Multi-effect sodium hyaluronate composite gel, preparation method thereof and application of multi-effect sodium hyaluronate composite gel in beauty

Abstract

The invention discloses a multi-effect sodium hyaluronate composite gel, a preparation method thereof and application of the multi-effect sodium hyaluronate composite gel in cosmetology, and belongs to the technical field of plastic surgery and general surgery implants. The multi-effect sodium hyaluronate composite gel is prepared from sodium hyaluronate gel and agarose mixed gel. The multi-effect sodium hyaluronate composite gel prepared by the invention not only can moisturize skin and supplement collagen, has the effects of whitening, resisting oxidation and resisting inflammation, but also effectively solves the problem that sodium hyaluronate, collagen and PDRN are easy to degrade, so that the beautifying effect is more long-acting, and the effect is more obvious. Therefore, the implant has better application prospect and value in the fields of plastic and aesthetic surgery and general surgical implants.

Description

Technical Field

[0001] The invention belongs to the technical field of plastic surgery and general surgical implants, and specifically relates to a multi-effect sodium hyaluronate composite gel, a preparation method thereof and application in cosmetology. Background Art

[0002] The main causes of skin aging can be divided into endogenous factors and exogenous factors. Endogenous factors are mainly natural aging, which is an aging process controlled by genetic factors. As age increases, the skin will gradually show symptoms of aging, such as wrinkles and sagging. This is an inevitable natural phenomenon. Exogenous factors include adverse environmental stimulation, lack of sleep, lack of exercise and bad living habits. Long-term exposure to sunlight, cold wind, polluted air, as well as bad living habits such as staying up late, lack of exercise, smoking and drinking, will accelerate the process of skin aging. These factors will destroy the skin's protective layer, causing the skin to lose moisture and elasticity, and accelerate aging. The process of skin aging is usually accompanied by the loss of hyaluronic acid, type I collagen, type III collagen and elastin in the dermis of the skin. Elastin generally stops growing at around 25 years old, and its loss rate is faster than collagen. Supplementing hyaluronic acid and collagen to the skin can delay aging to a certain extent and improve skin condition.

[0003] Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleotides with a molecular weight between 50KDa and 1500KDa. It is derived from trout or salmon sperm DNA, and its DNA fragments are most similar to the human body. PDRN is a patented registered drug with multiple activities: tissue repair, anti-ischemia, anti-inflammatory, hydration and moisturizing. Studies have found that PDRN can be used as a fibroblast growth stimulator in wound repair; furthermore, the nucleotides formed by the decomposition of PDNA bind to adenosine A2A receptors, bidirectionally regulate related inflammatory and anti-inflammatory factors, and can play a sustained anti-inflammatory role, while reducing the time required for healing and promoting human cell regeneration. When used for injection beauty, it can activate the skin's self-healing ability, stimulate the continuous production of growth factors inside the skin, and promote fibroblast activity. Studies have shown that when PDRN and hyaluronic acid are used in combination, the effect of promoting wound healing is significantly higher than that of hyaluronic acid alone, showing the effect of mutual promotion between the two.

[0004] Patent CN118356527A discloses a sodium hyaluronate-polydeoxyribonucleotide composite solution, which is a simple compounding of polydeoxyribonucleotides and cross-linked sodium hyaluronate gel, but the free polydeoxyribonucleotides degrade quickly. Patent CN118845531A discloses a PDRN-sodium hyaluronate composite solution, which is a simple compounding of PDRN and non-cross-linked sodium hyaluronate. Both PDRN and non-cross-linked sodium hyaluronate have the problem of rapid degradation. Patent CN118873432A discloses a PDRN-non-cross-linked collagen composition, which only physically mixes PDRN and collagen, and cannot solve the problem of easy degradation of the two. Patent CN118356527A discloses a PDRN-cross-linked collagen composition, which cross-links PDRN and collagen through a cross-linking agent. Although it alleviates the problem of easy degradation of PDRN and collagen to a certain extent, the preparation process of the product is relatively complicated, and the product is more suitable for filling and wrinkle removal, and the application direction is different. Patent CN118490890A discloses an anti-wrinkle compound injection, which physically mixes non-cross-linked hyaluronic acid and collagen, but still has the problem of rapid degradation of free hyaluronic acid and collagen. Patent CN118078652A discloses a wrinkle-removing subcutaneous fixed-point layering technology stimulation repair liquid, which physically mixes cross-linked HA gel, animal collagen and recombinant collagen, but still has the problem of rapid degradation of collagen.

[0005] Therefore, how to solve the problem of easy degradation of sodium hyaluronate, collagen and PDRN is an important challenge faced by the current existing technology. Summary of the invention

[0006] The invention provides a multi-effect sodium hyaluronate composite gel, wherein the composite gel comprises a sodium hyaluronate gel and an agarose mixed gel.

[0007] The above multi-effect sodium hyaluronate composite gel is prepared by the following method:

[0008] (1) Preparation of Sodium Hyaluronate Gel

[0009] BDDE is added to a sodium hydroxide solution and mixed to obtain a BDDE solution; sodium hyaluronate and the BDDE solution are then mixed evenly to perform cross-linking; after the cross-linking is completed, the mixture is placed in a PBS buffer solution for swelling, and after the swelling is completed, the water is drained, homogenized, and sterilized to obtain a sodium hyaluronate gel;

[0010] (2) Preparation of agarose mixed gel

[0011] Disperse recombinant humanized type I collagen, recombinant humanized type III collagen, PDRN and agarose powder in a PBS buffer solution, mix evenly, sterilize, and after cooling to room temperature, homogenize to obtain an agarose mixed gel;

[0012] (3) Mixing

[0013] The sodium hyaluronate gel and the agarose mixed gel are mixed and stirred evenly to obtain a multi-effect sodium hyaluronate composite gel.

[0014] In the preparation method of the above multi-effect sodium hyaluronate composite gel, each raw material is selected from the following parts:

[0015] BDDE 0.03-0.04 parts, sodium hydroxide solution 10-20 parts, sodium hyaluronate 3-8 parts, recombinant type I humanized collagen 0.32-2.56 parts, recombinant type III humanized collagen 0.32-2.56 parts, PDRN 0.32-2.56 parts, agarose powder 0.08-0.2 parts;

[0016] When the component is a solid component, the portion number represents grams; when the component is a liquid component, the portion number represents milliliters; in actual applications, the portion number can be enlarged or reduced in proportion to the portion number.

[0017] In a specific embodiment, each raw material is selected from the following parts:

[0018] 0.033 parts of BDDE, 20 parts of sodium hydroxide solution, 5 parts of sodium hyaluronate, 1.2 parts of recombinant humanized type I collagen, 1.2 parts of recombinant humanized type III collagen, 1.2 parts of PDRN, and 0.12 parts of agarose powder;

[0019] When the component is a solid component, the portion number represents grams; when the component is a liquid component, the portion number represents milliliters; in actual applications, the portion number can be enlarged or reduced in proportion to the portion number.

[0020] In the preparation method of the multi-effect sodium hyaluronate composite gel, the mass concentration of the sodium hydroxide solution is selected from 0.5% to 3.0%, preferably 2%.

[0021] In the preparation method of the above-mentioned multi-effect sodium hyaluronate composite gel, the cross-linking conditions are selected from: cross-linking in a water bath at 25-75°C for 3-24 hours; preferably: cross-linking in a water bath at 25°C for 22 hours.

[0022] In the preparation method of the above-mentioned multi-effect sodium hyaluronate composite gel, the sterilization is selected from: sterilization at 115-125° C. for 10-20 min; preferably: sterilization at 121° C. for 15 min.

[0023] In the preparation method of the multi-effect sodium hyaluronate composite gel, the swelling time is selected from 16 to 24 hours, preferably 20 hours.

[0024] In the preparation method of the multi-effect sodium hyaluronate composite gel, the homogenization refers to passing the gel through a 500-800 mesh sieve on a pressurized homogenizer to obtain a gel with uniform particle size.

[0025] In the preparation method of the multi-effect sodium hyaluronate composite gel, in the step (3), the stirring conditions are: stirring time 4 to 10 hours, stirring speed 70 to 100 r / min.

[0026] In the present invention, the volume of the PBS buffer solution used for swelling is 10 to 50 times the weight of the cross-linked sodium hyaluronate.

[0027] The multi-effect sodium hyaluronate composite gel prepared by the invention is a clear and transparent flowing liquid.

[0028] The present invention provides application of the multi-effect sodium hyaluronate composite gel in preparing products with injectable cosmetic effects.

[0029] The beneficial effects of the present invention are:

[0030] The multi-effect sodium hyaluronate composite gel prepared by the present invention can not only hydrate and moisturize the skin, supplement collagen, and has whitening, anti-oxidation and anti-inflammatory effects, but also effectively solves the problem of easy degradation of sodium hyaluronate, collagen and PDRN, making the beauty effect more long-lasting, and thus has good application prospects and value in the fields of cosmetic surgery and general surgical implants.

[0031] A certain concentration of agarose is dissolved at high temperature and becomes a gel after cooling to room temperature. The higher the agarose content, the greater the gel strength, that is, the harder it is. Within the appropriate concentration range, a colorless, transparent, soft, and elastic agarose gel can be obtained, which is very similar to sodium hyaluronate gel. Agarose gel has a porous structure. In the process of liquid to gelation, collagen and PDRN can be coated on the inside or outside of the pore. Because there is no enzyme in the body that can degrade agarose, the degradation rate is relatively slow. In the process of slow degradation of agarose, the collagen and PDRN coated by agarose are gradually released, continuously providing nutrition to the skin, and effectively solving the problem of easy degradation of free collagen and PDRN. This method is simple and easy, and does not require chemical cross-linking or morphological changes of the substance. Micro-cross-linking of sodium hyaluronate prolongs the degradation time of sodium hyaluronate, and the amount of cross-linking agent is small, which reduces the probability of adverse reactions. At the same time, agarose gel and sodium hyaluronate gel with micro-particle size also have micro-filling effects. In addition, the present invention also proves that the product has stronger anti-oxidation effect after moist heat sterilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a physical picture of agarose composite gel;

[0033] Figure 2 It is an injectable multi-effect sodium hyaluronate composite gel. DETAILED DESCRIPTION

[0034] In the present invention, injection-grade sodium hyaluronate powder (Huaxi Biotechnology Co., Ltd.), BDDE (Aladdin Chemical Reagent Co., Ltd.), recombinant type I humanized collagen (Chengdu Yunxi Biotechnology Co., Ltd.), recombinant type III humanized collagen (Chengdu Yunxi Biotechnology Co., Ltd.), PDRN (Ruijiming (Shandong) Biotechnology Co., Ltd.), and agarose powder (Hunan Wokai Biotechnology Co., Ltd.).

[0035] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0036] Example 1

[0037] The steps for preparing the multi-effect sodium hyaluronate composite gel are as follows:

[0038] (1) Preparation of Sodium Hyaluronate Gel

[0039] 0.033 g of BDDE was added to 20 mL of sodium hydroxide solution (concentration of 2%), and mixed to obtain a BDDE solution; then 5 g of injection-grade sodium hyaluronate powder and the BDDE solution were added to a centrifuge tube and stirred evenly; the centrifuge tube was placed in a 25°C water bath for crosslinking for 22 hours; the gel was then placed in a phosphate buffer solution (pH = 7.0) prepared with injection water for swelling and washing (swelling and washing are carried out simultaneously, ion exchange is continuously carried out during swelling, sodium hydroxide and free BDDE diffuse into the solution and are gradually washed away), each time for 4 hours, a total of 5 operations, the volume of PBS buffer solution used each time is 800 mL, and the water is drained after the last swelling is completed; 500 mesh sieve is used for homogenization, and sterilization is carried out at 121°C for 15 minutes to obtain a sodium hyaluronate gel.

[0040] (2) Preparation of agarose mixed gel

[0041] 1.2 g of recombinant humanized type I collagen, 1.2 g of recombinant type III humanized collagen, 1.2 g of PDRN and 0.12 g of agarose powder were dispersed in 40 g of phosphate buffer solution (PH = 7.0) prepared with water for injection, mixed evenly, put into a sterilizer, sterilized at 121 ° C for 15 min, dissolved, cooled to room temperature, and homogenized with an 800 mesh sieve to obtain an agarose mixed gel coated with collagen and PDRN.

[0042] Agarose mixed gel actual picture Figure 1 shown.

[0043] (3) Mixing and filling

[0044] The sodium hyaluronate gel and agarose mixed gel are stirred in a sterile environment, mixed evenly, and aseptically filled into a sterile prefilled syringe to obtain a multi-effect sodium hyaluronate composite gel for injection.

[0045] The actual picture of multi-effect sodium hyaluronate composite gel is as follows Figure 2 shown.

[0046] Comparative Example 1

[0047] Compared with Example 1, in this comparative example, when preparing the multi-effect sodium hyaluronate composite gel, the sodium hyaluronate was not cross-linked, and the recombinant type I humanized collagen, the recombinant type III humanized collagen and the PDRN were not coated by the agarose gel.

[0048] The steps for preparing the multi-effect sodium hyaluronate composite gel are as follows:

[0049] (1) Preparation of sodium hyaluronate mixed solution

[0050] Add 5 g of sodium hyaluronate powder, 1.2 g of recombinant humanized type I collagen, 1.2 g of recombinant humanized type III collagen and 1.2 g of PDRN to 600 g of PBS buffer solution (pH=7.0) prepared with water for injection, stir evenly, and sterilize at 121° C. for 15 min.

[0051] (2) Preparation of agarose gel

[0052] Disperse 0.12 g of agarose powder in 40 g of phosphate buffer solution (pH = 7.0) prepared with water for injection, mix well, put into a sterilizer, sterilize at 121°C for 15 min, dissolve it, cool to room temperature, and homogenize with an 800-mesh sieve to obtain agarose gel.

[0053] (3) Mixing and filling

[0054] The sodium hyaluronate mixed solution and agarose gel are stirred in a sterile environment, mixed evenly, and aseptically filled into a sterile pre-filled syringe to obtain a multi-effect sodium hyaluronate composite gel for injection.

[0055] Comparative Example 2

[0056] Compared with Example 1, in this comparative example, the sodium hyaluronate was not cross-linked when preparing the multi-effect sodium hyaluronate composite gel.

[0057] The steps for preparing the multi-effect sodium hyaluronate composite gel are as follows:

[0058] (1) Preparation of Sodium Hyaluronate Solution

[0059] Add 5 g of sodium hyaluronate powder into 600 g of PBS buffer solution (PH=7.0) prepared with water for injection, stir evenly, and sterilize at 121° C. for 15 min.

[0060] (2) Preparation of agarose mixed gel

[0061] 1.2 g of recombinant humanized type I collagen, 1.2 g of recombinant type III humanized collagen, 1.2 g of PDRN, and 0.12 g of agarose powder were dispersed in 40 g of phosphate buffer solution (PH = 7.0) prepared with water for injection, mixed evenly, put into a sterilizer, sterilized at 121°C for 15 min, dissolved, cooled to room temperature, and homogenized through an 800-mesh sieve to obtain an agarose gel coated with collagen and PDRN.

[0062] (3) Mixing and filling

[0063] The sodium hyaluronate solution and agarose mixed gel are stirred in a sterile environment, mixed evenly, and aseptically filled into a sterile pre-filled syringe to obtain a multi-effect sodium hyaluronate composite gel for injection.

[0064] Comparative Example 3

[0065] Compared with Example 1, in this comparative example, when preparing the multi-effect sodium hyaluronate composite gel, the recombinant type I humanized collagen, the recombinant type III humanized collagen and the PDRN were not coated by the agarose gel.

[0066] The steps for preparing the multi-effect sodium hyaluronate composite gel are as follows:

[0067] (1) Preparation of sodium hyaluronate mixed gel

[0068] 0.033 g BDDE was added to 20 mL sodium hydroxide solution (concentration of 2%), and mixed to obtain BDDE solution; then 5 g injection-grade sodium hyaluronate powder and BDDE solution were added to a centrifuge tube and stirred evenly; the centrifuge tube was placed in a 25°C water bath for crosslinking for 22 hours; the gel was then placed in a phosphate buffer solution (pH = 7.0) prepared with injection water for swelling and washing, each time for 4 hours, for a total of 5 operations, and the volume of PBS buffer solution used each time was 800 mL. After the last swelling was completed, the water was drained; 500 mesh sieve was used for homogenization, and sterilized at 121°C for 15 minutes to obtain sodium hyaluronate gel; then 1.2 g recombinant type I humanized collagen, 1.2 g recombinant type III humanized collagen, and 1.2 g PDRN were added, stirred for 4 hours, and sterilized at 121°C for 15 minutes to obtain a sodium hyaluronate mixed gel.

[0069] (2) Preparation of agarose gel

[0070] Disperse 0.12 g of agarose powder in 40 g of phosphate buffer solution (PH=7.0) prepared with water for injection, mix well, put into a sterilizer, sterilize at 121°C for 15 min, dissolve it, cool to room temperature, and homogenize with an 800-mesh sieve to obtain agarose gel.

[0071] (3) Mixing and filling

[0072] The sodium hyaluronate mixed gel and agarose gel are stirred in a sterile environment, mixed evenly, and aseptically filled into a sterile prefilled syringe to obtain a multi-effect sodium hyaluronate composite gel for injection.

[0073] Comparative Example 4

[0074] Compared with Example 1, in this comparative example, when preparing the multi-effect sodium hyaluronate composite gel, no recombinant humanized type I collagen, recombinant humanized type III collagen and PDRN were added.

[0075] The steps for preparing sodium hyaluronate composite gel are as follows:

[0076] (1) Preparation of Sodium Hyaluronate Gel

[0077] 0.033 g of BDDE was added to 20 mL of sodium hydroxide solution (concentration of 2%), and mixed to obtain a BDDE solution; then 5 g of injection-grade sodium hyaluronate powder and the BDDE solution were added to a centrifuge tube and stirred evenly; the centrifuge tube was placed in a 25°C water bath for crosslinking for 22 hours; the gel was then placed in a phosphate buffer solution (pH = 7.0) prepared with injection water for swelling and washing, each time for 4 hours, for a total of 5 times, and the volume of the PBS buffer solution used each time was 800 mL. After the last swelling was completed, the water was drained; homogenization was performed through a 500-mesh sieve, and sterilization was performed at 121°C for 15 minutes to obtain a sodium hyaluronate gel.

[0078] (2) Preparation of agarose gel

[0079] Disperse 0.12 g of agarose powder in 40 g of phosphate buffer solution (PH=7.0) prepared with water for injection, mix well, put into a sterilizer, sterilize at 121°C for 15 min, dissolve it, cool to room temperature, and homogenize with an 800-mesh sieve to obtain agarose gel.

[0080] (3) Mixing and filling

[0081] The sodium hyaluronate gel and agarose gel are stirred in a sterile environment, mixed evenly, and aseptically filled into a sterile prefilled syringe to obtain a multi-effect sodium hyaluronate composite gel for injection.

[0082] Comparative Example 5

[0083] Compared with Example 1, in this comparative example, when preparing the multi-effect sodium hyaluronate composite gel, the recombinant type I humanized collagen, the recombinant type III humanized collagen and the PDRN were not coated with agarose gel, and the collagen and the like were not sterilized by wet heat.

[0084] The steps for preparing the multi-effect sodium hyaluronate composite gel are as follows:

[0085] (1) Preparation of sodium hyaluronate mixed gel

[0086] 0.033 g BDDE was added to 20 mL sodium hydroxide solution (concentration of 2%), and mixed to obtain BDDE solution; then 5 g injection-grade sodium hyaluronate powder and BDDE solution were added to a centrifuge tube and stirred evenly; the centrifuge tube was placed in a 25°C water bath for crosslinking for 22 h; the gel was then placed in a phosphate buffer solution (pH = 7.0) prepared with injection water for swelling and washing, each time for 4 h, for a total of 5 times, and the volume of PBS buffer solution used each time was 800 mL. After the last swelling was completed, the water was drained; 500 mesh sieve was used for homogenization, and sterilized at 121°C for 15 min to obtain sodium hyaluronate gel; then 1.2 g recombinant type I humanized collagen, 1.2 g recombinant type III humanized collagen, and 1.2 g PDRN were added, and stirred for 4 h to obtain a sodium hyaluronate mixed gel.

[0087] (2) Preparation of agarose gel

[0088] Disperse 0.12 g of agarose powder in 40 g of phosphate buffer solution (PH=7.0) prepared with water for injection, mix well, put into a sterilizer, sterilize at 121°C for 15 min, dissolve it, cool to room temperature, and homogenize with an 800-mesh sieve to obtain agarose gel.

[0089] (3) Mixing and filling

[0090] The sodium hyaluronate mixed gel and agarose gel are mixed, stirred evenly, and filled into a pre-filled syringe to obtain a multi-effect sodium hyaluronate composite gel.

[0091] 1. In vitro degradation test

[0092] An appropriate amount of deoxyribonuclease I (freeze-dried powder containing calcium chloride, derived from bovine pancreas) was dissolved in a phosphate buffer solution of pH = 7 to obtain an enzyme solution of deoxyribonuclease I (enzyme activity of 50U / mL). 3mL of mixed enzyme solution was added to 0.12g of the gel samples of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively, and vortexed to mix so that the composite gel concentration in the initial enzymatic solution was about 40mg / mL. The reactants were placed in a 37°C water bath shaker, 50μL of the test solution was sampled every 10min, 3mL of phosphate buffer (pH7.0) was added to the test solution for dilution, 0.22μm filter membrane was filtered, and the absorbance at 260nm was immediately measured using an ultraviolet spectrophotometer. At 260nm, the absorption value of the PDRN degradation product is reflected, which reflects the degree of PDRN degradation. Phosphate buffer (pH = 7.0) was used as a blank control. The end point of the enzymatic hydrolysis is represented by the fact that the absorbance value no longer changes.

[0093] The changes in absorbance values ​​of each sample are shown in Table 1:

[0094] Table 1 Absorbance changes of each sample

[0095]

[0096] As shown in Table 1, the free PDRN in Comparative Examples 1 and 3 has been substantially degraded completely at the fourth sampling; the absorbance values ​​of the samples in Example 1 and Comparative Example 2 are very small and grow slowly. It is possible that a small amount of PDRN exposed to the outer end is degraded, while the PDRN coated in agarose gel is not easily degraded. The experimental results fully illustrate that PDRN can be effectively coated with agarose gel to prolong the degradation time. At the same time, it can also be shown that agarose coating is successful, so it is speculated that collagen is also coated.

[0097] 2. Dynamic viscosity test

[0098] The dynamic viscosity of the samples was measured by TADHR rheometer at 25°C and a shear rate of 3.14-200 / s. The viscosity values ​​of Example 1 and Comparative Examples 1-5 are shown in Table 2:

[0099] Table 2 Viscosity values ​​of each sample

[0100]

[0101] Dynamic viscosity characterizes the frictional resistance of the fluid when it flows, and is positively correlated with the concentration of the substance in the fluid. The solution of hyaluronic acid, collagen and PDRN has a certain viscosity, and the viscosity increases after the hyaluronic acid is cross-linked, but the concentrations of collagen and PDRN in the present invention are relatively low, so the viscosity of the injection gel in the present invention is mainly determined by the concentration of hyaluronic acid or cross-linked sodium hyaluronate.

[0102] As can be seen from Table 2, compared with Example 1, the dynamic viscosity of Comparative Example 1 and Comparative Example 2 is slightly smaller. The main reason is that the hyaluronic acid in Example 1 is cross-linked and the viscosity increases; the viscosity of Comparative Example 1 is slightly larger than that of Comparative Example 2, and the dynamic viscosity of Comparative Example 3 and Comparative Example 5 is slightly larger than that of Example 1. The reason is that in Comparative Example 1, Comparative Example 3 and Comparative Example 5, collagen and PDRN are not coated with agarose, the concentration of the substance in the fluid increases, and the dynamic viscosity increases.

[0103] 3. Antioxidant Effect-ABTS + Free radical scavenging effect

[0104] 0.0206 g ABTS +Dissolve in 5 mL of distilled water, then add 0.0035 g of potassium persulfate, mix well, react in the dark for 12 hours, dilute with distilled water until the absorbance at a wavelength of 734 nm is 0.7 ± 0.02, and adjust to zero with distilled water. Then put 0.3 g of the sample of Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5 into a centrifuge tube, heat at 95°C for 30 minutes to dissolve the gel and release the active substance. After heating, keep the sample at 60°C, add 4 mL of ABTS working solution, shake well, let stand at 60°C in a dark environment for 10 minutes, cool to room temperature, centrifuge, and take the supernatant to measure the absorbance at a wavelength of 734 nm.

[0105] Calculate ABTS as follows + Clearance rate:

[0106]

[0107] In the formula, A0 represents the absorbance of ABTS without adding sample; A1 represents the absorbance of sample without adding ABTS; A2 represents the absorbance after mixing sample with ABTS;

[0108] The measured results are shown in Table 3:

[0109] Table 3 Effect of each group of samples on ABTS + · Free radical scavenging rate %

[0110] Sample name Example 1 Comparative Example 3 Comparative Example 4 Comparative Example 5 Clearance % 97.49 98.03 58.90 71.20

[0111] From the results in Table 3, it can be seen that agarose-coated recombinant humanized type I collagen, recombinant humanized type III collagen, and PDRN will not remove ABTS from the product. + · The effect of free radicals; By comparing the test results of Example 1, Comparative Example 3 and Comparative Example 4, it can be seen that after sodium hyaluronate gel is compounded with recombinant type I humanized collagen, recombinant type III humanized collagen and PDRN, the effect of free radicals on ABTS + The free radical scavenging rate was significantly improved, indicating that collagen, PDRN and HA gel synergized, and also proved that the invented product had a strong antioxidant effect. According to the results of Comparative Examples 3 and 5, it was also found that after wet heat sterilization, the product helped to + The free radical scavenging effect was significantly enhanced. It is speculated that after moist heat sterilization, collagen is degraded into peptides with shorter peptide chains, which are more likely to react with free radicals and show stronger antioxidant effects.

[0112] IV. Clinical Trials

[0113] Sixty volunteers aged 18 or above who had a need to improve facial skin were selected as subjects, ranging in age from 22 to 55 years old, with an average age of (35.16±5.41) years old.

[0114] Population selection criteria:

[0115] (1) Aged 18 or above;

[0116] (2) Subjects who have a strong desire to improve the current status of facial skin;

[0117] (3) Subjects who agreed not to use other cosmetic treatments related to the study during the study;

[0118] (4) Subjects who understand and comply with the trial requirements and can complete the entire follow-up process;

[0119] (5) Voluntarily participate in this clinical study and sign the informed consent.

[0120] Exclusion criteria:

[0121] (1) Subjects are known to be allergic to sodium hyaluronate, PDRN, collagen, or phosphate; Subjects with a history of clinically significant skin allergies or systemic allergies;

[0122] (2) Those taking anticoagulant drugs or having severe coagulation disorders;

[0123] (3) Subjects with facial scars, ulcers, skin infections, a history of granulomas, or other active skin diseases or malignant skin diseases, which may affect the judgment of the treatment effect;

[0124] (4) Subjects have a history of severe diseases of important organs or autoimmune diseases caused by HIV infection;

[0125] (5) Women who are pregnant or breastfeeding; female subjects of childbearing age who do not agree to take medically approved contraceptive measures (such as oral contraceptives, condoms, etc.) during the trial;

[0126] (6) Those with excessively high expectations or neuropsychiatric diseases;

[0127] (7) Subjects who have participated in other clinical trials within 90 days before the screening period;

[0128] (8) Cosmetic treatment of the facial treatment area: non-surgical injection treatments such as skin fillers in the past year, botulinum toxin injections in the past 6 months, laser treatment of the face in the past 6 months, facial skin peeling in the past 6 months, facial plastic surgery, blepharoplasty, brow lift, etc. in the past 6 months.

[0129] (9) Subjects deemed by the researcher to be unsuitable for participation in this trial study.

[0130] 60 subjects were randomly divided into two groups, 30 in each group, one group was the test group and the other group was the control group. After washing the face before injection, the subjects applied the numbing cream for 30 minutes and then washed off the numbing cream. The test group was subcutaneously injected with the sodium hyaluronate composition of Example 1, and the control group was subcutaneously injected with the sodium hyaluronate composition of Comparative Example 4, both by machine injection. After the injection, the subjects applied a refrigerated medical mask for 20 minutes.

[0131] The treatment lasted for 30 days, with the drug given once at the beginning of each cycle. After three consecutive cycles of treatment, the skin moisture content, facial pore size, skin elasticity, firmness, improvement rate of facial fine lines depth and distribution area, improvement rate of facial red blood streak distribution and skin chromaticity change of the experimental group and the control group were evaluated.

[0132] The test results are as follows:

[0133] Compared with the control group, the main efficacy indicators of the test group were significantly improved, including a 25.56% difference in skin moisture content, an 8.90% difference in facial pore size improvement, a 33.01% difference in skin elasticity, a 13.59% difference in skin firmness improvement, a 19.04% difference in facial fine lines improvement, a 19.78% difference in facial redness improvement, and an 11.05% difference in skin chromaticity change. No adverse events that may be related to the test device were observed.

[0134] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A multi-effect sodium hyaluronate composite gel, characterized in that: The composite gel comprises a mixed gel of sodium hyaluronate gel and agarose.

2. The method for preparing the multi-effect sodium hyaluronate composite gel according to claim 1, characterized in that: The steps include: (1) Preparation of Sodium Hyaluronate Gel Add BDDE to a sodium hydroxide solution and mix well to prepare a BDDE solution; then mix the sodium hyaluronate and the BDDE solution evenly to perform cross-linking; After the cross-linking is completed, the mixture is placed in a PBS buffer solution for swelling. After the swelling is completed, the water is drained, homogenized, and sterilized to obtain a sodium hyaluronate gel; (2) Preparation of agarose mixed gel Disperse recombinant humanized type I collagen, recombinant humanized type III collagen, PDRN and agarose powder in a PBS buffer solution, mix evenly, sterilize, and after cooling to room temperature, homogenize to obtain an agarose mixed gel; (3) Mixing The sodium hyaluronate gel and the agarose mixed gel are mixed and stirred evenly to obtain a multi-effect sodium hyaluronate composite gel.

3. The preparation method according to claim 2, characterized in that: The ingredients are selected from the following quantities: 0.03-0.04 parts of BDDE, 10-20 parts of sodium hydroxide solution, 3-8 parts of sodium hyaluronate, 0.32-2.56 parts of recombinant humanized type I collagen, 0.32-2.56 parts of recombinant humanized type III collagen, 0.32-2.56 parts of PDRN, and 0.08-0.2 parts of agarose powder.

4. The preparation method according to claim 2, characterized in that: The mass concentration of the sodium hydroxide solution is selected from 0.5% to 3.0%.

5. The preparation method according to claim 2, characterized in that: The cross-linking conditions are selected from: cross-linking in a water bath at 25 to 75° C. for 3 to 24 hours.

6. The preparation method according to claim 2, characterized in that: The sterilization is selected from: sterilization at 115-125° C. for 10-20 min.

7. The preparation method according to claim 2, characterized in that: The swelling time is selected from 16 to 24 hours.

8. The preparation method according to claim 2, characterized in that: The homogenization refers to passing the gel through a 500-800 mesh screen on a pressurized homogenizer to obtain a gel with uniform particle size.

9. The preparation method according to claim 2, characterized in that: In the step (3), the stirring conditions are: stirring time 4 to 10 hours, stirring speed 70 to 100 r / min.

10. Use of the multi-effect sodium hyaluronate composite gel according to claim 1 or the multi-effect sodium hyaluronate composite gel prepared by the method according to any one of claims 2 to 9 in preparing products with injectable cosmetic effects.

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