Modified hyaluronic acid with antibacterial and whitening effects and application of modified hyaluronic acid in medical beauty filler

Abstract

The invention belongs to the technical field of biological materials, and relates to modified hyaluronic acid with antibacterial and whitening effects and application of the modified hyaluronic acid in a medical beauty filler. 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 a hyaluronic acid, and in particular to a modified hyaluronic acid with antibacterial and whitening effects and its application in medical beauty fillers. Background Art

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a high-molecular-weight mucopolysaccharide composed of disaccharide units composed of glucuronic acid and acetylglucosamine. It is the main component of the extracellular matrix and extracellular matrix of cells and has an important influence on the physiological functions of cells. Hyaluronic acid is widely distributed in the epithelium, connective tissue and nervous tissue of vertebrates, especially in the skin tissue, joint fluid and eyeball.

[0003] At present, hyaluronic acid fillers have been widely used in the treatment of wrinkles, scars and facial contour defects. They are divided into two types: single-phase and bi-phase according to the degree of cross-linking. Single-phase fillers have good stability and no particles. They are prepared by changing the quantitative ratio of high molecular weight hyaluronic acid and low molecular weight hyaluronic acid. They have low strength, are easily changed by external forces, and have no particle texture. Bi-phase fillers are the most widely used products on the market. They are composed 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 to play a lubricating role, so that the suspension can be injected into the tissue through a fine needle.

[0004] Sodium hyaluronate has a certain antibacterial effect, which is mainly achieved by binding to fibrin and interfering with the function of bacterial surface adhesion factors. However, this antibacterial effect is relatively weak and mainly targets certain Gram-positive and Gram-negative bacteria. For other types of bacteria, or when the number of bacteria is large, the antibacterial effect of sodium hyaluronate is inadequate. The effect of sodium hyaluronate in whitening is also relatively limited. Although it can indirectly improve the problem of uneven skin tone and dullness by moisturizing and promoting skin cell regeneration, it cannot directly inhibit the production of melanin or accelerate the metabolism of melanin. Therefore, it is difficult to achieve significant whitening effects by relying solely on sodium hyaluronate. Sodium hyaluronate has a certain antioxidant effect, which can neutralize free radicals and reduce the damage to the skin caused by environmental factors. However, this antioxidant effect is relatively weak and is mainly limited to the surface of the skin. When the skin is subjected to strong oxidative stress, such as ultraviolet radiation, environmental pollution, etc., the antioxidant effect of sodium hyaluronate is insufficient.

[0005] In view of the above problems, the present invention adopts ultraviolet light reduction technology, uses hyaluronic acid aqueous solution as a polymer dispersion platform, constructs a silver nanoparticle dispersion system, and converts it into HA-Ag powder after drying treatment, which cooperates with carboxymethyl chitosan to give the product excellent antibacterial properties. Microcapsule 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 strengthening the whitening effect, vitamin C can reduce the oxidized astaxanthin, restore its antioxidant activity, and further amplify the antioxidant effect. Summary of the invention

[0006] In view of the above problems, the present invention provides a modified hyaluronic acid with antibacterial and whitening effects and its application in medical beauty fillers. The specific preparation steps are as follows:

[0007] S1. Preparation of HA-Ag composite material: at room temperature, weigh 0.1-0.3g sodium hyaluronate and dissolve it in 100mL distilled water, stir evenly to obtain HA aqueous solution, and prepare 0.5mol / L silver nitrate solution; take 95mL HA solution and 5mL 0.5mol / L silver nitrate solution, mix them, stir for 5h under light-proof conditions, irradiate with ultraviolet light for 10-30min, and obtain HA-Ag complex aqueous solution; obtain HA-Ag complex powder by freeze drying;

[0008] S2. Disperse 5-10 g of chitosan in 50-100 mL of isopropanol, perform ultrasonic treatment 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 times 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 4-6 h, then filter to obtain a solid product, dry and wash with ethanol for 3-5 times to remove impurities, and obtain carboxymethyl chitosan;

[0009] S3, extracting astaxanthin: taking 1.1-2.4g of Haematococcus pluvialis and dissolving it in 26-30mL of ethyl acetate under shading conditions, extracting by breaking the wall in a high-speed stirrer at 40°C for 60min to obtain a mixed solution, vacuum filtering to obtain a crude astaxanthin extract, repeating twice, combining the filtrate, concentrating by rotary evaporation to obtain a crude astaxanthin oleoresin, vacuum drying in a freeze dryer for 24h to evaporate the residual solvent to obtain astaxanthin oleoresin, and storing in a dark place;

[0010] S4, weigh 0.8-1.1g of glyceryl monostearate, 0.9-1.2g of sucrose fatty acid ester and 1.2-2.4g of carboxymethyl chitosan obtained in step S2, disperse them in 60-80mL of 90°C distilled water, cool to 60°C, add 1.7-2.5g of HA-Ag complex powder obtained in step S1, 0.4-0.6g of vitamin C, 0.28-0.36g of arbutin and 3.6-4.8g of maltodextrin, stir evenly at high speed and cool to room temperature to obtain a mixed emulsion, take 1.0-1.4g of astaxanthin oleoresin obtained in step S3 and dissolve it in the mixed emulsion, emulsify and shear at high speed at 10000r / min for 8-12min, and after the emulsification is completed, spray 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 PBS, add 0.1-0.3 mL phenoxyethanol and 0.3-0.5 g nicotine amide, stir for 10-15 min, adjust the pH to 7.35-7.45 with 10% citric acid after full dissolution, ultrasonicate for 10-20 min, and then stir in water for 30-40 min, set the temperature to 20-40 ° C, and wait until a complete sol is formed to obtain the final modified hyaluronic acid microcapsules with antibacterial and whitening effects.

[0012] 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.

[0013] Preferably, in step S2, the amount of chitosan used is 8 g, isopropanol is 80 mL, 38% NaOH solution is 30 mL, 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 for 4 times.

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

[0015] 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.

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

[0017] 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 nicotine amide used is 0.4 g.

[0018] 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 to 30 min.

[0019] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is:

[0020] 1. The silver ion added in the present invention is a broad-spectrum antibacterial agent, which has a strong killing effect on a variety of bacteria, viruses and fungi. When it is used as a microcapsule wall material, it can directly play an antibacterial role; carboxymethyl chitosan is a natural polymer compound that has antibacterial properties against a variety of bacteria. When it is used as a microcapsule wall material, it can play the following roles to enhance the antibacterial effect; sodium hyaluronate is a glucuronic acid that has the effects of moisturizing, lubricating and repairing the skin. Although it does not directly have antibacterial ability, when it is used as a microcapsule wall material, it can play the following roles to indirectly enhance the antibacterial effect; 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 a variety of bacteria; the antibacterial activity and pH adjustment ability of carboxymethyl chitosan can further enhance the antibacterial effect. This complementary effect enables the microcapsule to have a wider antibacterial spectrum and stronger antibacterial ability. These three materials may produce a synergistic effect in the microcapsule, jointly destroying the cell structure and metabolic process of bacteria, thereby enhancing the antibacterial effect.

[0021] 2. Vitamin C added in the present 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 to be one of the strongest antioxidant single molecules found in nature so far. It can remove a variety of free radicals, thereby inhibiting lipid peroxidation reactions caused by free radicals and achieving antioxidant effects. Astaxanthin also has an anti-ultraviolet effect, which can reduce the increased secretion of lipid free radicals due to sunlight exposure, and help delay skin aging; vitamin C can regenerate oxidized astaxanthin to restore its antioxidant activity.

[0022] 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, and by reducing the activity of tyrosinase, the amount of melanin generated is reduced, thereby reducing skin pigmentation. At the same time, vitamin C can promote the production of collagen, making the skin smoother and tighter. Arbutin can make melanin more evenly distributed on the skin surface and reduce spots. Nicotinamide is a derivative of vitamin B3, which can interfere with the signal channel between keratinocytes and melanocytes and reduce the production and transport of melanin. Arbutin inhibits the production of melanin, vitamin C protects the skin from damage by free radicals, and nicotine amide promotes skin metabolism. The three work together to significantly brighten the skin tone and reduce spots and freckles.

[0023] 4. The present invention uses microencapsulation technology to encapsulate astaxanthin in wall materials such as sodium hyaluronate, which can effectively isolate 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 substance, is used as the wall material of astaxanthin microcapsules, which can improve the water solubility of astaxanthin to a certain extent, making it easier to use in the fields of cosmetics, medical beauty, health care products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative labor.

[0025] Figure 1 and Figure 2 The morphology of astaxanthin microcapsules made of modified sodium hyaluronate under SEM observation;

[0026] Figure 3 It is a comparison diagram of the diameters of the inhibition zones of Example 1, Comparative Example 1 and Comparative Example 2;

[0027] Figure 4 is the free radical scavenging rate of Example 2, Comparative Example 3 and Comparative Example 4;

[0028] Figure 5 It is a line graph of the inhibition rate of tyrosinase in Example 3 and Comparative Examples 5 to 7. DETAILED DESCRIPTION

[0029] 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 in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the content of the present invention and are not used to limit the present invention.

[0030] In the present 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.; vitamin C was purchased from Henan Kuoyuan Chemical Products Co., Ltd., and the purity was ≥99%;

[0031] Example 1

[0032] 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, and cooperates with carboxymethyl chitosan. The long polymer chain of carboxymethyl chitosan can precipitate and flocculate bacterial cells, and the disinfection factors (such as NH 3+ ) can gather on the surface of bacteria and attract anions in the cell wall of microorganisms, thereby hindering the metabolism and reproduction of microorganisms. Silver ions can further penetrate the cell membrane and react with components in the cell, leading to bacterial death. This synergistic effect makes the antibacterial effect of the composite antibacterial agent more significant.

[0033] The specific implementation steps are as follows:

[0034] 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 HA aqueous solution, and prepare 0.5 mol / L silver nitrate solution; take 95 mL of HA aqueous solution and 5 mL of 0.5 mol / L silver nitrate solution, mix them, stir for 5 h in a dark environment, and irradiate with ultraviolet light for 10 min to obtain HA-Ag complex aqueous solution; obtain HA-Ag complex powder by freeze drying;

[0035] 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 times 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, then filter to obtain a solid product, dry and wash with ethanol 5 times to remove impurities, and obtain carboxymethyl chitosan;

[0036] S3, extracting astaxanthin: taking 1.1g of Haematococcus pluvialis and dissolving it in 26mL of ethyl acetate under shading conditions, extracting the mixture by breaking the wall in a high-speed stirrer at 40°C for 60min to obtain a mixed solution, vacuum filtering to obtain a crude astaxanthin extract, repeating twice, combining the filtrate, concentrating by rotary evaporation to obtain a crude astaxanthin oleoresin, vacuum drying in a freeze dryer for 24h to evaporate the residual solvent to obtain astaxanthin oleoresin, and storing in a dark place;

[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, disperse them in 60mL of 90°C distilled water, cool to 60°C, 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 evenly at high speed and cool to room temperature to obtain a mixed emulsion, take 1.0g of astaxanthin oleoresin obtained in step S3 and dissolve it in the mixed emulsion, emulsify and shear at high speed at 10000r / min for 8min, and after the emulsification is completed, atomize and dry at an inlet air 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;

[0038] S5. Dissolve the prepared astaxanthin microcapsules in 50 mL PBS, add 0.1 mL phenoxyethanol and 0.3 g nicotine amide, and stir for 10 min. After fully dissolved, adjust the pH to 7.35 with 10% citric acid, ultrasonicate for 10 min, and then stir in water for 30 min. Set the temperature to 20 ° C. When a complete sol is formed, the final modified hyaluronic acid 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 the other steps were the same as those in Example 1;

[0040] 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;

[0041] Antimicrobial activity test:

[0042] 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 process was completed on a clean bench without other bacteria. The bacterial culture solution consisted of peptone (1.0%, w / v), yeast powder (0.5%, w / v), sodium chloride (1.0%, w / v), agar powder (1.5%, w / v) was added to the culture solution, and the remaining components 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 according to a volume ratio of 1 / 100 to the culture solution, and placed in a shaking table at 37°C for 12 hours. 80 mL of culture medium was placed in a cell culture dish, and after solidification, 100 ul of bacterial solution was dripped on the surface and evenly smeared. 20 ul of the circular sheets (with a diameter of 10 mm) of the sodium hyaluronate microcapsules prepared in Example 1, Comparative Example 1 and Comparative Example 2 were evenly smeared on the culture medium containing bacteria. The culture medium was placed in an incubator at 37 ° C. After 24 hours, 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.

[0043] Table 1 Diameters of inhibition zones of Example 1, Comparative Example 1 and Comparative Example 2

[0044]

[0045] It can be seen from the test data 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 the joint use of the two improves the antibacterial effect of sodium hyaluronate microcapsules.

[0046] Example 2

[0047] This embodiment illustrates that the present invention introduces microencapsulation technology, selects astaxanthin as the capsule core component, and relies on the protection of sodium hyaluronate to extend the stable existence time of astaxanthin, greatly improving the antioxidant capacity of the product. At the same time, vitamin C can reduce oxidized astaxanthin to restore its antioxidant activity.

[0048] The specific implementation steps are as follows:

[0049] 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 a HA aqueous solution, and prepare a 0.5 mol / L silver nitrate solution; take 95 mL of HA solution and 5 mL of 0.5 mol / L silver nitrate solution, mix them, stir for 5 h in a dark environment, and irradiate with ultraviolet light for 20 min to obtain a HA-Ag complex aqueous solution; and obtain a HA-Ag complex powder by freeze drying;

[0050] S2. Disperse 8 g of chitosan in 80 mL of isopropanol, ultrasonicate for 8 min, add 30 mL of 38% NaOH solution, stir and mix evenly, add 20 g of chloroacetic acid in 4 times 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 5 h, then filter to obtain a solid product, dry and wash with ethanol 5 times to remove impurities, and obtain carboxymethyl chitosan;

[0051] S3, extracting astaxanthin: taking 1.8g of Haematococcus pluvialis and dissolving it in 28mL of ethyl acetate under shading conditions, extracting the mixture by breaking the wall in a high-speed stirrer at 40°C for 60min to obtain a mixed solution, vacuum filtering to obtain a crude astaxanthin extract, repeating twice, combining the filtrate, concentrating by rotary evaporation to obtain a crude astaxanthin oleoresin, vacuum drying in a freeze dryer for 24h to evaporate the residual solvent to obtain astaxanthin oleoresin, and storing in a dark place;

[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, disperse them in 70mL of distilled water at 90°C, cool to 60°C, 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 evenly at high speed and cool to room temperature to obtain a mixed emulsion, take 1.2g of astaxanthin oleoresin obtained in step S3 and dissolve it in the mixed emulsion, emulsify and shear at high speed at 10000r / min for 10min, and after the emulsification is completed, atomize and dry at an inlet air temperature of 180°C to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material;

[0053] S5. Dissolve the prepared astaxanthin microcapsules in 80 mL PBS, add 0.2 mL phenoxyethanol and 0.4 g nicotine amide, and stir for 12 min. After fully dissolved, adjust the pH to 7.45 with 10% citric acid, ultrasonicate for 15 min, and then stir in hydrothermal for 35 min. Set the temperature to 30 ° C. When a complete sol is formed, the final modified hyaluronic acid microcapsules with antibacterial and whitening effects can be obtained.

[0054] Comparative Example 3: In step S4, vitamin C is replaced with β-carotene, and the other steps are the same as those in Example 2.

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

[0056] Antioxidant effect test

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

[0058] In this experiment, the ability of astaxanthin microcapsules to scavenge DPPH free radicals was determined by colorimetry. 1 mL of sodium hyaluronate microcapsules prepared using Example 2, Comparative Example 3 and Comparative Example 4 were respectively transferred into 20 mL test tubes, 1 mL of 0.25 mM DPPH ethanol solution was added, the mixture was shaken thoroughly, reacted at room temperature in the dark for 30 min, and then poured into a cuvette. 1 mL of anhydrous ethanol and 1 mL of sample solution were used as zeroing solutions, and the absorbance was measured at a wavelength of 517 nm and the absorbance value was recorded as A1.

[0059] 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 at room temperature away from light 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.

[0060] The above steps were repeated three times and the data were recorded respectively. The clearance rate was calculated according to the following formula:

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

[0062] A1 represents the sample absorbance value, and A2 represents the blank absorbance value;

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

[0064] Add 5mM pH7.4 potassium phosphate buffer solution to the 7mM ABTS solution that has been left overnight to dilute it so that the absorbance measured at 734nm is between 0.75-0.80 and it can be used. Among them, 7mM ABTS solution: take 19.2mg of ABTS in a 20mL centrifuge tube, add 5mL of distilled water and 88μl of 140mM potassium persulfate solution, vortex until it is completely dissolved, and refrigerate for 12-16h away from light for later use; 5mM pH7.4 potassium phosphate buffer: accurately weigh 1.36g of potassium dihydrogen phosphate, add 79mL of 0.1mol / L sodium hydroxide solution, and dilute to a 200mL volumetric flask with distilled water after it is completely dissolved.

[0065] 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 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.

[0066] 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, 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.

[0067] The above steps were repeated three times and the data were recorded respectively. The clearance rate was calculated according to the following formula:

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

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

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

[0071] Pipette 1 mL of 0.75 mmol / L ethanol solution of o-phenanthroline 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, incubate at 37°C for 60 min, use distilled water as zeroing reagent, read the absorbance at 536 nm, record as A p Value; replace 1mL H2O2 in the above step with 1mL distilled water, and the measured absorbance value is recorded 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, respectively, and the absorbance value was measured and recorded as A s .

[0072] The above test was repeated three times, and the clearance 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 rate of Example 2, Comparative Example 3 and Comparative Example 4

[0075]

[0076] 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, 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 vitamin C and arbutin are added to the capsule wall of the present invention. Vitamin C and arbutin can inhibit the activity of tyrosinase, and by reducing the activity of tyrosinase, the amount of melanin generated is reduced, thereby 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.

[0079] The specific implementation steps are as follows:

[0080] 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 a HA aqueous solution, and prepare a 0.5 mol / L silver nitrate solution; take 95 mL of HA solution and 5 mL of 0.5 mol / L silver nitrate solution, mix them, stir for 5 h in a dark environment, and irradiate with ultraviolet light for 30 min to obtain a HA-Ag complex aqueous solution; and obtain a HA-Ag complex powder by freeze drying;

[0081] S2. Disperse 10 g of chitosan in 100 mL of isopropanol, ultrasonicate for 10 min, add 35 mL of 38% NaOH solution, stir and mix evenly, add 22 g of chloroacetic acid in 4 times 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 6 h, then filter to obtain a solid product, dry and wash with ethanol 3 times to remove impurities, and obtain carboxymethyl chitosan;

[0082] S3, extracting astaxanthin: taking 2.4g of Haematococcus pluvialis and dissolving it in 30mL of ethyl acetate under shading conditions, extracting the mixture by breaking the wall in a high-speed stirrer at 40°C for 60min to obtain a mixed solution, vacuum filtering to obtain a crude astaxanthin extract, repeating twice, combining the filtrate, concentrating by rotary evaporation to obtain a crude astaxanthin oleoresin, vacuum drying in a freeze dryer for 24h to evaporate the residual solvent to obtain astaxanthin oleoresin, and storing in a dark place;

[0083] S4, weighing 1.1g of glyceryl monostearate, 1.2g of sucrose fatty acid ester and 2.4g of carboxymethyl chitosan obtained in step S2, dispersing them in 80mL of 90°C distilled water, cooling to 60°C, adding 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, stirring at high speed and cooling to room temperature to obtain a mixed emulsion, taking 1.4g of astaxanthin oleoresin obtained in step S3 and dissolving it in the mixed emulsion, emulsifying and shearing at high speed at 10000r / min for 12min, and atomizing and drying at an inlet air temperature of 180°C after the emulsification to obtain astaxanthin microcapsules with modified sodium hyaluronate as the wall material;

[0084] S5. Dissolve the prepared astaxanthin microcapsules in 100 mL PBS, add 0.3 mL phenoxyethanol and 0.5 g nicotine amide, and stir for 15 min. After fully dissolved, adjust the pH to 7.40 with 10% citric acid, ultrasonicate for 20 min, and then stir in hydrothermal for 40 min. Set the temperature to 40 °C and wait until a complete sol is formed to obtain the final modified hyaluronic acid microcapsules with antibacterial and whitening effects.

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

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

[0087] Comparative Example 7: Vitamin C was not added in step S4, and the other steps were the same as those in Example 3;

[0088] Tyrosinase inhibition assay

[0089] S1. Material preparation

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

[0091] Preparation of PBS buffer: Weigh 7.8 g of sodium dihydrogen phosphate and 17.918 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

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

[0093] Example 3 and Comparative Examples 5 to 7 Preparation of sample solution: Accurately weigh the test sample and dilute it with PBS buffer to a solution with a mass fraction of 5%.

[0094] Preparation of tyrosinase solution: dissolve 25000U of tyrosinase in 250mL of pure water to prepare a tyrosinase solution with an enzyme activity of 100U / mL, divide the solution into 1.5mL EP tubes, fill each tube with 1mL and store in a -40℃ refrigerator.

[0095] S2. Tyrosinase activity assay

[0096] 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; the test tube rack was placed in a 37°C constant temperature water bath for 10 to 15 minutes; tyrosinase was added to the tubes of the A1 and B1 groups in sequence, shaken, and the reaction was stopped after 15 minutes. The plates were spotted in sequence on a 96-well plate, and the absorbance was measured with an enzyme 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:

[0097] 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;

[0098] Table 3 Reaction solution composition

[0099]

[0100] The experimentally measured inhibition rate data are shown in Table 4;

[0101] Table 4 Inhibition rate of tyrosinase in Example 3 and Comparative Examples 5 to 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 can be seen that vitamin C and arbutin are added to the capsule wall of the present invention, and vitamin C and arbutin can reduce the activity of tyrosinase, reduce the production of melanin, and thus reduce skin pigmentation.

Claims

1. A method for preparing modified hyaluronic acid with antibacterial and whitening effects, characterized in that: The specific preparation steps are as follows: S1. Preparation of HA-Ag composite material: at room temperature, weigh 0.1-0.3g sodium hyaluronate and dissolve it in 100mL distilled water, stir evenly to obtain HA aqueous solution; prepare 0.5mol / L silver nitrate solution, take 95mL HA solution and 5mL 0.5mol / L silver nitrate solution and mix them, stir for 5h in dark conditions, irradiate with ultraviolet light for 10-30min to obtain HA-Ag complex aqueous solution; obtain HA-Ag complex powder by freeze drying; S2. Disperse 5-10 g of chitosan in 50-100 mL of isopropanol, perform ultrasonic treatment 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 times 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 4-6 h, then filter to obtain a solid product, dry and wash with ethanol for 3-5 times to remove impurities, and obtain carboxymethyl chitosan; S3, extracting astaxanthin: taking 1.1-2.4g of Haematococcus pluvialis and dissolving it in 26-30mL of ethyl acetate under shading conditions, extracting by breaking the wall in a high-speed stirrer at 40°C for 60min to obtain a mixed solution, vacuum filtering to obtain a crude astaxanthin extract, repeating twice, combining the filtrate, concentrating by rotary evaporation to obtain a crude astaxanthin oleoresin, vacuum drying in a freeze dryer for 24h to evaporate the residual solvent to obtain astaxanthin oleoresin, and storing in a dark place; S4, weigh 0.8-1.1g of glyceryl monostearate, 0.9-1.2g of sucrose fatty acid ester and 1.2-2.4g of carboxymethyl chitosan obtained in step S2, disperse them in 60-80mL of 90°C distilled water, cool to 60°C, add 1.7-2.5g of HA-Ag complex powder obtained in step S1, 0.4-0.6g of vitamin C, 0.28-0.36g of arbutin and 3.6-4.8g of maltodextrin, stir evenly at high speed and cool to room temperature to obtain a mixed emulsion, take 1.0-1.4g of astaxanthin oleoresin obtained in step S3 and dissolve it in the mixed emulsion, emulsify and shear at high speed at 10000r / min for 8-12min, and after the emulsification is completed, spray dry 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 50-100 mL PBS, add 0.1-0.3 mL phenoxyethanol and 0.3-0.5 g nicotine amide, stir for 10-15 min, adjust the pH to 7.35-7.45 with 10% citric acid after full dissolution, ultrasonicate for 10-20 min, and then stir in water for 30-40 min, set the temperature to 20-40 ° C, and wait until a complete sol is formed to obtain the final modified hyaluronic acid microcapsules with antibacterial and whitening effects.

2. The method for preparing a modified hyaluronic acid having 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 band is UVB, and the irradiation time is 20 min.

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

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

5. The method for preparing a modified hyaluronic acid having antibacterial and whitening effects according to claim 1, characterized in that: In the 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.

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

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

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

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

10. The modified hyaluronic acid microcapsules with antibacterial and whitening effects as claimed in claim 9 are used as cosmetic injection filling agents.

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