Sodium hyaluronate composite solution for relieving skin aging and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a sodium hyaluronate composite solution for relieving skin aging and a preparation method thereof, the sodium hyaluronate composite solution comprises sodium hyaluronate, taurine, proline, glutamic acid, glycine and valine; sodium hyaluronate has a specific molecular weight, the composite solution has a specific pH value and osmotic pressure, and all the components have a synergistic effect, so that a moist environment is provided for skin, free radicals are removed, collagen synthesis is promoted, and aging phenomena such as skin laxity and wrinkles are improved. The composition can maintain skin moisture for a long time, scavenge free radicals, resist oxidation and promote collagen synthesis to improve skin aging, the preparation process guarantees stable components, the solution is matched with the physiological environment of the skin, and the composition is non-irritant, safe to use and good in anti-aging effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a sodium hyaluronate composite solution for alleviating skin aging and its preparation method. Background Technology

[0002] The skin, the largest organ in the human body, serves as the primary defense against external stimuli such as ultraviolet rays and pollutants, and is also the most direct external manifestation of aging. Skin aging is not a single process, but rather the result of the combined effects of endogenous aging and exogenous damage: endogenous aging occurs naturally with age, manifested as a slowdown in cell metabolism and a decline in fibroblast proliferation, leading to a reduction in extracellular matrix synthesis; exogenous damage, primarily caused by ultraviolet radiation, air pollution, and life stress, induces a large accumulation of reactive oxygen species in the skin, accelerating the degradation and loss of hyaluronic acid and collagen denaturation, ultimately leading to typical aging symptoms such as skin laxity, deepening wrinkles, reduced elasticity, and decreased hydration. Therefore, relying solely on a single functional ingredient cannot fundamentally alleviate skin aging; a multi-dimensional synergistic regulation involving "replenishing the extracellular matrix, clearing oxidative damage, and promoting collagen regeneration" is necessary.

[0003] Hyaluronic acid, as a core component of the extracellular matrix, is a common raw material for exogenous skin hydration due to its strong water-locking ability. However, when used alone, it can only improve dry skin and cannot solve the problems of oxidative damage and collagen loss. Taurine, as an essential sulfur-containing non-protein amino acid, has been proven by modern research to have significant antioxidant and anti-fatigue effects, and can scavenge excess free radicals to reduce cell damage. However, it has not been effectively integrated into hyaluronic acid-based anti-aging formulas to exert a synergistic effect. In addition, collagen synthesis depends on specific precursor substances: proline and glycine are the core building blocks of the collagen triple helix structure, glutamate provides energy metabolism support for collagen synthesis, and valine can regulate protein synthesis efficiency. The collagen-promoting effects of these amino acids have been verified, but existing formulas mostly do not combine them with hyaluronic acid and taurine, making it difficult to form a complete anti-aging chain.

[0004] Existing technologies have significant limitations: For example, the hyaluronic acid gel for water-light injection disclosed in CN 106074213A uses only sodium hyaluronate as the core, combined with basic excipients such as glycerin and mannitol, lacking the design for regulating fibroblast proliferation and scavenging free radicals, resulting in a single anti-aging dimension; although the water-light treatment composition in CN 105233270A uses multi-molecular weight sodium hyaluronate compound and even adds collagen, it focuses on moisturizing function and does not integrate the antioxidant effect of taurine and the collagen-promoting effect of amino acids, thus failing to achieve multi-component synergy; at the same time, some existing preparation processes do not control the dissolved oxygen content, which leads to easy oxidation and degradation of amino acids and sodium hyaluronate, or the sterilization process destroys the molecular structure of sodium hyaluronate, further weakening the product's effectiveness and storage stability, making it difficult to meet the clinical demand for long-acting and multi-effect anti-aging products. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a sodium hyaluronate composite solution for alleviating skin aging and its preparation method.

[0006] (II) Technical Solution A sodium hyaluronate complex solution for alleviating skin aging comprises the following components and amounts: sodium hyaluronate 2-15 mg / mL, taurine 0.05-2 mg / mL, proline 0.05-1 mg / mL, glutamic acid 0.05-1 mg / mL, glycine 0.05-1 mg / mL, and valine 0.05-1 mg / mL; wherein the molecular weight of the sodium hyaluronate is 0.5 × 10⁻⁶. 6 -3.5×10 6 The pH of the composite solution is 6-8 and the osmotic pressure is 250-350 mOsmol / kg. Sodium hyaluronate provides a moist microenvironment for the proliferation of skin fibroblasts while maintaining skin hydration; taurine removes excess reactive oxygen free radicals in the skin and blocks oxidative stress-mediated aging pathways; proline, glutamic acid, and glycine, as key precursors for collagen synthesis, synergistically generate a glycine-proline-hydroxyproline tripeptide structure, while valine regulates protein synthesis efficiency to promote collagen deposition. Furthermore, these four substances, along with taurine, synergistically enhance antioxidant and collagen-promoting effects, jointly improving skin laxity and wrinkles.

[0007] Preferably, the sodium hyaluronate content is 3-9 mg / mL, and at this content, the molecular weight of the sodium hyaluronate is 1.5 × 10⁻⁶. 6 -3.0×10 6 Sodium hyaluronate in the Dalton molecular weight range forms a stable hydration layer within the skin.

[0008] Preferably, the taurine content is 0.1-1 mg / mL; and the proline, glutamic acid, glycine, and valine content are each independently 0.1-0.4 mg / mL.

[0009] Preferably, the pH of the composite solution is 6.2-7.8 and the osmotic pressure is 300-350 mOsmol / kg, which is a range that matches the physiological environment of human skin.

[0010] Preferably, the sodium hyaluronate in the component has a molecular weight of 2 × 10⁻⁶. 6 The compound solution contains 8 mg / mL of Dalton, 0.2 mg / mL of taurine, 0.2 mg / mL of proline, 0.15 mg / mL of glutamic acid, 0.15 mg / mL of glycine, and 0.1 mg / mL of valine; the pH of the compound solution is 7.2 and the osmotic pressure is 300 mOsmol / kg.

[0011] Preferably, the preparation method of the sodium hyaluronate complex solution for alleviating skin aging includes the following steps: S1, continuously introduce nitrogen gas into the injection water for 10-20 minutes at a flow rate of 0.5-1 L / min, so that the dissolved oxygen content in the water is below 100 ppm; S2, under nitrogen protection and constant temperature of 25-30℃, add sodium hyaluronate, taurine, proline, glutamic acid, glycine and valine to the injection water after step S1 treatment in sequence, and stir at 50-100r / min for 10-30min until completely dissolved. S3, the pH of the solution was adjusted to 6-8 using phosphate buffer, and the osmotic pressure was adjusted to 250-350 mOsmol / kg using sodium chloride; S4. The solution is brought to a final volume with water for injection. After final volume, it is filtered through a 0.22μm microporous membrane to remove impurities with a particle size ≥0.22μm. S5. Fill the filtered solution into pre-filled syringes, controlling the filling accuracy to ±0.1mL; S6. Sterilize the filled solution at 125℃ for 5 minutes, achieving a sterility level of 10 after sterilization. -6 A sterile sodium hyaluronate composite solution was obtained.

[0012] Preferably, in step S1, the dissolved oxygen content in the injected water is less than 50 ppm after nitrogen is introduced, and the dissolved oxygen content is monitored in real time using a portable dissolved oxygen meter to ensure that the dissolved oxygen does not rise above 100 ppm throughout the process.

[0013] Preferably, in step S2, a magnetic stirrer is used during the stirring process, and the stirring speed is gradually adjusted according to the dissolution process: 50 r / min for the first 5 minutes, and 80-100 r / min for the subsequent 15-25 minutes.

[0014] Preferably, in step S6, the sterilization process uses a pulsed vacuum sterilizer. Before sterilization, the vacuum is first drawn to -0.08 to -0.09 MPa and maintained for 5 minutes. Then, saturated steam is introduced to raise the temperature to 125°C. After sterilization, the temperature is gradually reduced to below 40°C.

[0015] Preferably, the sodium hyaluronate composite solution is used in medical aesthetic treatments related to skin anti-aging to achieve skin moisturizing, anti-oxidative damage, and promotion of collagen regeneration.

[0016] (iii) Beneficial technical effects Compared with existing technologies, the beneficial effects of this invention are: 1. Sodium hyaluronate not only maintains skin hydration for a long time, preventing the aggravation of fine lines caused by dryness, but also provides a moist microenvironment for fibroblast proliferation, supporting normal cell metabolism; Taurine can specifically remove excess reactive oxygen free radicals in the skin, block the cellular aging pathway mediated by oxidative stress, and reduce the damage of free radicals to collagen fibers; Proline, glutamic acid, and glycine, as key precursors for collagen synthesis, can target and promote the formation of collagen triple helix structure, while valine further regulates protein synthesis efficiency to increase collagen deposition. Moreover, the four amino acids form a synergistic effect with taurine, significantly enhancing the antioxidant and collagen-promoting effects, achieving a simultaneous "hydration-antioxidant-regeneration" effect, and fundamentally improving signs of aging such as skin laxity and wrinkles.

[0017] 2. By strictly controlling the dissolved oxygen content in the injection water under nitrogen protection, the active ingredients such as amino acids and sodium hyaluronate can be prevented from being degraded and rendered ineffective due to oxidation, thus significantly extending the product's shelf life. The high-temperature rapid sterilization process at 125℃ for 5 minutes ensures that the product meets high standards of sterility while minimizing damage to the molecular structure of sodium hyaluronate, maintaining its water-locking ability and cell support function. This process resolves the contradiction between "sterilization effect and activity retention" in existing processes, ensuring the long-term effectiveness of the product.

[0018] 3. The pH and osmotic pressure range of the composite solution are precisely matched to the physiological environment of human skin, which can significantly reduce the risk of skin irritation and avoid adverse reactions such as redness and stinging after use; all components are natural substances or essential nutrients that are tolerable to the human body, without cytotoxicity, and can ensure the normal survival and proliferation of fibroblasts. While achieving highly effective anti-aging, it also takes into account the safety of long-term use and is suitable for various skin care scenarios such as water light therapy. Attached Figure Description

[0019] Figure 1 This is a process flow diagram of a method for preparing a sodium hyaluronate composite solution to alleviate skin aging, as disclosed in this invention. Figure 2 The results of MTT assay for detecting the proliferation activity of L-929 cells in the fibroblast proliferation experiment of the examples and comparative examples are shown. Figure 3 These are the ROS staining results for the antioxidant efficacy of the examples and comparative examples; Figure 4 These are the results of β-galactosidase staining in the examples and comparative examples for detecting senescent fibroblasts; Figure 5 These are the results of the relative collagen content in mouse skin at different time points in the examples and comparative examples. Detailed Implementation

[0020] according to Figures 1 to 5 The specific embodiments of the present invention are as follows: The technical solution of the present invention will be further described in detail below with reference to specific embodiments. The scope of protection of the present invention is not limited to the following embodiments; all equivalent transformations or modifications made based on the technical solution of the present invention fall within the scope of protection of the present invention.

[0021] Raw material description Sodium hyaluronate: molecular weight coverage 0.5×10 6 -3.5×10 6 Dalton, with a molecular weight of 1.5 × 10⁻⁶. 6 -3.0×10 6 The components of Dalton shall account for no less than 90% and the purity shall be no less than 99.0%.

[0022] Taurine: purity not less than 99.5%, meeting food-grade standards.

[0023] Proline, glutamic acid, glycine, and valine: all have a purity of not less than 99.5%, meeting food-grade standards.

[0024] Water for injection: Meets the standards for water for injection in the 2025 edition of the Pharmacopoeia of the People's Republic of China, with an electrical conductivity not exceeding 1.3 μS / cm (25℃) and an endotoxin content not exceeding 0.25 EU / mL.

[0025] Phosphate buffer: prepared from sodium dihydrogen phosphate and disodium hydrogen phosphate, with a pH range of 5.5 to 8.5.

[0026] Sodium chloride: analytical grade, purity not less than 99.5%.

[0027] Example 1 Formula composition: Sodium hyaluronate 3mg / mL, molecular weight 3.0×106 Dalton; taurine 0.1 mg / mL; proline 0.1 mg / mL; glutamic acid 0.1 mg / mL; glycine 0.1 mg / mL; valine 0.1 mg / mL. The pH of the composite solution is 6.2, and the osmotic pressure is 250 mOsmol / kg.

[0028] Preparation process: Nitrogen gas was continuously introduced into the injection water for 10 minutes at a flow rate of 0.8 L / min. The dissolved oxygen content in the water was monitored in real time using a portable dissolved oxygen meter until it dropped to 45 ppm.

[0029] Under nitrogen protection, the ambient temperature is controlled at a constant 28°C. Sodium hyaluronate, taurine, proline, glutamic acid, glycine, and valine are added sequentially to the nitrogen-treated injection water in the prescribed amounts.

[0030] Use a magnetic stirrer to stir. Stir at 50 rpm for the first 5 minutes, then increase the speed to 90 rpm for the next 20 minutes, and continue stirring until all components are completely dissolved.

[0031] Add phosphate buffer to adjust the pH of the solution to 6.2; add sodium chloride to adjust the osmotic pressure of the solution to 250 mOsmol / kg.

[0032] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0033] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0034] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.085 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 125°C and held for 5 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 38°C, resulting in a sterile sodium hyaluronate composite solution with a sterility assurance level of 10. -6 .

[0035] Example 2 Formula composition: Sodium hyaluronate 9 mg / mL, molecular weight 1.5 × 10⁻⁶ 6 Dalton; taurine 1 mg / mL; proline 0.4 mg / mL; glutamic acid 0.4 mg / mL; glycine 0.4 mg / mL; valine 0.4 mg / mL. The pH of the composite solution is 7.8, and the osmotic pressure is 350 mOsmol / kg.

[0036] Preparation process: Nitrogen gas was continuously introduced into the injection water for 20 minutes at a flow rate of 1 L / min. The dissolved oxygen content in the water was monitored in real time using a portable dissolved oxygen meter until it dropped to 50 ppm.

[0037] Under nitrogen protection, the ambient temperature is controlled at a constant 30°C. Sodium hyaluronate, taurine, proline, glutamic acid, glycine, and valine are added sequentially to the nitrogen-treated injection water in the prescribed amounts.

[0038] Use a magnetic stirrer to stir at 50 rpm for the first 5 minutes, then increase the speed to 100 rpm for the next 25 minutes, and continue stirring until all components are completely dissolved.

[0039] Add phosphate buffer to adjust the pH of the solution to 7.5; add sodium chloride to adjust the osmotic pressure of the solution to 350 mOsmol / kg.

[0040] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0041] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0042] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.09 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 125°C and held for 5 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 35°C, resulting in a sterile sodium hyaluronate composite solution with a sterility assurance level of 10. -6 .

[0043] Example 3 Formula composition: Sodium hyaluronate 8 mg / mL, molecular weight 2.2 × 10⁻⁶ 6 Dalton; taurine 0.2 mg / mL; proline 0.2 mg / mL; glutamic acid 0.15 mg / mL; glycine 0.15 mg / mL; valine 0.1 mg / mL. The pH of the composite solution is 7.2, and the osmotic pressure is 300 mOsmol / kg.

[0044] Preparation process: Nitrogen gas was continuously introduced into the injection water for 15 minutes at a flow rate of 0.5 L / min. The dissolved oxygen content in the water was monitored in real time using a portable dissolved oxygen meter until it dropped to 40 ppm.

[0045] Under nitrogen protection, the ambient temperature is controlled at a constant 26°C. Sodium hyaluronate, taurine, proline, glutamic acid, glycine, and valine are added sequentially to the nitrogen-treated injection water in the prescribed amounts.

[0046] Use a magnetic stirrer to stir. Stir at 50 rpm for the first 5 minutes, then increase the speed to 80 rpm for the next 15 minutes, and continue stirring until all components are completely dissolved.

[0047] Add phosphate buffer to adjust the pH of the solution to 7.2; add sodium chloride to adjust the osmotic pressure of the solution to 300 mOsmol / kg.

[0048] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0049] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0050] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.08 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 125°C and held for 5 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 36°C, resulting in a sterile sodium hyaluronate composite solution with a sterility assurance level of 10. -6 .

[0051] Example 4 Formula composition: Sodium hyaluronate 15mg / mL, molecular weight 3.5×10 6 Daltons; taurine 2 mg / mL; proline 1 mg / mL; glutamic acid 1 mg / mL; glycine 1 mg / mL; valine 1 mg / mL. The pH of the composite solution is 8, and the osmotic pressure is 350 mOsmol / kg.

[0052] Preparation process: Nitrogen gas was continuously introduced into the injection water for 15 minutes at a flow rate of 0.5 L / min. The dissolved oxygen content in the water was monitored in real time using a portable dissolved oxygen meter until it dropped to 40 ppm.

[0053] Under nitrogen protection, the ambient temperature is controlled at a constant 26°C. Sodium hyaluronate, taurine, proline, glutamic acid, glycine, and valine are added sequentially to the nitrogen-treated injection water in the prescribed amounts.

[0054] Use a magnetic stirrer to stir. Stir at 50 rpm for the first 5 minutes, then increase the speed to 80 rpm for the next 15 minutes, and continue stirring until all components are completely dissolved.

[0055] Add phosphate buffer to adjust the pH of the solution to 7.2; add sodium chloride to adjust the osmotic pressure of the solution to 350 mOsmol / kg.

[0056] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0057] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0058] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.08 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 125°C and held for 5 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 37°C, resulting in a sterile sodium hyaluronate composite solution with a sterility assurance level of 10. -6 .

[0059] Comparative Example 1 Formula composition: Contains only sodium hyaluronate 3 mg / mL, with a molecular weight of 3.0 × 10⁻⁶. 6 Dalton. The pH of the composite solution is 6.2, and the osmotic pressure is 250 mOsmol / kg.

[0060] Preparation process: Nitrogen gas was continuously introduced into the injection water for 10 minutes at a flow rate of 0.8 L / min. The dissolved oxygen content in the water was monitored in real time using a portable dissolved oxygen meter until it dropped to 45 ppm.

[0061] Under nitrogen protection, the ambient temperature is controlled at a constant 28°C, and the prescribed amount of sodium hyaluronate is added to the nitrogen-treated injection water.

[0062] Use a magnetic stirrer to stir. Stir at 50 rpm for the first 5 minutes, then adjust the speed to 90 rpm for the next 20 minutes, and continue stirring until the sodium hyaluronate is completely dissolved.

[0063] Add phosphate buffer to adjust the pH of the solution to 6.2; add sodium chloride to adjust the osmotic pressure of the solution to 250 mOsmol / kg.

[0064] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0065] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0066] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.085 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 125°C and held for 5 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 38°C, resulting in a sterile sodium hyaluronate solution with a sterility assurance level of 10. -6 .

[0067] Comparative Example 2 Formula composition: Sodium hyaluronate 3mg / mL, molecular weight 3.0×10 6 Daltons; taurine 0.1 mg / mL. The pH of the composite solution is 6.2, and the osmotic pressure is 250 mOsmol / kg.

[0068] Preparation process: Nitrogen gas was continuously introduced into the injection water for 10 minutes at a flow rate of 0.8 L / min. The dissolved oxygen content in the water was monitored in real time using a portable dissolved oxygen meter until it dropped to 45 ppm.

[0069] Under nitrogen protection, the ambient temperature is controlled at a constant 28°C. Sodium hyaluronate and taurine are added sequentially to the nitrogen-treated injection water according to the formula.

[0070] Use a magnetic stirrer to stir. Stir at 50 rpm for the first 5 minutes, then increase the speed to 90 rpm for the next 20 minutes, and continue stirring until all components are completely dissolved.

[0071] Add phosphate buffer to adjust the pH of the solution to 6.2; add sodium chloride to adjust the osmotic pressure of the solution to 250 mOsmol / kg.

[0072] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0073] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0074] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.085 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 125°C and held for 5 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 38°C, resulting in a sterile sodium hyaluronate composite solution with a sterility assurance level of 10. -6 .

[0075] Comparative Example 3 Formula composition: Sodium hyaluronate 8 mg / mL, molecular weight 2.2 × 10⁻⁶ 6 Dalton; proline 0.15 mg / mL; glutamic acid 0.15 mg / mL; glycine 0.15 mg / mL; valine 0.1 mg / mL. The pH of the composite solution is 7.2, and the osmotic pressure is 300 mOsmol / kg.

[0076] Preparation process: Nitrogen gas was continuously introduced into the injection water for 15 minutes at a flow rate of 0.5 L / min. The dissolved oxygen content in the water was monitored in real time using a portable dissolved oxygen meter until it dropped to 40 ppm.

[0077] Under nitrogen protection, the ambient temperature is controlled at a constant 26°C. Sodium hyaluronate, proline, glutamic acid, glycine, and valine are added sequentially to the nitrogen-treated injection water.

[0078] Use a magnetic stirrer to stir. Stir at 50 rpm for the first 5 minutes, then increase the speed to 80 rpm for the next 15 minutes, and continue stirring until all components are completely dissolved.

[0079] Add phosphate buffer to adjust the pH of the solution to 7.2; add sodium chloride to adjust the osmotic pressure of the solution to 300 mOsmol / kg.

[0080] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0081] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0082] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.08 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 125°C and held for 5 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 36°C, resulting in a sterile sodium hyaluronate composite solution with a sterility assurance level of 10. -6 .

[0083] Comparative Example 4: Formula: Sodium hyaluronate 8 mg / mL, molecular weight 2.2 × 10⁻⁶ 6 Dalton, taurine 0.2 mg / mL, proline 0.2 mg / mL, glutamic acid 0.15 mg / mL, glycine 0.15 mg / mL, valine 0.1 mg / mL.

[0084] Add the prescribed amounts of sodium hyaluronate, taurine, proline, glutamic acid, glycine, and valine sequentially to the injection water.

[0085] Use a magnetic stirrer to stir. Stir at 50 rpm for the first 5 minutes, then increase the speed to 80 rpm for the next 15 minutes, and continue stirring until all components are completely dissolved.

[0086] Add phosphate buffer to adjust the pH of the solution to 7.2; add sodium chloride to adjust the osmotic pressure of the solution to 300 mOsmol / kg.

[0087] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0088] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0089] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.08 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 125°C and held for 5 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 36°C, resulting in a sterile sodium hyaluronate composite solution with a sterility assurance level of 10. -6 .

[0090] Comparative Example 5: Formula: Sodium hyaluronate 8 mg / mL, molecular weight 2.2 × 10⁻⁶ 6 Dalton, taurine 0.2 mg / mL, proline 0.2 mg / mL, glutamic acid 0.15 mg / mL, glycine 0.15 mg / mL, valine 0.1 mg / mL.

[0091] Nitrogen gas was continuously introduced into the injection water for 15 minutes at a flow rate of 0.5 L / min. The dissolved oxygen content in the water was monitored in real time using a portable dissolved oxygen meter until it dropped to 40 ppm.

[0092] Under nitrogen protection, the ambient temperature is controlled at a constant 26°C. Sodium hyaluronate, taurine, proline, glutamic acid, glycine, and valine are added sequentially to the nitrogen-treated injection water in the prescribed amounts.

[0093] Use a magnetic stirrer to stir. Stir at 50 rpm for the first 5 minutes, then increase the speed to 80 rpm for the next 15 minutes, and continue stirring until all components are completely dissolved.

[0094] Add phosphate buffer to adjust the pH of the solution to 7.2; add sodium chloride to adjust the osmotic pressure of the solution to 300 mOsmol / kg.

[0095] The solution was brought to a final volume of 1000 mL with water for injection. After bringing the final volume, the solution was filtered through a 0.22 μm microporous membrane to remove impurities with a particle size of not less than 0.22 μm.

[0096] The filtered solution was filled into pre-filled syringes with a filling accuracy controlled within ±0.1 mL.

[0097] The filled solution was sterilized using a pulsed vacuum sterilizer. Before sterilization, a vacuum was drawn to -0.08 MPa and maintained for 5 minutes. Then, saturated steam was introduced, and the temperature was raised to 121°C and held for 15 minutes. After sterilization, a gradient cooling method was used to lower the solution temperature to below 36°C, resulting in a sterile sodium hyaluronate composite solution with a sterility assurance level of 10. -6 .

[0098] Comparative Efficacy Test between Example and Comparative Example 1. Fibroblast proliferation experiment This study investigated the effects of sodium hyaluronate composite solution on the proliferation capacity of fibroblasts using CCK-8 and MTT assays.

[0099] In the MTT assay, the cell proliferation rates of fibroblasts in Examples 1-3 and Comparative Examples 1-3 increased to 114.9±3.6%, 120.7±4.3%, 128.7±2.2%, 105.0±2.1%, 104.5±1.8%, and 111.1±4.6%, respectively, which were higher than those in the negative control group, indicating that all six examples had good biocompatibility. Furthermore, compared with Comparative Examples 1, 2, and 3, the complete formulations of Examples 1, 2, and 3 showed a more significant effect in promoting the proliferation of skin fibroblasts, specifically as follows: Figure 2 As shown.

[0100] The proliferation rates of the groups containing sodium hyaluronate, taurine, and four amino acids (Examples 1-3) were significantly higher than those of the control group: Example 3 showed the best proliferation effect, increasing by 28.7% compared to the negative control group; Examples 1 and 2 showed increases of 14.9% and 20.7%, respectively; while the proliferation rates of Comparative Example 1 containing only sodium hyaluronate and Comparative Example 2 containing sodium hyaluronate and taurine only increased by 4.5%-5.0%; Comparative Example 3 containing sodium hyaluronate and four amino acids but lacking taurine, although higher than Comparative Examples 1-2, was still lower than that of Examples 1-3.

[0101] 2. Antioxidant effects This study used skin fibroblasts as a model to assess their antioxidant capacity by measuring the relative quantification of free radical ROS in fibroblasts in the presence of sodium hyaluronate complex solution.

[0102] Fluorescence staining results showed that the DCF fluorescence intensity of the Example 1-3 groups was significantly lower than that of the model group, with the lowest fluorescence intensity in Example 2. This indicates that the complete formulation can effectively remove excess ROS in cells, block oxidative stress-mediated aging pathways, and has clear and potent antioxidant activity.

[0103] The antioxidant performance of some component groups was significantly worse than that of the complete formulation group: Comparative Example 1, containing only sodium hyaluronate, and Comparative Example 3, containing sodium hyaluronate and four amino acids, showed no significant difference in DCF fluorescence intensity compared to the model group, and did not exhibit antioxidant efficacy; Comparative Example 2, containing sodium hyaluronate and taurine, showed significantly higher DCF fluorescence intensity than Example 1, indicating a substantial weakening of antioxidant activity, as detailed below. Figure 3 As shown.

[0104] Analysis of the differences in formulations shows that in the complete formulation of Example 1, the four amino acids can form a synergistic effect with taurine—taurine directly removes ROS, while amino acids provide raw materials for the synthesis of antioxidant enzymes and enhance enzyme activity. The two work together to improve antioxidant efficiency. In contrast, Example 2 lacks amino acid components and relies solely on taurine for antioxidant effects, which are limited. This confirms that the synergistic effect of each component is the core guarantee for the product's strong antioxidant effect.

[0105] 3. Endogenous anti-aging efficacy testing This study constructed mouse dermal fibroblasts by simulating endogenous aging stimulation with hydrogen peroxide, detected the expression of aging-related marker β-galactosidase in fibroblasts by staining, and evaluated the anti-aging efficacy of sodium hyaluronate complex solution by calculating the positive rate of cell senescence.

[0106] After co-incubation with the aging model group in Examples 1, 2, and 3, the positive rates of senescent cells were 54.0±3.6%, 37.7±4.5%, and 39.7±4.7%, respectively; while the positive rate of senescent cells in the model group was as high as 73.7±7.6%. There were statistically significant differences between the complete formula group and the model group (P<0.05). Example 2 showed the best anti-aging effect, with a positive rate 36% lower than the model group. Specifically... Figure 4 As shown.

[0107] The anti-aging effects of single-component or partial-component groups were significantly inferior to those of the complete formula group: Comparative Example 1, which contained only sodium hyaluronate, did not show any clear anti-aging activity; Comparative Example 2, which contained sodium hyaluronate and taurine, and Comparative Example 3, which contained sodium hyaluronate and four amino acids, although containing some of the active ingredients of the present invention, had a significantly higher positive rate of senescent cells than Examples 1-3, and the anti-aging effect was greatly reduced.

[0108] In the complete formulations of Examples 1-3, sodium hyaluronate, taurine, and proline, along with two other amino acids, form a synergistic system that can effectively inhibit the aging process of dermal fibroblasts. However, in Comparative Examples 1-3, the lack of taurine or key components of the four amino acids disrupts the synergistic network, resulting in a significant decrease in anti-aging efficacy.

[0109] 4. Animal efficacy test A D-galactose-induced aging model was established in mice. After removing the hair from both sides of the spine of the experimental animals and disinfecting with povidone-iodine, intradermal injections were performed approximately 1 cm from the spine on both sides of the midline. The control group received saline injections. The injection dose was 50 μL per site, with at least 1 cm between adjacent sites on the same side. The wound was disinfected after each injection. Injections were repeated every two weeks for a total of three times. Collagen content at the injection sites was measured using a collagen content assay kit at 1, 2, 3, and 4 weeks after the last injection.

[0110] Compared with the control group, groups 1, 2, and 3 of Examples all significantly increased the collagen content in mouse skin within 4 weeks after injection (P<0.05), and exhibited a sustained synergistic effect. One week after the last injection, the collagen content in the three groups increased by 30.1%, 45.2%, and 39.1% relative to the control group, respectively; two weeks after the last injection, the increases were 31.0%, 48.0%, and 23.3%, respectively; three weeks after the last injection, the increases were 26.6%, 37.2%, and 19.5%, respectively; and four weeks after the last injection, the relative increases remained at 22.8%, 27.7%, and 18.8%, respectively. (Details are as follows...) Figure 5 As shown.

[0111] Although Comparative Example 3 showed a significant increase in collagen content 1-2 weeks after injection, it returned to normal levels comparable to the control group from the 3rd week onwards; Comparative Examples 1 and 2 did not show any effect in promoting skin collagen synthesis. Analysis of the differences in component formulations indicates that the addition of taurine plays a crucial role in maintaining the stability of newly formed collagen and prolonging its duration of action.

[0112] 5. Product performance stability test This experiment aims to verify the effects of nitrogen purging and high-temperature short-time sterilization on product stability during product preparation.

[0113] The sodium hyaluronate composite solutions prepared using the methods of Example 3 and Comparative Example 4 were selected and placed in an environment of 25°C and 75% RH. The properties of the formulation under accelerated conditions were tested, and the specific data are shown in Table 1 below.

[0114] Table 1

[0115] Table 1 shows that controlling dissolved oxygen in the solvent using nitrogen during product preparation significantly improves product stability and provides favorable conditions for long-term storage. In contrast, Example 4, where dissolved oxygen was not controlled during preparation, showed a decreasing trend in total amino acid content, intrinsic viscosity, and transmittance during accelerated testing, further demonstrating the impact of dissolved oxygen in the formulation on product performance stability.

[0116] Sodium hyaluronate composite solutions prepared according to the methods of Example 3 and Comparative Example 5 were selected to observe the effect of different sterilization conditions on product performance. The specific experimental data are shown in Table 2 below. Table 2

[0117] Observation of experimental data shows that high-temperature short-time sterilization can not only ensure product sterility and ensure that all product parameters meet technical requirements, but also cause less degradation and damage to the molecular weight of sodium hyaluronate, which is conducive to improving the effectiveness of the product.

[0118] The overall performance comparison between the embodiments and the comparative embodiments is shown in the table below: Table 3 .

[0119] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A sodium hyaluronate complex solution for alleviating skin aging, characterized in that, It comprises the following components and amounts: sodium hyaluronate 2-15 mg / mL, taurine 0.05-2 mg / mL, proline 0.05-1 mg / mL, glutamic acid 0.05-1 mg / mL, glycine 0.05-1 mg / mL, and valine 0.05-1 mg / mL; the molecular weight of the sodium hyaluronate is 0.5 × 10⁻⁶. 6 -3.5×10 6 The pH of the composite solution is 6-8 and the osmotic pressure is 250-350 mOsmol / kg. Sodium hyaluronate provides a moist microenvironment for the proliferation of skin fibroblasts while maintaining skin hydration; taurine removes excess reactive oxygen free radicals in the skin and blocks oxidative stress-mediated aging pathways. Proline, glutamic acid, and glycine, as key precursors for collagen synthesis, work together to form a glycine-proline-hydroxyproline tripeptide structure. Valine regulates protein synthesis efficiency to promote collagen deposition. Furthermore, these four substances, along with taurine, synergistically enhance antioxidant and collagen-promoting effects, thus improving skin laxity and wrinkles.

2. The sodium hyaluronate composite solution for alleviating skin aging according to claim 1, characterized in that, The sodium hyaluronate content is 3-9 mg / mL, and at this content, the molecular weight of sodium hyaluronate is 1.5 × 10⁻⁶. 6 -3.0×10 6 Sodium hyaluronate in the Dalton molecular weight range forms a stable hydration layer within the skin.

3. The sodium hyaluronate composite solution for alleviating skin aging according to claim 1, characterized in that, The taurine content is 0.1-1 mg / mL; the proline, glutamic acid, glycine, and valine content are each 0.1-0.4 mg / mL.

4. The sodium hyaluronate composite solution for alleviating skin aging according to claim 1, characterized in that, The composite solution has a pH of 6.2-7.8 and an osmotic pressure of 300-350 mOsmol / kg, a range that matches the physiological environment of human skin.

5. The sodium hyaluronate composite solution for alleviating skin aging according to claim 1, characterized in that, The sodium hyaluronate in the component has a molecular weight of 2×10⁻⁶. 6 The compound solution contains 8 mg / mL of Dalton, 0.2 mg / mL of taurine, 0.2 mg / mL of proline, 0.15 mg / mL of glutamic acid, 0.15 mg / mL of glycine, and 0.1 mg / mL of valine; the pH of the compound solution is 7.2 and the osmotic pressure is 300 mOsmol / kg.

6. A method for preparing a sodium hyaluronate composite solution for alleviating skin aging as described in any one of claims 1-5, characterized in that, Includes the following steps: S1, continuously introduce nitrogen gas into the injection water for 10-20 minutes at a flow rate of 0.5-1 L / min, so that the dissolved oxygen content in the water is below 100 ppm; S2, under nitrogen protection and constant temperature of 25-30℃, add sodium hyaluronate, taurine, proline, glutamic acid, glycine and valine to the injection water after step S1 treatment in sequence, and stir at 50-100r / min for 10-30min until completely dissolved. S3, the pH of the solution was adjusted to 6-8 using phosphate buffer, and the osmotic pressure was adjusted to 250-350 mOsmol / kg using sodium chloride; S4. The solution is brought to a final volume with water for injection. After final volume, it is filtered through a 0.22μm microporous membrane to remove impurities with a particle size ≥0.22μm. S5. Fill the filtered solution into pre-filled syringes, controlling the filling accuracy to ±0.1mL; S6. Sterilize the filled solution at 125℃ for 5 minutes, achieving a sterility level of 10 after sterilization. -6 A sterile sodium hyaluronate composite solution was obtained.

7. The method for preparing the sodium hyaluronate composite solution for alleviating skin aging according to claim 6, characterized in that, In step S1, after nitrogen is introduced, the dissolved oxygen content in the injected water is less than 50 ppm, and the dissolved oxygen content is monitored in real time using a portable dissolved oxygen meter to ensure that the dissolved oxygen does not rise back to above 100 ppm throughout the process.

8. The method for preparing the sodium hyaluronate composite solution for alleviating skin aging according to claim 6, characterized in that, In step S2, a magnetic stirrer is used during the stirring process, and the stirring speed is gradually adjusted according to the dissolution process: 50 r / min for the first 5 minutes, and 80-100 r / min for the subsequent 15-25 minutes.

9. The method for preparing the sodium hyaluronate composite solution for alleviating skin aging according to claim 6, characterized in that, In step S6, the sterilization process uses a pulsed vacuum sterilizer. Before sterilization, the vacuum is first drawn to -0.08 to -0.09 MPa and maintained for 5 minutes. Then, saturated steam is introduced to raise the temperature to 125°C. After sterilization, the temperature is gradually reduced to below 40°C.

10. An application of the sodium hyaluronate composite solution as described in any one of claims 1-5 in the field of medical aesthetics, characterized in that, The composite solution is used in medical aesthetic treatments related to skin anti-aging to achieve skin moisturizing, anti-oxidative damage, and promotion of collagen regeneration.

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