfate A / resveratrol micelles prepared by the above method exhibit better antioxidant properties, cell damage repair effects, and structural stability.

[0058] Preferably, the ultrasonic conditions include: a power of 150-250W, which can be 150W, 170W, 190W, 210W, 230W, 250W, or any value within any two of the above ranges; a frequency of 30-50kHz, which can be 30kHz, 35kHz, 40kHz, 45kHz, 50kHz, or any value within any two of the above ranges; and a time of 20-40min, which can be 20min, 25min, 30min, 35min, 40min, or any value within any two of the above ranges. Controlling the ultrasonic conditions within the above ranges can further improve the yield and structural stability of chondroitin sulfate A / resveratrol micelles.

[0059] According to the present invention, preferably, the mixing step includes: adding a solution containing resveratrol dropwise to the solution containing chondroitin sulfate A. This can further improve the reaction effect of resveratrol and chondroitin sulfate A, thereby further improving the yield of chondroitin sulfate A / resveratrol micelles.

[0060] From the perspective of further improving the yield of chondroitin sulfate A / resveratrol micelles, preferably, the concentration of resveratrol in the solution containing resveratrol is 2-4 mg / mL, which can be 2 mg / mL, 3 mg / mL, 4 mg / mL, or any value within any two of the above ranges; the concentration of chondroitin sulfate A in the solution containing chondroitin sulfate is 3-8 mg / mL, which can be 3 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, or any value within any two of the above ranges.

[0061] Preferably, the preparation step of the chondroitin sulfate A / resveratrol micelles further includes: dialysis of the obtained mixture, wherein the dialysis bag used for dialysis has a molecular weight cutoff of 3500-4000 Da, which can be 3500 Da, 3600 Da, 3700 Da, 3800 Da, 3900 Da, 4000 Da, or any value within any two of the above ranges. Controlling the molecular weight of the chondroitin sulfate A / resveratrol micelles within the above range can further improve the antioxidant effect and cell oxidative damage repair effect of the chondroitin sulfate A / resveratrol micelles.

[0062] In order to further improve the antioxidant effect and cell oxidative damage repair effect of the prepared chondroitin sulfate A / resveratrol micelles, preferably, the amount of resveratrol used is 3-8 mg relative to 100 mg of chondroitin sulfate A, which can be 3 mg, 4 mg, 6 mg, 8 mg, or any value within any two of the above ranges.

[0063] Preferably, the antioxidant repair serum further contains sodium hyaluronate, butylene glycol, vitamin B5, and water. More preferably, based on the total mass of the antioxidant repair serum (100%), the sodium hyaluronate content is 0.1-0.3%, which can be 0.1%, 0.2%, 0.3%, or any value within any two of the above ranges; the butylene glycol content is 3-5%, which can be 3%, 4%, 5%, or any value within any two of the above ranges; the vitamin B5 content is 0.8-1.2%, which can be 0.8%, 0.9%, 1%, 1.1%, 1.2%, or any value within any two of the above ranges; the balance is water. Through the synergistic effect of the specific amounts of vitamin B5, sodium hyaluronate, succinic acid, and chondroitin sulfate A / resveratrol micelles mentioned above, the antioxidant properties and stability of the antioxidant repair serum can be further improved.

[0064] According to the present invention, the sodium hyaluronate used in the present invention can be a low molecular weight sodium hyaluronate with a molecular weight of 200,000 to 400,000 Daltons.

[0065] The antioxidant repair serum provided by this invention can be obtained by mixing the above-mentioned components.

[0066] The present invention will be described in detail below through examples. In the following examples, the protease was purchased from Yuanye Biotechnology Co., Ltd.

[0067] Preparation and purification of chondroitin sulfate A 1. Raw material pretreatment and crude polysaccharide extraction Raw material processing: Select tracheal cartilage from healthy cattle, remove fat, connective tissue, and bone tissue, and clean with physiological saline. Chop or homogenize the cartilage into pieces approximately 2mm in size. 3 Small pieces are used to increase the contact area for subsequent enzymatic hydrolysis.

[0068] Enzymatic extraction: Hydrolysis was performed using a protease at pH 7.0±1 and 50℃ for 8 hours to release glycosaminoglycan chains. After enzymatic hydrolysis, the protease was inactivated by heating at 100℃ for 10 minutes, and the mixture was centrifuged (8000×g, 20 minutes) to remove insoluble residues and collect the supernatant.

[0069] Preliminary purification: The supernatant was decolorized and small molecule impurities were removed by passing it through a macroporous adsorption resin. Ethanol fractionation precipitation was then performed: 3 times the volume of pre-cooled anhydrous ethanol was slowly added to the supernatant, and the mixture was allowed to stand overnight at 4°C. The precipitate was collected by centrifugation to obtain crude polysaccharides (mainly a mixture of chondroitin sulfate).

[0070] 2. Ion exchange column chromatography purification Column preparation: (1) Column packing: DEAE-Sepharose Fast Flow (strong anion exchange medium).

[0071] (2) Column specifications: The column bed volume (CV) is 20 times the dry weight of crude polysaccharide, and the column height to diameter ratio is 10:1.

[0072] (3) Pretreatment: Rinse with 0.5M NaOH, 0.5M HCl and deionized water in sequence, and finally equilibrate with starting buffer (20mM Tris-HCl, pH 8.0) until pH and conductivity are stable.

[0073] Sample loading and elution: (1) Dissolve the crude polysaccharide in the starting buffer, filter it through a 0.22 μm filter membrane and then load it onto the column. The amount of sample loaded should not exceed 50% of the column binding capacity.

[0074] (2) Use linear or step NaCl gradient elution and monitor UV absorption and conductivity.

[0075] (3) CS-A was eluted in the range of 0.5-0.8M NaCl, and the eluted fractions corresponding to the main peak were collected.

[0076] 3. Desalination and drying Dialysis desalination The collected CS-A eluent was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in deionized water at 4°C for 24 hours, with the water changed every 6 hours, until the conductivity of the dialysate was close to that of deionized water.

[0077] Concentration and freeze drying The dialyzed solution can be reduced to a viscous state by rotary evaporation (≤40℃).

[0078] The powder was transferred to a freeze-drying bottle, pre-frozen at -80°C for 6 hours, and then freeze-dried for 24 hours to obtain a white to off-white fluffy powder.

[0079] Infrared spectroscopy and HPLC analysis confirmed that its sulfation site is mainly located at the C-4 position of N-acetylgalactosamine.

[0080] Preparation Example 1 (1) Weigh 100 mg of chondroitin sulfate A (CS-A) and dissolve it in 20 mL of deionized water to obtain a solution containing chondroitin sulfate A.

[0081] Weigh 5 mg of resveratrol and dissolve it in 2 mL of anhydrous ethanol to obtain a solution containing resveratrol.

[0082] (2) Under ultrasonic stirring (200W, 40kHz), the solution containing resveratrol was slowly added dropwise to the solution containing chondroitin sulfate A. After continuous sonication for 30 minutes, the solution was placed in a dialysis bag (MWCO 3500Da) and dialyzed in pure water for 24 hours to remove ethanol and unencapsulated free resveratrol, yielding the product as shown. Figure 1 As shown, chondroitin sulfate / resveratrol nanomicelle powder was obtained by freeze-drying.

[0083] Preparation Example 2 (1) Weigh 60 mg of chondroitin sulfate A (CS-A) and dissolve it in 20 mL of deionized water to obtain a solution containing chondroitin sulfate A.

[0084] Weigh 4 mg of resveratrol and dissolve it in 2 mL of anhydrous ethanol to obtain a solution containing resveratrol.

[0085] (2) Under ultrasonic stirring (150W, 30kHz), the solution containing resveratrol was slowly added dropwise to the solution containing chondroitin sulfate A. After continuous ultrasonication for 40min, the solution was placed in a dialysis bag (MWCO 3800Da) and dialyzed in pure water for 24 hours to remove ethanol and unencapsulated free resveratrol. After freeze drying, chondroitin sulfate / resveratrol nanomicelle powder was obtained.

[0086] Preparation Example 3 (1) Weigh 120 mg of chondroitin sulfate A (CS-A) and dissolve it in 20 mL of deionized water to obtain a solution containing chondroitin sulfate A.

[0087] Weigh 8 mg of resveratrol and dissolve it in 2 mL of anhydrous ethanol to obtain a solution containing resveratrol.

[0088] (2) Under ultrasonic stirring (550W, 50kHz), the solution containing resveratrol was slowly added dropwise to the solution containing chondroitin sulfate A. After continuous ultrasonication for 20min, the solution was placed in a dialysis bag (MWCO 4000Da) and dialyzed in pure water for 24 hours to remove ethanol and unencapsulated free resveratrol. After freeze drying, chondroitin sulfate / resveratrol nanomicelle powder was obtained.

[0089] Preparation Example 4 Nanomicelle powder was prepared according to the method of Preparation Example 1, except that resveratrol was replaced with curcumin.

[0090] Preparation Example 5 Nanomicelle powder was prepared according to the method of Preparation Example 1, except that resveratrol was replaced with quercetin.

[0091] Preparation Example 6 Nanomicelle powder was prepared according to the method of Preparation Example 1, except that chondroitin sulfate A was replaced with chondroitin sulfate C.

[0092] Example 1 The chondroitin sulfate / resveratrol nanomicelle powder prepared in Preparation Example 1 was used to prepare composite solutions of different concentrations. The scavenging rate was determined by the DPPH method, and the removal effect on superoxide anion free radicals at a concentration of 2.0 mg / mL was determined by the pyrogallol autoxidation method. The DPPH free radical scavenging ability data are shown in Table 1, and the superoxide anion free radical scavenging ability is shown in Table 2.

[0093] Table 1. Comparative Test Results of DPPH Free Radical Scavenging Capacity

[0094] Table 2 Results of Superoxide Anion Scavenging Capacity Test

[0095] As shown in Tables 1 and 2, the chondroitin sulfate A / resveratrol micelles of the present invention exhibit excellent clearance rates even at low concentrations (0.5 mg / mL), significantly superior to single CS-A / positive control VC and other control groups, demonstrating a synergistic effect between chondroitin sulfate A and resveratrol in the chondroitin sulfate A / resveratrol micelles. Furthermore, it exhibits the strongest clearance ability against superoxide anions (IC50 = 0.65 mg / mL), demonstrating good superoxide anion clearance capacity.

[0096] Example 2 Human skin fibroblasts (HSF) were cultured, and an oxidative damage model was established using H2O2. MTT (cell viability), SOD activity, and MDA content were measured and recorded in Table 3.

[0097] MTT:RAW264.7 cells were cultured in DMEM medium containing 10 wt% FBS and 1 wt% penicillin antibiotics at 37°C in a 5% CO2 incubator until 80% confluence, and then a cell suspension was prepared. The cells were then sputtered at a rate of 1 × 10⁻⁶ cells / mL. 4 Cells were seeded per well in 96-well plates and cultured for 24 hours. The culture medium was then replaced with a medium containing different drugs, while the control group received an equal volume of medium. After 24 hours of incubation, cell viability was determined using the MTT assay.

[0098] SOD: Superoxide dismutase (SOD) activity was assessed using the WST-8 assay. RAW264.7 cells were seeded in 6-well plates and treated with different drugs after LPS stimulation. After cell lysis, the supernatant was collected by centrifugation, and protein concentration was determined by the BCA method. SOD activity was calculated according to the kit instructions.

[0099] MDA: Malondialdehyde (MDA) content was assessed using the thiobarbituric acid (TBA) method. Cells were treated as described above, lysed, and centrifuged to collect the supernatant. The MDA content was calculated according to the kit instructions.

[0100] Table 3

[0101] Example 3-1 The CS-A / resveratrol micelle lyophilized powder prepared in Preparation Example 1, low molecular weight sodium hyaluronate, butylene glycol, vitamin B5 and water were mixed evenly to obtain an antioxidant repair essence.

[0102] The antioxidant repair essence contains 2.0 wt% CS-A / resveratrol micelle freeze-dried powder, 0.2 wt% low molecular weight sodium hyaluronate, 4.0 wt% butylene glycol, and 1.0% vitamin B5.

[0103] Example 3-2 The CS-A / resveratrol micelle lyophilized powder prepared in Preparation Example 2, low molecular weight sodium hyaluronate, butylene glycol, vitamin B5 and water were mixed evenly to obtain an antioxidant repair essence.

[0104] The antioxidant repair essence contains 3.0 wt% CS-A / resveratrol micelle freeze-dried powder, 0.3 wt% low molecular weight sodium hyaluronate, 3.0 wt% butylene glycol, and 0.8% vitamin B5.

[0105] Example 3-3 The CS-A / resveratrol micelle lyophilized powder prepared in Preparation Example 3, low molecular weight sodium hyaluronate, butylene glycol, vitamin B5 and water were mixed evenly to obtain an antioxidant repair essence.

[0106] The antioxidant repair essence contains 1.0 wt% CS-A / resveratrol micelle freeze-dried powder, 0.1 wt% low molecular weight sodium hyaluronate, 5.0 wt% butylene glycol, and 1.2% vitamin B5.

[0107] Comparative Example 3-1 The serum was prepared according to the method in Example 3-1, except that the CS-A / resveratrol micelle lyophilized powder was replaced with vitamin C.

[0108] Comparative Example 3-2 The essence was prepared according to the method of Example 3-1, except that the CS-A / resveratrol micelle lyophilized powder prepared in Preparation Example 1 was replaced with the nano micelles prepared in Preparation Example 4.

[0109] Comparative Example 3-3 The essence was prepared according to the method of Example 3-1, except that the CS-A / resveratrol micelle lyophilized powder prepared in Preparation Example 1 was replaced with the nano micelles prepared in Preparation Example 5.

[0110] Comparative Examples 3-4 The essence was prepared according to the method of Example 3-1, except that the CS-A / resveratrol micelle lyophilized powder prepared in Preparation Example 1 was replaced with the nano micelles prepared in Preparation Example 6.

[0111] Comparative Examples 3-5 The essence was prepared according to the method of Example 3-1, except that the CS-A / resveratrol micelle lyophilized powder was replaced with CS-A and resveratrol in a mass ratio of 20:1.

[0112] Test case Stability test: The serum was placed in a 45℃ constant temperature chamber for accelerated aging test. The results are shown in Tables 4-5.

[0113] Resveratrol retention test: UV-Vis spectrophotometry Sample pretreatment: Solution A (total resveratrol): Accurately measure 0.5 mL of the essence, dilute with methanol to 5 mL and shake vigorously to completely destroy the micelles, then bring the volume to a certain level. Allow to stand or centrifuge and collect the supernatant.

[0114] Solution B (Free Resveratrol): Accurately measure 0.5 mL of the extract and dilute with 5 mL of pure water, ensuring the integrity of the micelle structure. Then centrifuge at high speed (15,000 rpm, 30 minutes). The micelles form a dense layer; carefully collect the supernatant, which contains the free drug.

[0115] UV inspection: Dilute 1 mL of solution A and solution B with the corresponding solvent (methanol or water) to 10 mL, and measure the absorbance at a wavelength of 306 nm.

[0116] Standard samples with concentrations of 0.01-10 mg / mL were prepared using resveratrol standards in methanol and aqueous solutions. The absorbance at different concentrations (0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL) was then measured, and a standard curve was plotted.

[0117] calculate: The total resveratrol concentration was calculated using the absorbance of solution A.

[0118] The concentration of free resveratrol was calculated using the absorbance of solution B.

[0119] Micellar encapsulation concentration = total concentration - free concentration.

[0120] Retention rate calculation method: (Micelle encapsulation concentration after placement / Initial micelle encapsulation concentration) × 100% Resveratrol Retention Rate Test: Determination of Sulfate Content Sample preparation: Take 1.0 mL of the essence sample and hydrolyze it in a boiling water bath (100℃) for 5 h with 2.0 mL of 4M hydrochloric acid solution to completely release the sulfate groups on CS-A into sulfate ions.

[0121] Colorimetric determination: Take 2 mL of hydrolysate, add 3 mL of freshly prepared barium chloride-gelatin reagent, mix, and immediately vortex vigorously for 30 seconds. After standing at room temperature for 5 minutes, measure the absorbance.

[0122] Preparation of barium chloride-gelatin reagent: Add 5 mL of 0.5 M barium chloride solution to 5 mL of 1 wt% gelatin solution, then add 0.5 g of sodium chloride, and finally dilute with pure water to a total volume of 50 mL and mix thoroughly.

[0123] Standard curve: A standard curve for sulfate content was prepared using potassium sulfate standard solution (0-200 μg / mL).

[0124] Calculate the retention rate: The sulfate content in fresh and aged samples was measured and converted into CS-A content.

[0125] CS-A retention rate (%) = (sulfate content after storage / initial sulfate content) × 100% Table 4. Comparison of Product Stability under Accelerated High Temperature Test at 45℃ - 1 (30 days)

[0126] Table 5. Comparison of Product Stability under Accelerated High Temperature Test at 45℃ - 2 (30 days)

[0127] The results in Tables 4 and 5 show that the traditional VC formulation and the formulation provided in the comparative example showed obvious oxidative discoloration after two weeks, while the formulation of the present invention remained stable during the 30-day high-temperature test, which proves the protective effect of the CS-A carrier on the active ingredients.

[0128] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Application of chondroitin sulfate A / resveratrol micelles in the preparation of antioxidant products.

2. The application according to claim 1, characterized in that, The antioxidant product is an antioxidant product that scavenge DPPH free radicals and / or superoxide anion free radicals.

3. Application of chondroitin sulfate A / resveratrol micelles in the preparation of cell oxidative damage repair products.

4. The application according to any one of claims 1 to 3, characterized in that, The preparation steps of the chondroitin sulfate A / resveratrol micelles include: mixing a solution containing resveratrol and a solution containing chondroitin sulfate A under ultrasonic conditions.

5. The application according to claim 4, characterized in that, The ultrasound conditions include: power of 150-250W, frequency of 30-50kHz, duration of 20-40min, and / or, The mixing step includes: adding a solution containing resveratrol dropwise to the solution containing chondroitin sulfate A; Preferably, the concentration of resveratrol in the solution containing resveratrol is 2-4 mg / mL, and the concentration of chondroitin sulfate A in the solution containing chondroitin A is 3-6 mg / mL.

6. The application according to claim 4, characterized in that, The preparation steps of the chondroitin sulfate A / resveratrol micelles further include: dialysis of the obtained mixture, wherein the dialysis bag used for the dialysis has a molecular weight cutoff of 3500-4000 Da; and / or, Preferably, the amount of resveratrol used is 3-8 mg relative to 100 mg of chondroitin sulfate A.

7. The application according to any one of claims 1 to 3, characterized in that, The product is selected from at least one of topical skin care products, health products, and pharmaceuticals.

8. An antioxidant repair serum, characterized in that, The antioxidant repair serum contains chondroitin sulfate A / resveratrol micelles.

9. The antioxidant repair serum according to claim 8, characterized in that, Based on the total mass of the antioxidant repair essence as 100%, the content of chondroitin sulfate A / resveratrol micelles is 1-3%.

10. The antioxidant repair serum according to claim 8 or 9, characterized in that, The antioxidant repair serum also contains sodium hyaluronate, butylene glycol, vitamin B5, and water; Preferably, based on the total mass of the antioxidant repair essence as 100%, the content of sodium hyaluronate is 0.1-0.3%, the content of butylene glycol is 3-5%, and the content of vitamin B5 is 0.8-1.2%.

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