Modified hyaluronic acid compound with whitening, freckle removing and antioxidant effects and application of modified hyaluronic acid compound in cosmetics and medical cosmetology

Abstract

The invention belongs to the technical field of biological materials, and relates to a modified hyaluronic acid compound with whitening, freckle removing and antioxidant effects and application of the modified hyaluronic acid compound in cosmetics and medical cosmetology. According to the preparation method, an ultraviolet irradiation reduction technology is adopted, a hyaluronic acid aqueous solution is used as a polymer dispersion platform, a silver nano colloidal particle dispersion system is constructed, the silver nano colloidal particle dispersion system is converted into HA-Ag powder after being dried, and the HA-Ag powder and carboxymethyl chitosan are mutually cooperated, so that the product is endowed with excellent antibacterial performance. A microcapsule technology is introduced, astaxanthin is selected as a capsule core component, and by virtue of protection of sodium hyaluronate, the stable existence time of astaxanthin is prolonged, and the oxidation resistance of the product is greatly improved. The vitamin C and the arbutin are blended into the microcapsule wall material, and then the microcapsule wall material is mixed with the nicotine amide for use, so that the vitamin C can reduce oxidized astaxanthin while the whitening effect is enhanced, the antioxidant activity of the oxidized astaxanthin is recovered, and the antioxidant effect is further amplified.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials and relates to hyaluronic acid, and in particular to a modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects and its application in cosmetics and medical cosmetology. Background Art

[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 extracellular matrix and extracellular matrix of cells and has a significant impact on cellular physiological functions. Hyaluronic acid is widely distributed in the epithelium, connective tissue, and nervous tissue of vertebrates, and is particularly abundant in skin tissue, joint fluid, and the eye.

[0003] Currently, hyaluronic acid fillers are widely used to treat wrinkles, scars, and facial contour defects. They are categorized into single-phase and biphasic types, depending on the degree of cross-linking. Single-phase fillers are stable and particle-free. They are prepared by varying the ratio of high-molecular-weight hyaluronic acid to low-molecular-weight hyaluronic acid. They have low strength, are easily affected by external forces, and lack a granular texture. Biphasic fillers, the most widely used products on the market, consist of stable hyaluronic acid gel particles and uncross-linked hyaluronic acid. The cross-linked hyaluronic acid particles are suspended in the uncross-linked hyaluronic acid, acting as a lubricant and enabling the suspension to be injected into tissues through a fine needle.

[0004] Sodium hyaluronate has a certain antibacterial effect, primarily 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 bacterial numbers are high, sodium hyaluronate's antibacterial effect is insufficient. Its whitening effect is also relatively limited. While it can indirectly improve uneven skin tone and dullness by moisturizing and promoting skin cell regeneration, it does not directly inhibit melanin production or accelerate melanin metabolism. Therefore, achieving significant whitening effects solely with sodium hyaluronate is difficult. Sodium hyaluronate has a certain antioxidant effect, neutralizing free radicals and mitigating environmental damage to the skin. However, this antioxidant effect is relatively weak and primarily limited to the surface layer of the skin. When the skin is subjected to severe oxidative stress, such as ultraviolet radiation or environmental pollution, the antioxidant effect of sodium hyaluronate becomes insufficient.

[0005] To address the above issues, the present invention adopts ultraviolet light reduction technology, uses a hyaluronic acid aqueous solution as a polymer dispersion platform, constructs a silver nanoparticle dispersion system, and converts it into HA-Ag powder after drying. It works synergistically with carboxymethyl chitosan to give the product excellent antibacterial properties. Microencapsulation technology is introduced, and astaxanthin is selected as the capsule core component. Relying on the protection of sodium hyaluronate, the stable existence time of astaxanthin is extended, and the antioxidant capacity of the product is greatly improved. Vitamin C and arbutin are incorporated into the microcapsule wall material and then mixed with nicotine amide. While enhancing the whitening effect, vitamin C can reduce oxidized astaxanthin, restore its antioxidant activity, and further amplify the antioxidant effect. Summary of the Invention

[0006] To address the above problems, the present invention provides a modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects and its application in cosmetics and medical cosmetology. The specific preparation steps are as follows: S1. Preparation of HA-Ag composite material: At room temperature, weigh 0.1-0.3 g of sodium hyaluronate and dissolve it in 100 mL of distilled water. Stir evenly to obtain an HA aqueous solution, and prepare a 0.5 mol / L silver nitrate solution. Mix 95 mL of the HA solution and 5 mL of the 0.5 mol / L silver nitrate solution, stir in the dark for 5 h, and irradiate with ultraviolet light for 10-30 min to obtain an HA-Ag complex aqueous solution. Freeze-dry to obtain HA-Ag complex powder. S2. Disperse 5-10 g of chitosan in 50-100 mL of isopropanol and sonicate for 5-10 min. Add 25-35 mL of 38% NaOH solution and stir to mix evenly. Add 18-22 g of chloroacetic acid in 4 portions within 1 h. Stir continuously during the addition process and gradually raise the temperature to 60°C. Then, react at a constant temperature of 60°C for 4-6 h. Filter to obtain the solid product. After drying, wash with ethanol 3-5 times to remove impurities to obtain carboxymethyl chitosan. S3. Extracting astaxanthin: Dissolve 1.1-2.4 g of Haematococcus pluvialis in 26-30 mL of ethyl acetate under light-shielding conditions, perform wall-breaking extraction in a high-speed blender at 40°C for 60 min to obtain a mixed solution, vacuum filter to obtain a crude astaxanthin extract, repeat twice, combine the filtrates, and concentrate by rotary evaporation to obtain a crude astaxanthin oleoresin. Dry in a freeze dryer under vacuum for 24 h to remove the residual solvent to obtain astaxanthin oleoresin, and store in the dark. S4, weighing 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, dispersing them in 60-80 mL of 90° C. distilled water, cooling to 60° C., adding 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, stirring at high speed and cooling to room temperature to obtain a mixed emulsion, taking 1.0-1.4 g of astaxanthin oleoresin obtained in step S3 and dissolving it in the mixed emulsion, emulsifying and shearing at high speed at 10,000 r / min for 8-12 min, and atomizing and drying at an inlet air temperature of 180° C. after the emulsification is completed, to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material; S5. Stir and dissolve the prepared astaxanthin microcapsules in 50-100 mL PBS, add 0.1-0.3 mL phenoxyethanol and 0.3-0.5 g nicotine amide, stir for 10-15 min, and after sufficient dissolution, adjust the pH to 7.35-7.45 with 10% citric acid. Ultrasonicate for 10-20 min, then stir in hydrothermal solution for 30-40 min at a temperature of 20-40 °C until a complete sol is formed to obtain the final modified hyaluronic acid complex microcapsules with antibacterial and whitening effects.

[0007] Preferably, in step S1, the amount of sodium hyaluronate used is 0.2 g, the ultraviolet band is UVB, and the irradiation time is 20 min.

[0008] Preferably, in step S2, the amount of chitosan used is 8 g, isopropanol is 80 mL, 38% NaOH solution is 30 mL, and chloroacetic acid is 10 mL. Ultrasonic treatment is performed for 8 min, the reaction is stirred for 5 h, and the mixture is washed with ethanol 4 times.

[0009] Preferably, in step S3, the amount of Haematococcus pluvialis used is 1.8 g and the amount of ethyl acetate used is 28 mL.

[0010] Preferably, in step S4, the amount of glyceryl monostearate is 0.9 g, the amount of sucrose fatty acid ester is 1.1 g, the amount of carboxymethyl chitosan is 1.8 g, and the amount of distilled water is 70 mL.

[0011] Preferably, in step S4, the amount of HA-Ag complex powder is 2.1 g, the amount of vitamin C is 0.32 g, the amount of maltodextrin is 4.2 g, and the amount of astaxanthin oleoresin is 1.2 g.

[0012] Preferably, in step S5, the amount of PBS used is 80 mL, the amount of phenoxyethanol used is 0.2 mL, and the amount of nicotineamide used is 0.4 g.

[0013] Preferably, in step S5, the stirring time is 12 min, the pH is adjusted to 7.40, the ultrasound is performed for 15 min, the hydrothermal stirring is performed for 35 min, and the temperature is set for 30 min.

[0014] By adopting the above technical solution, the technical progress achieved by the present invention is: 1. The silver ions incorporated into the present invention are broad-spectrum antimicrobial agents with potent antimicrobial activity against a wide range of bacteria, viruses, and fungi. When used as a microcapsule wall material, they can directly exert their antimicrobial activity. Carboxymethyl chitosan is a natural polymer with antimicrobial properties against a wide range of bacteria. When used as a microcapsule wall material, they can enhance the antimicrobial effect through the following effects. Sodium hyaluronate, a glucuronic acid, has moisturizing, lubricating, and skin-repairing properties. Although it does not possess direct antimicrobial activity itself, its use as a microcapsule wall material can indirectly enhance the antimicrobial effect through the following effects: Sodium hyaluronate's moisturizing and lubricating properties maintain a stable internal microcapsule environment; the broad-spectrum antimicrobial activity of silver ions can kill a wide range of bacteria; and the antimicrobial activity and pH-adjusting properties of carboxymethyl chitosan further enhance the antimicrobial effect. This complementary effect gives the microcapsules a broader antimicrobial spectrum and stronger antimicrobial potency. These three materials may work synergistically within the microcapsules, disrupting bacterial cell structure and metabolic processes, thereby enhancing the antimicrobial effect.

[0015] 2. The vitamin C added to the present invention, also known as ascorbic acid, is a powerful antioxidant. It can neutralize free radicals and reduce the damage caused by oxidative stress to the body. Astaxanthin is a natural antioxidant with very strong antioxidant activity. It is considered to be one of the strongest antioxidant single molecules discovered in nature to date. It can scavenge various free radicals, thereby inhibiting lipid peroxidation reactions caused by free radicals, achieving antioxidant effects. Astaxanthin also has anti-ultraviolet effects, which can reduce the increased secretion of lipid free radicals due to sunlight exposure, helping to delay skin aging. Vitamin C can regenerate oxidized astaxanthin, restoring its antioxidant activity.

[0016] 3. The present invention adds vitamin C and arbutin to the capsule wall and mixes them with nicotine amide to form a comprehensive whitening system. Vitamin C and arbutin can inhibit the activity of tyrosinase, reducing melanin production by reducing tyrosinase activity, thereby reducing skin pigmentation. Vitamin C can also promote collagen production, making the skin smoother and firmer. Arbutin can make melanin more evenly distributed on the skin surface, reducing spots. Nicotine amide is a derivative of vitamin B3, which can interfere with the signaling pathway between keratinocytes and melanocytes, reducing the production and transport of melanin. Arbutin inhibits melanin production, vitamin C protects the skin from damage by free radicals, and nicotine amide promotes skin metabolism. The three together can significantly brighten the skin tone and reduce spots and freckles.

[0017] 4. The present invention uses microencapsulation technology to encapsulate astaxanthin in a wall material 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. However, sodium hyaluronate, a water-soluble polymer, can be used as the wall material of astaxanthin microcapsules to improve the water solubility of astaxanthin to a certain extent, making it easier to use in cosmetics, medical beauty, health care products and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 and Figure 2 The morphology of astaxanthin microcapsules made of modified sodium hyaluronate under SEM observation; Figure 3 This is a comparison chart of the inhibition zone diameters of Example 1, Comparative Example 1, and Comparative Example 2; Figure 4 is the free radical scavenging rate of Example 2, Comparative Example 3 and Comparative Example 4; Figure 5 It is a line graph showing the inhibition rates of Example 3 and Comparative Examples 5 to 7 on tyrosinase. DETAILED DESCRIPTION

[0020] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is 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 used to explain the present invention and are not intended to limit the present invention.

[0021] In the present invention, sodium hyaluronate (CAS: 9067-32-7) was purchased from Ruichengkang Pharmaceutical Technology (Shaanxi) Co., Ltd.; chitosan (DD>90%, 200 mPa·s) was purchased from Shandong Weifang Haizhiyuan Biological Products Co., Ltd.; arbutin was purchased from Fubo Chemical Co., Ltd.; vitamin C was purchased from Henan Kuoyuan Chemical Products Co., Ltd., all with a purity of ≥99%; Example 1

[0022] This example illustrates that the capsule wall of the present invention uses hyaluronic acid aqueous solution as a polymer dispersion platform to construct a silver nanoparticle dispersion system, which is converted into HA-Ag powder after drying. The HA-Ag powder cooperates with carboxymethyl chitosan. The long polymer chain of carboxymethyl chitosan can precipitate and flocculate bacterial cells. At the same time, the disinfection factors (such as NH 3+ ) can accumulate on bacterial surfaces, attracting anions in the microbial cell wall, thereby hindering their metabolism and reproduction. 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 combined antimicrobial agent's antibacterial effect even more significant.

[0023] The specific implementation steps are as follows: S1. Preparation of HA-Ag composite material: At room temperature, weigh 0.1 g of sodium hyaluronate and dissolve it in 100 mL of distilled water. Stir evenly to obtain an HA aqueous solution, and prepare a 0.5 mol / L silver nitrate solution. Take 95 mL of the HA aqueous solution and 5 mL of the 0.5 mol / L silver nitrate solution, mix them, stir for 5 h in the dark, and irradiate with ultraviolet light for 10 min to obtain an HA-Ag complex aqueous solution. The HA-Ag complex powder is obtained by freeze-drying. S2. Disperse 5 g of chitosan in 50 mL of isopropanol, ultrasonicate for 5 min, add 25 mL of 38% NaOH solution, stir and mix evenly, add 18 g of chloroacetic acid in 4 portions within 1 h, stir continuously during the addition process and gradually raise the temperature to 60 °C, then react at a constant temperature of 60 °C for 4 h, filter to obtain the solid product, dry and wash with ethanol 5 times to remove impurities, and then obtain carboxymethyl chitosan; S3. Extracting astaxanthin: Dissolve 1.1 g of Haematococcus pluvialis in 26 mL of ethyl acetate under light-shielding conditions, perform wall-breaking extraction in a high-speed blender at 40°C for 60 min to obtain a mixed solution, vacuum filter to obtain a crude astaxanthin extract, repeat twice, combine the filtrates, and concentrate by rotary evaporation to obtain a crude astaxanthin oleoresin. Dry in a freeze dryer under vacuum for 24 h to remove the residual solvent to obtain astaxanthin oleoresin, and store in the dark; S4, weighing 0.8 g of glyceryl monostearate, 0.9 g of sucrose fatty acid ester and 1.2 g of carboxymethyl chitosan obtained in step S2, dispersing them in 60 mL of 90° C. distilled water, cooling to 60° C., adding 1.7 g of HA-Ag complex powder obtained in step S1, 0.4 g of vitamin C, 0.28 g of arbutin and 3.6 g of maltodextrin, stirring at high speed and cooling to room temperature to obtain a mixed emulsion, taking 1.0 g of astaxanthin oleoresin obtained in step S3 and dissolving it in the mixed emulsion, emulsifying and shearing at high speed at 10,000 r / min for 8 min, and after the emulsification is completed, atomizing and drying are performed at an air inlet temperature of 180° C. to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material; astaxanthin microcapsules with modified sodium hyaluronate as the wall material are as follows: Figure 1 、 2 As shown, it appears as a round capsule under SEM observation; S5. Dissolve the prepared astaxanthin microcapsules in 50 mL of PBS, add 0.1 mL of phenoxyethanol and 0.3 g of nicotine amide, and stir for 10 min. After sufficient dissolution, adjust the pH to 7.35 with 10% citric acid, ultrasonicate for 10 min, and then stir in hydrothermal solution for 30 min at a temperature of 20 °C. When a complete sol is formed, the final modified hyaluronic acid composite microcapsules with antibacterial and whitening effects are obtained.

[0024] Comparative Example 1: The HA-Ag complex powder in step S4 was replaced with sodium hyaluronate, and the other steps were the same as in Example 1; Comparative Example 2: The carboxymethyl chitosan in step S4 was replaced with chitosan, and the other steps were the same as those in Example 1;

[0025] The antibacterial properties of the 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. The experimental processes were all completed on a clean bench free of other bacteria. The bacterial culture solution consisted of peptone (1.0%, w / v), yeast powder (0.5%, w / v), and sodium chloride (1.0%, w / v). Agar powder (1.5%, w / v) was added to the culture solution, and the remaining ingredients were deionized water to obtain a solid culture medium. After the configuration was completed, it was sterilized in a high-temperature and high-pressure autoclave. The strain was added to the test tube at a volume ratio of 1 / 100 to the culture solution and placed in a shaker at 37°C for 12 h. 80 mL of culture medium was placed in a cell culture dish. After solidification, 100 μl of bacterial solution was dripped onto the surface of each dish and evenly spread. Circular sheets (10 mm in diameter) evenly spread with 20 μl of sodium hyaluronate microcapsules prepared in Example 1, Comparative Example 1, and Comparative Example 2 were placed on the culture medium containing bacteria. The culture medium was placed in an incubator at 37 ° C. After 24 h, the size of the inhibition zone was observed and recorded. Each group was repeated 3 times. The experimental data obtained are shown in Tables 1 and Figure 3 shown.

[0026] Table 1 Diameters of inhibition zones of Example 1, Comparative Example 1 and Comparative Example 2

[0027] The test data show that both silver ions and carboxymethyl chitosan in the HA-Ag composite powder have antibacterial effects, and the antibacterial effect is better when the two are used synergistically. This confirms that silver ions and carboxymethyl chitosan have a synergistic effect in antibacterial effects, and their combined use improves the antibacterial effect of sodium hyaluronate microcapsules.

[0028] Example 2

[0029] This example illustrates the introduction of microencapsulation technology in the present invention, the selection of astaxanthin as the capsule core component, and the extension of the stable existence time of astaxanthin under the protection of sodium hyaluronate, which greatly enhances the antioxidant capacity of the product. At the same time, vitamin C can reduce oxidized astaxanthin and restore its antioxidant activity.

[0030] The specific implementation steps are as follows: S1. Preparation of HA-Ag composite material: At room temperature, weigh 0.2 g of sodium hyaluronate and dissolve it in 100 mL of distilled water. Stir evenly to obtain an HA aqueous solution, and prepare a 0.5 mol / L silver nitrate solution. Mix 95 mL of the HA solution and 5 mL of the 0.5 mol / L silver nitrate solution, stir in the dark for 5 h, and irradiate with ultraviolet light for 20 min to obtain an HA-Ag complex aqueous solution. The HA-Ag complex powder is obtained by freeze-drying. S2. Disperse 8 g of chitosan in 80 mL of isopropanol, sonicate for 8 min, add 30 mL of 38% NaOH solution, stir and mix evenly, add 20 g of chloroacetic acid in 4 portions within 1 h, continue stirring during the addition process and gradually raise the temperature to 60°C, then react at a constant temperature of 60°C for 5 h, filter to obtain a solid product, dry and wash with ethanol 5 times to remove impurities, and obtain carboxymethyl chitosan; S3. Extracting astaxanthin: 1.8 g of Haematococcus pluvialis was dissolved in 28 mL of ethyl acetate under light-shielding conditions, and the mixture was subjected to wall-breaking extraction in a high-speed blender at 40° C. for 60 min to obtain a mixed solution. The mixture was vacuum-filtered to obtain a crude astaxanthin extract. This was repeated twice, and the filtrates were combined and concentrated by rotary evaporation to obtain a crude astaxanthin oleoresin. The crude astaxanthin oleoresin was then vacuum-dried in a freeze dryer for 24 h to remove the residual solvent and obtain the astaxanthin oleoresin, which was then stored in the dark. S4, weighing 2.1 g of glyceryl monostearate, 1.1 g of sucrose fatty acid ester and 1.8 g of carboxymethyl chitosan obtained in step S2, dispersing them in 70 mL of 90°C distilled water, cooling to 60°C, adding 2.1 g of HA-Ag complex powder obtained in step S1, 0.5 g of vitamin C, 0.32 g of arbutin and 4.2 g of maltodextrin, stirring at high speed and cooling to room temperature to obtain a mixed emulsion, taking 1.2 g of astaxanthin oleoresin obtained in step S3 and dissolving it in the mixed emulsion, emulsifying and shearing at high speed at 10000 r / min for 10 min, and after emulsification, performing atomization drying at an inlet air temperature of 180°C to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material; S5. Dissolve the prepared astaxanthin microcapsules in 80 mL of PBS, add 0.2 mL of phenoxyethanol and 0.4 g of nicotine amide, and stir for 12 min. After sufficient dissolution, adjust the pH to 7.45 with 10% citric acid, ultrasonicate for 15 min, and then stir in hydrothermal for 35 min at a temperature of 30 °C until a complete sol is formed. The final modified hyaluronic acid composite microcapsules with antibacterial and whitening effects are obtained.

[0031] Comparative Example 3: In step S4, vitamin C was replaced with β-carotene, and the other steps were the same as those in Example 2.

[0032] Comparative Example 4: In step S4, vitamin C was replaced with vitamin E, and the other steps were the same as those in Example 2;

[0033] Test of DPPH free radical scavenging ability This experiment used a colorimetric method to determine the DPPH free radical scavenging ability of astaxanthin microcapsules. 1 mL of sodium hyaluronate microcapsules prepared using Example 2, Comparative Example 3, and Comparative Example 4 were each transferred to a 20 mL test tube. 1 mL of a 0.25 mM DPPH ethanol solution was added and thoroughly shaken. The mixture was allowed to react in the dark at room temperature for 30 minutes before being poured into a cuvette. 1 mL of anhydrous ethanol and 1 mL of the sample solution were used as zeroing solutions. The absorbance was measured at a wavelength of 517 nm and recorded as A1.

[0034] Pipette 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, place in the dark at room temperature for 30 min, then pour into a cuvette. Use 1 mL of anhydrous ethanol and 1 mL of sample solvent (distilled water) as zero adjustment reagents, measure the absorbance at a wavelength of 517 nm and record the absorbance value as A2.

[0035] The above steps were repeated three times and the data were recorded respectively. The clearance rate was calculated according to the following formula: DPPH free radical scavenging rate (%) = [(A2-A1) / A2]*100% A1 represents the absorbance value of the sample, and A2 represents the absorbance value of the blank; Determination of ABTS free radical scavenging ability To prepare the overnight 7mM ABTS solution, dilute it with 5mM potassium phosphate buffer (pH 7.4) until the absorbance at 734nm is between 0.75-0.80. For the 7mM ABTS solution, place 19.2mg of ABTS in a 20mL centrifuge tube, add 5mL of distilled water and 88μl of 140mM potassium persulfate solution, vortex until completely dissolved, and refrigerate in the dark for 12-16 hours. For the 5mM potassium phosphate buffer (pH 7.4), accurately weigh 1.36g of potassium dihydrogen phosphate, add 79mL of 0.1mol / L sodium hydroxide solution, dissolve completely, and dilute to a 200mL volumetric flask with distilled water.

[0036] 1 mL of the sodium hyaluronate microcapsules prepared in Example 2, Comparative Example 3, and Comparative Example 4 were respectively transferred into 20 mL test tubes, 1 mL of the diluted ABTS solution was added, the mixture was thoroughly mixed and shaken, and the mixture was incubated at 30° C. for 60 min. The mixture was then poured into a cuvette, and 1 mL of distilled water and 1 mL of the sample solution were used as zero adjustment reagents. The absorbance was measured at a wavelength of 734 nm, and the absorbance value was recorded as A1.

[0037] Pipette 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. Pour into a cuvette and use 1 mL of distilled water and 1 mL of sample solvent (distilled water) as zero adjustment solutions. Measure the absorbance at a wavelength of 734 nm and record the absorbance value as A2.

[0038] The above steps were repeated three times and the data were recorded respectively. The clearance rate was calculated according to the following formula: ABTS free radical scavenging rate (%) = [(A2-A1) / A2]*100% A1 represents the absorbance value of the sample, and A2 represents the absorbance value of the blank.

[0039] Determination of hydroxyl radical scavenging ability Pipette 1 mL of 0.75 mmol / L 1-phenanthroline ethanol solution into 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, which is recorded as A. p Replace 1mL of H2O2 in the above step with 1mL of distilled water, and record the absorbance value as A B ; 1 mL of distilled water in the above steps was replaced by 1 mL of sodium hyaluronate microcapsules prepared in Example 2, Comparative Example 3 and Comparative Example 4, and the absorbance value was recorded as A s .

[0040] The above test was repeated three times, and the clearance formula is as follows: 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; Table 2 Free radical scavenging rates of Example 2, Comparative Example 3 and Comparative Example 4

[0041] From Table 2 and Figure 4 It can be seen that the present invention introduces microencapsulation technology, selects astaxanthin as the capsule core component, and adds vitamin C at the same time, which prolongs the stable existence time of astaxanthin and greatly improves the antioxidant capacity of the product.

[0042] Example 3

[0043] This example illustrates that vitamin C and arbutin are added to the capsule wall of the present invention. Vitamin C and arbutin can inhibit the activity of tyrosinase, thereby reducing the production of melanin and thus reducing skin pigmentation. At the same time, vitamin C can promote the production of collagen, making the skin smoother and firmer, and arbutin can make melanin more evenly distributed on the skin surface, reducing spots. Vitamin C has a certain reducing property and is easily oxidized and inactivated in the air, while arbutin is relatively stable and is not easily affected by the external environment.

[0044] The specific implementation steps are as follows: S1. Preparation of HA-Ag composite material: At room temperature, weigh 0.3 g of sodium hyaluronate and dissolve it in 100 mL of distilled water. Stir evenly to obtain an HA aqueous solution, and prepare a 0.5 mol / L silver nitrate solution. Mix 95 mL of the HA solution and 5 mL of the 0.5 mol / L silver nitrate solution, stir in the dark for 5 h, and irradiate with ultraviolet light for 30 min to obtain an HA-Ag complex aqueous solution. The HA-Ag complex powder is obtained by freeze-drying. S2. Disperse 10 g of chitosan in 100 mL of isopropanol and sonicate for 10 min. Add 35 mL of 38% NaOH solution and stir to mix evenly. Add 22 g of chloroacetic acid in four portions within 1 h. Stir continuously and gradually raise the temperature to 60°C during the addition process. Then, react at a constant temperature of 60°C for 6 h. Filter to obtain a solid product, dry it, and wash it three times with ethanol to remove impurities to obtain carboxymethyl chitosan. S3. Extracting astaxanthin: Dissolve 2.4 g of Haematococcus pluvialis in 30 mL of ethyl acetate under light-shielding conditions, perform wall-breaking extraction in a high-speed blender at 40° C. for 60 min to obtain a mixed solution, vacuum filter to obtain a crude astaxanthin extract, repeat twice, combine the filtrates, and concentrate by rotary evaporation to obtain a crude astaxanthin oleoresin. Vacuum dry the mixture in a freeze dryer for 24 h to remove the residual solvent and obtain astaxanthin oleoresin, which is then stored in the dark. S4, weighing 1.1 g of glyceryl monostearate, 1.2 g of sucrose fatty acid ester and 2.4 g of carboxymethyl chitosan obtained in step S2, dispersing them in 80 mL of 90° C. distilled water, cooling to 60° C., adding 2.5 g of HA-Ag complex powder obtained in step S1, 0.6 g of vitamin C, 0.36 g of arbutin and 4.8 g of maltodextrin, stirring at high speed and cooling to room temperature to obtain a mixed emulsion, taking 1.4 g of astaxanthin oleoresin obtained in step S3 and dissolving it in the mixed emulsion, emulsifying and shearing at high speed at 10,000 r / min for 12 min, and after the emulsification is completed, performing atomization drying at an inlet air temperature of 180° C. to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material; S5. Stir and dissolve the prepared astaxanthin microcapsules in 100 mL of PBS, add 0.3 mL of phenoxyethanol and 0.5 g of nicotine amide, and stir for 15 min. After sufficient dissolution, adjust the pH to 7.40 with 10% citric acid, ultrasonicate for 20 min, and then stir in hydrothermal for 40 min at 40 °C until a complete sol is formed. The final modified hyaluronic acid composite microcapsules with antibacterial and whitening effects are obtained.

[0045] Comparative Example 5: Nicotine amide was not added in step S5, and the other steps were the same as in Example 3; Comparative Example 6: Arbutin was not added in step S4, and the other steps were the same as those in Example 3; Comparative Example 7: Vitamin C was not added in step S4, and the other steps were the same as in Example 3; Tyrosinase inhibition test S1. Material preparation Tyrosinase powder, enzyme activity 25 000 U, Sigma; L-tyrosine, purity ≥99%, biochemical reagent, Shanghai JCBIO; Preparation of PBS buffer: Weigh 7.8 g of sodium dihydrogen phosphate and 17.91 g of disodium hydrogen phosphate and prepare aqueous solutions, dilute to 500 mL with distilled water, mix appropriately to prepare PBS buffer with pH = 6.8 and 0.1 mol / L To prepare 0.1 M hydrochloric acid: Accurately measure 0.431 mL of 36%-38% hydrochloric acid and dilute to 50 mL with distilled water. To prepare a 0.05% L-tyrosine solution: Weigh 0.05 g of L-tyrosine and dissolve it in 35 mL of 0.1 M hydrochloric acid. Add 65 mL of PBS buffer (pH 6.8). Example 3 and Comparative Examples 5-7 Sample solution preparation: accurately weigh the test sample and dilute it with PBS buffer to a solution with a mass fraction of 5%.

[0046] Preparation of tyrosinase solution: Dissolve 25,000 U of tyrosinase in 250 mL of pure water to prepare a tyrosinase solution with an enzyme activity of 100 U / mL. Dispense into 1.5 mL EP tubes, filling each tube with 1 mL and store in a -40°C refrigerator.

[0047] S2. Tyrosinase activity assay As shown in Table 3, L-tyrosine, sample solution, and PBS buffer were added to the tubes numbered A1, A2, B1, and B2 in sequence and mixed evenly; the test tube rack was placed in a 37°C constant temperature water bath for 10-15 minutes; tyrosinase was added to the tubes of groups A1 and B1 in sequence, shaken evenly, and the reaction was stopped after 15 minutes. The plates were spotted in sequence, and the absorbance was measured using a microplate reader and the inhibition rate was calculated. The results are shown in Tables 4 and Figure 5 The above test was repeated three times, and the inhibition rate was calculated as follows: Tyrosinase inhibition rate = [(A1-A2)-(B1-B2)] / (A1-A2) × 100%, where A1 is the absorbance of the blank sample with enzyme system; A2 is the absorbance of the blank sample without enzyme system; B1 is the absorbance of the sample group with enzyme system; B2 is the absorbance of the sample group without enzyme system; Table 3 Reaction solution composition

[0048] The experimentally measured inhibition rate data are shown in Table 4;

[0049] From Table 4 and Figure 5 It can be seen that the present invention adds vitamin C and arbutin to the capsule wall. Vitamin C and arbutin can reduce the activity of tyrosinase and the amount of melanin produced, thereby reducing skin pigmentation.

Claims

1. A modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects, characterized by: The specific preparation steps are as follows: S1. At room temperature, weigh 0.1-0.3 g of sodium hyaluronate and dissolve it in 100 mL of distilled water. Stir evenly to obtain an HA aqueous solution. Prepare a 0.5 mol / L silver nitrate solution, mix 95 mL of the HA solution and 5 mL of the 0.5 mol / L silver nitrate solution, stir in the dark for 5 h, and irradiate with ultraviolet light for 10-30 min to obtain an HA-Ag complex aqueous solution. Freeze-dry to obtain an HA-Ag complex powder. S2. Disperse 5-10 g of chitosan in 50-100 mL of isopropanol and sonicate for 5-10 min. Add 25-35 mL of 38% NaOH solution and stir to mix evenly. Add 18-22 g of chloroacetic acid in 4 portions within 1 h. Stir continuously during the addition process and gradually raise the temperature to 60°C. Then, react at a constant temperature of 60°C for 4-6 h. Filter to obtain the solid product. After drying, wash with ethanol 3-5 times to remove impurities to obtain carboxymethyl chitosan. S3. Dissolve 1.1-2.4 g of Haematococcus pluvialis in 26-30 mL of ethyl acetate under light-shielding conditions, perform wall-breaking extraction in a high-speed blender at 40°C for 60 min to obtain a mixed solution, vacuum filter to obtain a crude astaxanthin extract, repeat twice, combine the filtrates, and concentrate by rotary evaporation to obtain a crude astaxanthin oleoresin. Vacuum dry the mixture in a freeze dryer for 24 h to remove the residual solvent to obtain astaxanthin oleoresin, and store in a dark place. S4, weighing 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, dispersing them in 60-80 mL of 90° C. distilled water, cooling to 60° C., adding 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, stirring at high speed and cooling to room temperature to obtain a mixed emulsion, taking 1.0-1.4 g of astaxanthin oleoresin obtained in step S3 and dissolving it in the mixed emulsion, emulsifying and shearing at high speed at 10,000 r / min for 8-12 min, and atomizing and drying at an inlet air temperature of 180° C. after the emulsification is completed, to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material; S5. Stir and dissolve the prepared astaxanthin microcapsules in 50-100 mL PBS, add 0.1-0.3 mL phenoxyethanol and 0.3-0.5 g nicotine amide, stir for 10-15 min, and after sufficient dissolution, adjust the pH to 7.35-7.45 with 10% citric acid. Ultrasonicate for 10-20 min, then stir in hydrothermal solution for 30-40 min at a temperature of 20-40 °C until a complete sol is formed to obtain the final modified hyaluronic acid complex microcapsules with antibacterial and whitening effects.

2. The modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to claim 1, characterized in that: In step S1, the amount of sodium hyaluronate used is 0.2 g, the ultraviolet band is UVB, and the irradiation time is 20 min.

3. The modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to claim 2, characterized in that: In step S2, the amount of chitosan used is 8 g, isopropanol is 80 mL, 38% NaOH solution is 30 mL, and chloroacetic acid is 10 mL. Ultrasonic treatment is performed for 8 min, stirring is performed for 5 h, and the mixture is washed with ethanol 4 times.

4. The modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to claim 3, characterized in that: In step S3, the amount of Haematococcus pluvialis used is 1.8 g, and the amount of ethyl acetate used is 28 mL.

5. The modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to claim 4, characterized in that: In step S4, the amount of glyceryl monostearate used is 0.9 g, the amount of sucrose fatty acid ester is 1.1 g, the amount of carboxymethyl chitosan used is 1.8 g, and the amount of distilled water is 70 mL.

6. The modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to claim 5, characterized in that: In step S4, the amount of HA-Ag complex powder is 2.1 g, the amount of vitamin C is 0.32 g, the amount of maltodextrin is 4.2 g, and the amount of astaxanthin oleoresin is 1.2 g.

7. The modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to claim 6, characterized in that: In step S5, the amount of PBS used is 80 mL, the amount of phenoxyethanol is 0.2 mL, and the amount of nicotineamide is 0.4 g.

8. The modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to claim 7, characterized in that: In step S5, the stirring time is 12 min, the pH is adjusted to 7.40, the ultrasound is performed for 15 min, the hydrothermal stirring is performed for 35 min, and the temperature is set for 30 min.

9. Use of the modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to any one of claims 1 to 8 in the preparation of cosmetic injection filling agents.

10. Use of the modified hyaluronic acid complex with whitening, freckle removal and antioxidant effects according to any one of claims 1 to 8 in cosmetics.

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