A modified hyaluronic acid complex with antibacterial whitening effect and its application in medical and cosmetic fillers

Abstract

This invention belongs to the field of biomaterials technology and relates to a modified hyaluronic acid complex with antibacterial and whitening effects and its application in medical aesthetic fillers. This invention employs ultraviolet light reduction technology, using an aqueous hyaluronic acid solution as a polymer dispersion platform to construct a silver nanoparticle dispersion system. After drying, this system is converted into HA-Ag powder, which synergistically interacts with carboxymethyl chitosan to endow the product with excellent antibacterial properties. Microencapsulation technology is introduced, selecting astaxanthin as the core component. Relying on the protection of sodium hyaluronate, the stable existence time of astaxanthin is extended, significantly improving the product's antioxidant capacity. Vitamin C and arbutin are incorporated into the microcapsule wall material and then mixed with nicotinamide. While enhancing the whitening effect, vitamin C can reduce oxidized astaxanthin, restoring its antioxidant activity and further amplifying the antioxidant effect.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology and relates to hyaluronic acid, particularly to a modified hyaluronic acid complex with antibacterial and whitening effects and its application in medical aesthetic fillers. Background Technology

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a high-molecular-weight mucopolysaccharide composed of disaccharide units linked by glucuronic acid and acetylglucosamine. It is a major component of the intercellular matrix and extracellular matrix, playing a crucial role in cellular physiological functions. Hyaluronic acid is widely distributed in the epithelium, connective tissue, and nerve tissue of vertebrates, and is particularly abundant in skin tissue, synovial fluid, and the eye.

[0003] Currently, hyaluronic acid fillers are widely used in the treatment of wrinkles, scars, and facial contour defects. They are classified into monophasic and biphasic types based on the degree of cross-linking. Monophasic fillers offer good stability and are particle-free. They are prepared by altering the ratio of high-molecular-weight hyaluronic acid to low-molecular-weight hyaluronic acid. They have low strength, are easily altered by external forces, and lack a granular texture. Biphasic fillers are the most widely used products on the market. They consist of stable hyaluronic acid gel particles and uncross-linked hyaluronic acid. The cross-linked hyaluronic acid particles, suspended within the uncross-linked hyaluronic acid, act as a lubricant, allowing the suspension to be injected into tissues through a fine needle.

[0004] Sodium hyaluronate has some antibacterial properties, mainly through its binding to fibrin and interfering with the function of bacterial surface adhesion factors. However, this antibacterial effect is relatively weak and primarily targets certain Gram-positive and Gram-negative bacteria. For other types of bacteria, or when the bacterial count is high, sodium hyaluronate's antibacterial effect is less effective. Sodium hyaluronate's whitening effect is also relatively limited. While it can indirectly improve uneven skin tone and dullness through moisturizing and promoting skin cell regeneration, it cannot directly inhibit melanin production or accelerate melanin metabolism. Therefore, achieving significant whitening effects solely through sodium hyaluronate is quite difficult. Sodium hyaluronate has some antioxidant properties, neutralizing free radicals and reducing environmental damage to the skin. However, this antioxidant effect is relatively weak and mainly limited to the skin's surface. When the skin is subjected to strong oxidative stress, such as ultraviolet radiation or environmental pollution, the antioxidant effect of sodium hyaluronate becomes insufficient.

[0005] To address the above issues, this invention employs ultraviolet light reduction technology, using an aqueous hyaluronic acid solution as a polymer dispersion platform to construct a silver nanoparticle dispersion system. After drying, this system is converted into HA-Ag powder, which synergistically interacts with carboxymethyl chitosan to endow the product with excellent antibacterial properties. Microencapsulation technology is introduced, selecting astaxanthin as the core component. Protected by sodium hyaluronate, the stable existence time of astaxanthin is extended, significantly enhancing the product's antioxidant capacity. Vitamin C and arbutin are incorporated into the microcapsule wall material, and then mixed with nicotinamide. While enhancing the whitening effect, vitamin C can reduce oxidized astaxanthin, restoring its antioxidant activity and further amplifying the antioxidant efficacy. Summary of the Invention

[0006] To address the above problems, this invention provides a modified hyaluronic acid complex with antibacterial and whitening effects and its application in medical aesthetic fillers. The specific preparation steps are as follows:

[0007] Preparation of S1, HA-Ag composite material: At room temperature, 0.1-0.3 g of sodium hyaluronate was weighed and dissolved in 100 mL of distilled water, and stirred evenly to obtain an HA aqueous solution. A 0.5 mol / L silver nitrate solution was prepared. 95 mL of the HA solution and 5 mL of the 0.5 mol / L silver nitrate solution were mixed and stirred for 5 h under light-protected conditions. The mixture was then irradiated with ultraviolet light for 10-30 min to obtain an HA-Ag composite aqueous solution. The HA-Ag composite powder was obtained by freeze-drying.

[0008] S2. Disperse 5-10g of chitosan in 50-100mL of isopropanol, sonicate for 5-10min, add 25-35mL of 38% NaOH solution, stir and mix evenly, add 18-22g of chloroacetic acid in 4 portions over 1h, stirring continuously and gradually raising the temperature to 60℃ during the addition process, and then react at a constant temperature of 60℃ for 4-6h. Afterwards, filter to obtain the solid product, dry it, and wash it with ethanol 3-5 times to remove impurities to obtain carboxymethyl chitosan.

[0009] S3. Extraction of astaxanthin: Under light-protected conditions, 1.1–2.4 g of Haematococcus pluvialis was dissolved in 26–30 mL of ethyl acetate. The mixture was extracted at 40 °C in a high-speed stirrer for 60 min to break the cell wall and obtain a mixed solution. After vacuum filtration, the crude astaxanthin extract was obtained. The extraction was repeated twice, and the filtrates were combined. The crude astaxanthin oleoresin was obtained by rotary evaporation. The crude astaxanthin oleoresin was obtained by vacuum drying in a freeze dryer for 24 h to remove the residual solvent. It was then stored in the dark.

[0010] S4. Weigh 0.8–1.1 g of glyceryl monostearate, 0.9–1.2 g of sucrose fatty acid ester, and 1.2–2.4 g of carboxymethyl chitosan obtained in step S2 and disperse them in 60–80 mL of distilled water at 90 °C. After cooling to 60 °C, add 1.7–2.5 g of HA-Ag complex powder obtained in step S1, 0.4–0.6 g of vitamin C, 0.28–0.36 g of arbutin, and 3.6–4.8 g of maltodextrin. Stir at high speed until homogeneous and cool to room temperature to obtain a mixed emulsion. Dissolve 1.0–1.4 g of astaxanthin oleoresin obtained in step S3 in the mixed emulsion and emulsify and shear at 10000 r / min for 8–12 min. After emulsification, atomize and dry at an inlet air temperature of 180 °C to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material.

[0011] S5. Dissolve the prepared astaxanthin microcapsules in 50-100 mL of PBS, add 0.1-0.3 mL of phenoxyethanol and 0.3-0.5 g of nicotinamide, stir for 10-15 min, and adjust the pH to 7.35-7.45 with 10% citric acid. Sonicate for 10-20 min, and then stir hydrothermally for 30-40 min at a temperature of 20-40℃ until a complete sol is formed. The final modified hyaluronic acid complex microcapsules with antibacterial and whitening effects can be obtained.

[0012] Preferably, in step S1, the amount of sodium hyaluronate used is 0.2g, the ultraviolet band is UVB, and the irradiation time is 20min.

[0013] Preferably, in step S2, the amount of chitosan used is 8g, isopropanol is 80mL, 38% NaOH solution is 30mL, chloroacetic acid is 10mL, the mixture is sonicated for 8min, stirred for 5h, and washed 4 times with ethanol.

[0014] Preferably, in step S3, the amount of Haematococcus pluvialis used is 1.8g and the amount of ethyl acetate is 28mL.

[0015] Preferably, in step S4, the amount of glyceryl monostearate is 0.9g, sucrose fatty acid ester is 1.1g, carboxymethyl chitosan is 1.8g, and distilled water is 70mL.

[0016] Preferably, in step S4, the HA-Ag complex powder is 2.1g, vitamin C is 0.32g, maltodextrin is 4.2g, and astaxanthin oleoresin is 1.2g.

[0017] Preferably, in step S5, the amount of PBS used is 80 mL, phenoxyethanol is 0.2 mL, and nicotine amide is 0.4 g.

[0018] Preferably, in step S5, the stirring time is 12 min, the pH is adjusted to 7.40, the sonication is performed for 15 min, the hydrothermal stirring is performed for 35 min, and the temperature is set for 30 min.

[0019] The technological advancements achieved by this invention due to the adoption of the above technical solution are as follows:

[0020] 1. The silver ions added in this invention are a broad-spectrum antibacterial agent with strong bactericidal effects against various bacteria, viruses, and fungi. When used as a microcapsule wall material, they can directly exert an antibacterial effect. Carboxymethyl chitosan is a natural polymer compound with antibacterial properties against various bacteria. When used as a microcapsule wall material, it can enhance the antibacterial effect. Sodium hyaluronate is a dextran uronic acid with moisturizing, lubricating, and skin-repairing effects. Although it does not directly possess antibacterial ability, when used as a microcapsule wall material, it can indirectly enhance the antibacterial effect through the following functions: the moisturizing and lubricating effects of sodium hyaluronate can maintain the stability of the internal environment of the microcapsule; the broad-spectrum antibacterial effect of silver ions can kill various bacteria; and the antibacterial activity and pH-regulating ability of carboxymethyl chitosan can further enhance the antibacterial effect. This complementary effect gives the microcapsules a broader antibacterial spectrum and stronger antibacterial ability. These three materials may produce a synergistic effect in the microcapsules, jointly disrupting the bacterial cell structure and metabolic processes, thereby enhancing the antibacterial effect.

[0021] 2. The vitamin C added in this invention, also known as ascorbic acid, is a powerful antioxidant. It can neutralize free radicals and reduce the damage of oxidative stress to the body. Astaxanthin is a natural antioxidant with very strong antioxidant activity. It is considered one of the strongest antioxidant single-molecule substances discovered in nature to date. It can scavenge various free radicals, thereby inhibiting lipid peroxidation reactions caused by free radicals and achieving an antioxidant effect. Astaxanthin also has an anti-ultraviolet effect, which can reduce the increase in lipid free radicals secreted due to sun exposure and help delay skin aging. Vitamin C can regenerate oxidized astaxanthin and restore its antioxidant activity.

[0022] 3. This invention utilizes the addition of Vitamin C and arbutin to the capsule wall, combined with nicotine amide, to create a comprehensive whitening system. Vitamin C and arbutin inhibit tyrosinase activity, reducing melanin production and thus skin pigmentation. Simultaneously, Vitamin C promotes collagen production, resulting in smoother and firmer skin. Arbutin helps distribute melanin more evenly across the skin surface, reducing age spots. Nicotine amide, a derivative of Vitamin B3, interferes with signaling pathways between keratinocytes and melanocytes, reducing melanin production and transport. Arbutin inhibits melanin production, Vitamin C protects the skin from free radical damage, and nicotine amide promotes skin metabolism. The combined effect of these three ingredients significantly brightens skin tone and reduces age spots and freckles.

[0023] 4. This invention utilizes microencapsulation technology to encapsulate astaxanthin within wall materials such as sodium hyaluronate, effectively isolating it from external factors such as light and oxygen, thereby improving the stability of astaxanthin. Astaxanthin itself has poor water solubility, which limits its application in certain fields. Sodium hyaluronate, as a water-soluble polymer, can be used as the wall material for astaxanthin microcapsules, which can improve the water solubility of astaxanthin to a certain extent, making it easier to use in cosmetics, medical aesthetics, health products, and other fields. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 and Figure 2 The morphology of astaxanthin microcapsules with modified sodium hyaluronate as the wall material under SEM observation;

[0026] Figure 3 This is a comparison diagram of the diameter of the inhibition zone in Example 1, Comparative Example 1, and Comparative Example 2;

[0027] Figure 4 The free radical scavenging rates of Examples 2, 3, and 4 are shown.

[0028] Figure 5 This is a line graph showing the inhibition rate of tyrosinase in Examples 3 and Comparative Examples 5-7. Detailed Implementation

[0029] To make the technical problem to be solved, the technical solution, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the content of this invention and are not intended to limit this invention.

[0030] In this invention, sodium hyaluronate (CAS: 9067-32-7) was purchased from Ruichengkang Pharmaceutical Technology (Shaanxi) Co., Ltd.; chitosan (DD>90%, 200mPa·s) was purchased from Shandong Weifang Haizhiyuan Biological Products Co., Ltd.; arbutin was purchased from Fubo Chemical Co., Ltd.; and vitamin C was purchased from Henan Kuoyuan Chemical Products Co., Ltd., all with a purity ≥99%.

[0031] Example 1

[0032] This embodiment illustrates that in this invention, the capsule wall utilizes a hyaluronic acid aqueous solution as a polymeric dispersion platform to construct a silver nanoparticle dispersion system. After drying, it is converted into HA-Ag powder, which synergistically interacts with carboxymethyl chitosan. The long polymer chains of carboxymethyl chitosan can aggregate and flocculate bacterial cells, while the disinfecting factors (such as NH4+) on its molecular chains... 3+ Silver ions can accumulate on the surface of bacteria and attract anions in the cell wall, thus hindering the metabolism and reproduction of microorganisms. Silver ions, on the other hand, can further penetrate the cell membrane and react with intracellular components, leading to bacterial death. This synergistic effect makes the antibacterial effect of the compound antibacterial agent more significant.

[0033] The specific implementation steps are as follows:

[0034] Preparation of S1, HA-Ag composite material: At room temperature, 0.1 g of sodium hyaluronate was weighed and dissolved in 100 mL of distilled water, and stirred evenly to obtain an HA aqueous solution. A 0.5 mol / L silver nitrate solution was prepared. 95 mL of the HA aqueous solution and 5 mL of the 0.5 mol / L silver nitrate solution were mixed and stirred for 5 h under light-protected conditions. The mixture was then irradiated with ultraviolet light for 10 min to obtain an HA-Ag composite aqueous solution. The HA-Ag composite powder was obtained by freeze-drying.

[0035] S2. Disperse 5g of chitosan in 50mL of isopropanol, sonicate for 5min, add 25mL of 38% NaOH solution, stir and mix evenly, add 18g of chloroacetic acid in 4 portions over 1h, stirring continuously and gradually raising the temperature to 60℃ during the addition process, and then react at a constant temperature of 60℃ for 4h. Afterwards, filter to obtain the solid product, dry it, and wash it 5 times with ethanol to remove impurities, thus obtaining carboxymethyl chitosan.

[0036] S3. Extraction of astaxanthin: Under light-protected conditions, 1.1g of Haematococcus pluvialis was dissolved in 26mL of ethyl acetate. The mixture was extracted at 40℃ in a high-speed stirrer for 60min to break the cell wall and obtain a mixed solution. After vacuum filtration, the crude astaxanthin extract was obtained. The extraction was repeated twice, and the filtrates were combined. The crude astaxanthin oleoresin was obtained by rotary evaporation. The crude astaxanthin oleoresin was obtained by vacuum drying in a freeze dryer for 24h to remove the residual solvent. It was then stored in the dark.

[0037] S4. Weigh 0.8g of glyceryl monostearate, 0.9g of sucrose fatty acid ester, and 1.2g of carboxymethyl chitosan obtained in step S2 and disperse them in 60mL of distilled water at 90℃. After cooling to 60℃, add 1.7g of HA-Ag complex powder obtained in step S1, 0.4g of vitamin C, 0.28g of arbutin, and 3.6g of maltodextrin. Stir at high speed until homogeneous and cool to room temperature to obtain a mixed emulsion. Dissolve 1.0g of astaxanthin oleoresin obtained in step S3 in the mixed emulsion and emulsify and shear at 10000r / min for 8min. After emulsification, atomize and dry at an inlet air temperature of 180℃ to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material. The modified sodium hyaluronate astaxanthin microcapsules are shown below. Figure 1 , 2 As shown, it appears as a round capsule shape when observed under SEM.

[0038] S5. Dissolve the prepared astaxanthin microcapsules in 50 mL of PBS, add 0.1 mL of phenoxyethanol and 0.3 g of nicotinamide, stir for 10 min, and after complete dissolution, adjust the pH to 7.35 with 10% citric acid, sonicate for 10 min, and then stir hydrothermally for 30 min at 20 °C until a complete sol is formed. The final modified hyaluronic acid complex microcapsules with antibacterial and whitening effects can be obtained.

[0039] Comparative Example 1: The HA-Ag complex powder in step S4 was replaced with sodium hyaluronate, and all other steps were the same as in Example 1;

[0040] Comparative Example 2: The carboxymethyl chitosan in step S4 was replaced with chitosan, and all other steps were the same as in Example 1;

[0041] Antibacterial activity test:

[0042] The antibacterial properties of sodium hyaluronate microcapsules prepared in Example 1, Comparative Example 1, and Comparative Example 2 were tested using the agar inhibition zone diffusion method with Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC22922) as bacterial models. All experiments were conducted on a clean bench free of other microorganisms. The bacterial culture medium consisted of peptone (1.0%, w / v), yeast extract (0.5%, w / v), and sodium chloride (1.0%, w / v). Agar powder (1.5%, w / v) was added to the culture medium, with the remainder being deionized water, to obtain a solid culture medium. After preparation, the medium was sterilized in a high-temperature, high-pressure autoclave. The bacterial strain was added to test tubes at a volume ratio of 1 / 100 to the culture medium, and the tubes were incubated in a shaker at 37°C for 12 hours. Take 80 mL of culture medium into a cell culture dish, and after solidification, add 100 μL of bacterial suspension to the surface of each dish and spread it evenly. Place circular slices (10 mm in diameter) containing 20 μL of sodium hyaluronate microcapsules prepared in Example 1, Comparative Example 1, and Comparative Example 2, respectively, onto the culture medium containing bacteria. Incubate the medium at 37°C for 24 hours, and observe and record the size of the inhibition zone. Each group is repeated three times. The experimental data are shown in Table 1 and... Figure 3 As shown.

[0043] Table 1. Diameter of inhibition zones in Example 1, Comparative Example 1, and Comparative Example 2

[0044]

[0045] The test data shows that both silver ions and carboxymethyl chitosan in the HA-Ag complex powder have antibacterial effects. The antibacterial effect is better when the two are used together, which confirms that silver ions and carboxymethyl chitosan have a synergistic effect in antibacterial effect. The use of the two together improves the antibacterial effect of sodium hyaluronate microcapsules.

[0046] Example 2

[0047] This embodiment illustrates the introduction of microencapsulation technology in this invention, selecting astaxanthin as the core component. With the protection of sodium hyaluronate, the stable existence time of astaxanthin is extended, significantly improving the antioxidant capacity of the product. At the same time, vitamin C can reduce oxidized astaxanthin, restoring its antioxidant activity.

[0048] The specific implementation steps are as follows:

[0049] Preparation of S1, HA-Ag composite material: At room temperature, 0.2 g of sodium hyaluronate was weighed and dissolved in 100 mL of distilled water, and stirred evenly to obtain an HA aqueous solution. A 0.5 mol / L silver nitrate solution was prepared. 95 mL of the HA solution and 5 mL of the 0.5 mol / L silver nitrate solution were mixed and stirred for 5 h under light-protected conditions. The mixture was then irradiated with ultraviolet light for 20 min to obtain an HA-Ag composite aqueous solution. The HA-Ag composite powder was obtained by freeze-drying.

[0050] S2. Disperse 8g of chitosan in 80mL of isopropanol, sonicate for 8min, add 30mL of 38% NaOH solution, stir and mix evenly, add 20g of chloroacetic acid in 4 portions over 1h, stirring continuously and gradually raising the temperature to 60℃ during the addition process, and then react at a constant temperature of 60℃ for 5h. Afterwards, filter to obtain the solid product, dry it, and wash it 5 times with ethanol to remove impurities, thus obtaining carboxymethyl chitosan.

[0051] S3. Extraction of astaxanthin: Under light-protected conditions, 1.8g of Haematococcus pluvialis was dissolved in 28mL of ethyl acetate. The mixture was extracted at 40℃ in a high-speed stirrer for 60min to break the cell wall and obtain a mixed solution. After vacuum filtration, the crude astaxanthin extract was obtained. The extraction was repeated twice, and the filtrates were combined. The crude astaxanthin oleoresin was obtained by rotary evaporation. The crude astaxanthin oleoresin was obtained by vacuum drying in a freeze dryer for 24h to remove the residual solvent. It was then stored in the dark.

[0052] S4. Weigh 2.1g of glyceryl monostearate, 1.1g of sucrose fatty acid ester and 1.8g of carboxymethyl chitosan obtained in step S2 and disperse them in 70mL of distilled water at 90℃. After cooling to 60℃, add 2.1g of HA-Ag complex powder obtained in step S1, 0.5g of vitamin C, 0.32g of arbutin and 4.2g of maltodextrin. Stir at high speed until homogeneous and cool to room temperature to obtain a mixed emulsion. Dissolve 1.2g of astaxanthin oleoresin obtained in step S3 in the mixed emulsion and emulsify and shear at 10000r / min for 10min. After emulsification, atomize and dry at an air inlet temperature of 180℃ to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material.

[0053] S5. Dissolve the prepared astaxanthin microcapsules in 80 mL of PBS, add 0.2 mL of phenoxyethanol and 0.4 g of nicotinamide, stir for 12 min, and after complete dissolution, adjust the pH to 7.45 with 10% citric acid, sonicate for 15 min, and then stir hydrothermally for 35 min at 30 °C until a complete sol is formed. The final modified hyaluronic acid complex microcapsules with antibacterial and whitening effects can be obtained.

[0054] Comparative Example 3: In step S4, vitamin C was replaced with β-carotene. All other steps were the same as in Example 2.

[0055] Comparative Example 4: In step S4, vitamin C was replaced with vitamin E, and all other steps were the same as in Example 2;

[0056] Antioxidant effect test

[0057] (1) Test of DPPH free radical scavenging ability

[0058] This experiment used a colorimetric method to determine the ability of astaxanthin microcapsules to scavenge DPPH free radicals. 1 mL of sodium hyaluronate microcapsules prepared using Examples 2, 3, and 4 were transferred to 20 mL test tubes, and 1 mL of 0.25 mM DPPH ethanol solution was added. The tubes were shaken thoroughly and reacted at room temperature in the dark for 30 min. The mixture was then poured into a cuvette. 1 mL of anhydrous ethanol and 1 mL of the sample solution were used as the zeroing solution. The absorbance was measured at a wavelength of 517 nm, and the absorbance value (A1) was recorded.

[0059] Transfer 1 mL of sample solvent (distilled water) into a 20 mL test tube, add 1 mL of 0.25 mM DPPH ethanol solution, mix thoroughly and shake well. After standing at room temperature in the dark for 30 min, pour into a cuvette. Use 1 mL of anhydrous ethanol and 1 mL of sample solvent (distilled water) as zeroing reagents, measure the absorbance at a wavelength of 517 nm and record the absorbance value as A2.

[0060] The above steps were repeated three times, and the data was recorded each time. The clearance rate was calculated using the following formula:

[0061] DPPH free radical scavenging rate (%) = [(A2-A1) / A2]*100%

[0062] A1 represents the absorbance of the sample, and A2 represents the absorbance of the blank.

[0063] (2) Determination of ABTS free radical scavenging ability

[0064] Dilute the overnight 7mM ABTS solution with 5mM pH 7.4 potassium phosphate buffer solution until the absorbance at 734nm is between 0.75 and 0.80. Specifically: For the 7mM ABTS solution: add 19.2 mg of ABTS to a 20 mL centrifuge tube, add 5 mL of distilled water and 88 μl of 140 mM potassium persulfate solution, vortex until completely dissolved, and refrigerate in the dark for 12-16 hours. For the 5mM pH 7.4 potassium phosphate buffer solution: accurately weigh 1.36 g of potassium dihydrogen phosphate, add 79 mL of 0.1 mol / L sodium hydroxide solution, dissolve completely, and then dilute to a 200 mL volumetric flask with distilled water.

[0065] Take 1 mL of the sodium hyaluronate microcapsules prepared in Example 2, Comparative Example 3, and Comparative Example 4 into 20 mL test tubes, add 1 mL of diluted ABTS solution, mix thoroughly, incubate at 30 °C for 60 min, and then pour into a cuvette. Use 1 mL of distilled water and 1 mL of sample solution as zeroing reagents, measure the absorbance at a wavelength of 734 nm, and record the absorbance value as A1.

[0066] Transfer 1 mL of sample solvent (distilled water) into a 20 mL test tube, add 1 mL of diluted ABTS solution and mix well. Incubate at 30 °C for 60 min and pour into a cuvette. Use 1 mL of distilled water and 1 mL of sample solvent (distilled water) as the zeroing solution. Measure the absorbance at a wavelength of 734 nm and record the absorbance value as A2.

[0067] The above steps were repeated three times, and the data was recorded each time. The clearance rate was calculated using the following formula:

[0068] ABTS radical scavenging rate (%) = [(A2-A1) / A2]*100%

[0069] A1 represents the absorbance of the sample, and A2 represents the absorbance of the blank.

[0070] (3) Determination of hydroxyl radical scavenging ability

[0071] Transfer 1 mL of 0.75 mmol / L o-phenanthroline ethanol solution to a 20 mL test tube, add 2 mL of 0.2 M pH 7.4 PBS solution and 1 mL of distilled water, mix thoroughly, then add 1 mL of 0.75 mmol / L ferrous sulfate solution and 1 mL of 0.01% H2O2, shake well, and incubate at 37 °C for 60 min. Use distilled water as the zeroing reagent and read the absorbance at 536 nm, denoted as A. p Value; Replace 1 mL of H2O2 in the above steps with 1 mL of distilled water, and record the absorbance value as A. B ; Replace 1 mL of distilled water in the above steps with 1 mL of sodium hyaluronate microcapsules prepared in Example 2, Comparative Example 3, and Comparative Example 4 respectively, and record the absorbance value as A. s .

[0072] The above experiment was repeated three times, and the clearance rate formula is as follows:

[0073] Hydroxyl radical scavenging rate (%) = [(A s -A p ) / (A B -A p )]*100%; the obtained data are shown in Table 2 and Figure 4 As shown;

[0074] Table 2 Free radical scavenging rates of Example 2, Comparative Example 3 and Comparative Example 4

[0075]

[0076] From Table 2 and Figure 4 As can be seen, the present invention introduces microencapsulation technology, selects astaxanthin as the core component, and adds vitamin C, which prolongs the stable existence time of astaxanthin and greatly improves the antioxidant capacity of the product.

[0077] Example 3

[0078] This embodiment illustrates that the present invention adds vitamin C and arbutin to the capsule wall. Vitamin C and arbutin can inhibit the activity of tyrosinase. By reducing the activity of tyrosinase, the amount of melanin produced is reduced, thereby reducing skin pigmentation. At the same time, vitamin C can promote the production of collagen, making the skin smoother and firmer. Arbutin can make melanin more evenly distributed on the skin surface, reducing age spots. Vitamin C has a certain reducing property and is easily oxidized and deactivated in the air, while arbutin has relatively high stability and is not easily affected by the external environment.

[0079] The specific implementation steps are as follows:

[0080] Preparation of S1, HA-Ag composite material: At room temperature, 0.3 g of sodium hyaluronate was weighed and dissolved in 100 mL of distilled water, and stirred evenly to obtain an HA aqueous solution. A 0.5 mol / L silver nitrate solution was prepared. 95 mL of the HA solution and 5 mL of the 0.5 mol / L silver nitrate solution were mixed and stirred for 5 h in the dark. The mixture was then irradiated with ultraviolet light for 30 min to obtain an HA-Ag composite aqueous solution. The HA-Ag composite powder was obtained by freeze-drying.

[0081] S2. Disperse 10g of chitosan in 100mL of isopropanol, sonicate for 10min, add 35mL of 38% NaOH solution, stir and mix evenly, add 22g of chloroacetic acid in 4 portions over 1h, stirring continuously and gradually raising the temperature to 60℃ during the addition process, and then react at a constant temperature of 60℃ for 6h. Afterwards, filter to obtain the solid product, dry it, and wash it 3 times with ethanol to remove impurities to obtain carboxymethyl chitosan.

[0082] S3. Extraction of astaxanthin: Under light-protected conditions, 2.4 g of Haematococcus pluvialis was dissolved in 30 mL of ethyl acetate. The mixture was extracted at 40 °C in a high-speed stirrer for 60 min to break the cell wall and obtain a mixed solution. After vacuum filtration, the crude astaxanthin extract was obtained. The extraction was repeated twice, and the filtrates were combined. The crude astaxanthin oleoresin was obtained by rotary evaporation. The crude astaxanthin oleoresin was obtained by vacuum drying in a freeze dryer for 24 h to remove the residual solvent. It was then stored in the dark.

[0083] S4. Weigh 1.1g of glyceryl monostearate, 1.2g of sucrose fatty acid ester and 2.4g of carboxymethyl chitosan obtained in step S2 and disperse them in 80mL of distilled water at 90℃. After cooling to 60℃, add 2.5g of HA-Ag complex powder obtained in step S1, 0.6g of vitamin C, 0.36g of arbutin and 4.8g of maltodextrin. Stir at high speed until homogeneous and cool to room temperature to obtain a mixed emulsion. Dissolve 1.4g of astaxanthin oleoresin obtained in step S3 in the mixed emulsion and emulsify and shear at 10000r / min for 12min. After emulsification, atomize and dry at an air inlet temperature of 180℃ to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material.

[0084] S5. Dissolve the prepared astaxanthin microcapsules in 100mL PBS, add 0.3mL phenoxyethanol and 0.5g nicotinamide, stir for 15min, and after fully dissolving, adjust the pH to 7.40 with 10% citric acid, sonicate for 20min, and then stir hydrothermally for 40min at 40℃ until a complete sol is formed. The final modified hyaluronic acid complex microcapsules with antibacterial and whitening effects can be obtained.

[0085] Comparative Example 5: Nicotine amide was not added in step S5, and all other steps were the same as in Example 3;

[0086] Comparative Example 6: Arbutin was not added in step S4, and all other steps were the same as in Example 3;

[0087] Comparative Example 7: No vitamin C was added in step S4, and all other steps were the same as in Example 3;

[0088] Assay for inhibiting tyrosinase activity

[0089] S1. Material Preparation

[0090] Tyrosinase powder, enzyme activity 25,000U, Sigma; L-tyrosine, purity ≥99%, biochemical reagent, Shanghai JCBIO;

[0091] PBS buffer preparation: Weigh 7.8 g of sodium dihydrogen phosphate and 17.9 g of disodium hydrogen phosphate to prepare aqueous solutions separately. Dilute to 500 mL with distilled water. Mix thoroughly to prepare a 0.1 mol / L PBS buffer solution with pH 6.8.

[0092] Preparation of 0.1 mol hydrochloric acid: Accurately measure 0.431 mL of hydrochloric acid with a mass fraction of 36%–38%, and dilute to 50 mL with distilled water. Preparation of 0.05% L-tyrosine solution: Weigh 0.05 g of L-tyrosine and dissolve it in 35 mL of 0.1 mol hydrochloric acid, then add 65 mL of PBS buffer (pH 6.8).

[0093] Example 3, Preparation of sample solutions for comparative examples 5-7: Accurately weigh the test samples and dilute them with PBS buffer to a mass fraction of 5%.

[0094] Preparation of tyrosinase solution: Dissolve tyrosinase with an enzyme activity of 25000U in 250mL of pure water to prepare a tyrosinase solution with an enzyme activity of 100U / mL. Dispense the solution into 1.5mL EP tubes, filling each tube with 1mL and storing at -40℃.

[0095] S2, Tyrosinase Activity Experiment

[0096] As shown in Table 3, L-tyrosine, sample solution, and PBS buffer were added sequentially to tubes numbered A1, A2, B1, and B2 and mixed thoroughly. The tube racks were then placed in a 37°C water bath for 10–15 minutes. Tyrosinase was added sequentially to tubes in groups A1 and B1, shaken well, and the reaction was stopped after 15 minutes. The mixture was then spotted onto 96-well plates, and the absorbance was measured using a microplate reader. The inhibition rate was calculated. The results are shown in Table 4. Figure 5 As shown. The above experiment was repeated three times, and the inhibition rate was calculated using the following formula:

[0097] Tyrosinase inhibition rate = [(A1-A2)-(B1-B2)] / (A1-A2)×100%, where A1 is the absorbance of the blank sample with enzyme; A2 is the absorbance of the blank sample without enzyme; B1 is the absorbance of the sample group with enzyme; B2 is the absorbance of the sample group without enzyme.

[0098] Table 3 Composition of the reaction solution

[0099]

[0100] The inhibition rate data obtained from the experiment are shown in Table 4;

[0101] Table 4 shows the inhibition rates of tyrosinase in Examples 3 and Comparative Examples 5-7.

[0102] test sample Tyrosinase inhibition rate % Example 3 95.23±1.46 Comparative Example 5 86.23±2.78 Comparative Example 6 84.26±0.96 Comparative Example 7 81.25±1.56

[0103] From Table 4 and Figure 5 It is understood that the present invention adds vitamin C and arbutin to the capsule wall. Vitamin C and arbutin can reduce the activity of tyrosinase and reduce the amount of melanin produced, thereby reducing skin pigmentation.

Claims

1. A method for preparing a modified hyaluronic acid complex with antibacterial and whitening effects, characterized in that: The specific preparation steps are as follows: Preparation of S1 and HA-Ag composite materials: At room temperature, 0.1-0.3 g of sodium hyaluronate was weighed and dissolved in 100 mL of distilled water, and stirred evenly to obtain an HA aqueous solution; a 0.5 mol / L silver nitrate solution was prepared, and 95 mL of HA solution and 5 mL of 0.5 mol / L silver nitrate solution were mixed and stirred for 5 h under light-protected conditions, and then irradiated with ultraviolet light for 10-30 min to obtain an HA-Ag composite aqueous solution; HA-Ag composite powder was obtained by freeze-drying. S2. Disperse 5-10 g of chitosan in 50-100 mL of isopropanol, sonicate for 5-10 min, add 25-35 mL of 38% NaOH solution, stir and mix evenly, add 18-22 g of chloroacetic acid in 4 portions over 1 h, stirring continuously and gradually raising the temperature to 60℃ during the addition process, and then react at a constant temperature of 60℃ for 4-6 h. Afterward, filter to obtain the solid product, dry it, and wash it with ethanol 3-5 times to remove impurities to obtain carboxymethyl chitosan. S3. Extraction of astaxanthin: Under light-protected conditions, 1.1-2.4 g of Haematococcus pluvialis was dissolved in 26-30 mL of ethyl acetate. The mixture was extracted at 40 °C in a high-speed stirrer for 60 min to break the cell wall and obtain a mixed solution. After vacuum filtration, the crude astaxanthin extract was obtained. The extraction was repeated twice, and the filtrates were combined. The crude astaxanthin oleoresin was obtained by rotary evaporation. The crude astaxanthin oleoresin was obtained by vacuum drying in a freeze dryer for 24 h to remove the residual solvent. It was then stored in the dark. S4. Weigh 0.8-1.1 g of glyceryl monostearate, 0.9-1.2 g of sucrose fatty acid ester, and 1.2-2.4 g of carboxymethyl chitosan obtained in step S2 and disperse them in 60-80 mL of distilled water at 90℃. After cooling to 60℃, add 1.7-2.5 g of HA-Ag complex powder obtained in step S1, 0.4-0.6 g of vitamin C, 0.28-0.36 g of arbutin, and 3.6-4.8 g of maltodextrin. Stir at high speed until homogeneous and cool to room temperature to obtain a mixed emulsion. Dissolve 1.0-1.4 g of astaxanthin oleoresin obtained in step S3 in the mixed emulsion and emulsify and shear at 10000 r / min for 8-12 min. After emulsification, atomize and dry at an inlet air temperature of 180℃ to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material. S5. Dissolve the prepared astaxanthin microcapsules in 50-100 mL of PBS, add 0.1-0.3 mL of phenoxyethanol and 0.3-0.5 g of nicotinamide, stir for 10-15 min, and adjust the pH to 7.35-7.45 with 10% citric acid. Sonicate for 10-20 min, and then stir hydrothermally for 30-40 min at a temperature of 20-40 ℃ until a complete sol is formed. The final modified hyaluronic acid complex microcapsules with antibacterial and whitening effects can be obtained.

2. The method for preparing a modified hyaluronic acid complex with antibacterial and whitening effects according to claim 1, characterized in that, In step S1, the amount of sodium hyaluronate used is 0.2 g, the ultraviolet light band is UVB, and the irradiation time is 20 min.

3. The method for preparing a modified hyaluronic acid complex with antibacterial and whitening effects according to claim 1, characterized in that, In step S2, the amount of chitosan used is 8 g, isopropanol is 80 mL, 30 mL of 38% NaOH solution is 38 mL, chloroacetic acid is 10 mL, the mixture is sonicated for 8 min, stirred for 5 h, and washed 4 times with ethanol.

4. The method for preparing a modified hyaluronic acid complex with antibacterial and whitening effects according to claim 1, characterized in that, In step S3, the amount of Haematococcus pluvialis used is 1.8 g, and the amount of ethyl acetate is 28 mL.

5. The method for preparing a modified hyaluronic acid complex with antibacterial and whitening effects according to claim 1, characterized in that, In step S4, the amount of glyceryl monostearate is 0.9 g, sucrose fatty acid ester is 1.1 g, carboxymethyl chitosan is 1.8 g, and distilled water is 70 mL.

6. The method for preparing a modified hyaluronic acid complex with antibacterial and whitening effects according to claim 1, characterized in that, In step S4, the amount of HA-Ag complex powder is 2.1 g, vitamin C is 0.32 g, maltodextrin is 4.2 g, and astaxanthin oleoresin is 1.2 g.

7. The method for preparing a modified hyaluronic acid complex with antibacterial and whitening effects according to claim 1, characterized in that, In step S5, the amount of PBS used is 80 mL, phenoxyethanol is 0.2 mL, and nicotine amide is 0.4 g.

8. The method for preparing a modified hyaluronic acid complex with antibacterial and whitening effects according to claim 1, characterized in that, In step S5, the stirring time is 12 min, the pH is adjusted to 7.40, the sonication is performed for 15 min, the hydrothermal stirring is performed for 35 min, and the temperature is set for 30 min.

9. A modified hyaluronic acid complex microcapsule with antibacterial and whitening effects prepared by the preparation method according to any one of claims 1-8.

10. The modified hyaluronic acid complex microcapsules with antibacterial and whitening effects as described in claim 9, applied to cosmetic injection fillers.

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