A composite hyaluronic acid composition and use thereof

Abstract

The present application relates to the technical field of daily chemical industry, and particularly relates to a composite hyaluronic acid composition and application thereof.The composite hyaluronic acid composition comprises the following components in parts by weight: (1) 10-50 parts of high-molecular hyaluronic acid sodium; (2) 1-20 parts of acetylated hyaluronic acid sodium; (3) 2-30 parts of hyaluronic acid sodium cross-linked polymer; and (4) 0.1-10 parts of oligomeric hyaluronic acid and / or oligomeric hyaluronic acid sodium.The composite hyaluronic acid composition and skin care essence provided by the present application can improve the component compatibility, storage stability, freeze-thaw stability, viscosity retention rate, transparency retention rate and long-term moisturizing persistence of the hyaluronic acid system without significantly increasing the total amount of hyaluronic acid added.The technical scheme is particularly suitable for use in essence, gel, emulsion, face cream, spray, face mask, freeze-dried preparation, scalp care preparation and postoperative care preparation.

Description

Technical Field

[0001] This invention relates to the field of daily chemical technology, and in particular to a composite hyaluronic acid composition and its application. Background Technology

[0002] Hyaluronic acid (HA) and its sodium salt are among the most widely used moisturizing materials in the field of skin care. Hyaluronic acid molecules contain a large number of carboxyl, hydroxyl, and amide groups, and have a strong hydrophilic ability. They can increase the water content of the stratum corneum by binding free water and bound water, and are therefore widely used in serums, lotions, creams, freeze-dried preparations, and post-operative care products.

[0003] With the development of functional skincare products, single hyaluronic acid has gradually evolved into a complex system of multiple hyaluronic acid derivatives. For example, high molecular weight sodium hyaluronate is used to form a surface moisturizing film; low molecular weight hyaluronic acid is used to enhance skin penetration; acetylated hyaluronic acid is used to improve the affinity of the stratum corneum; and cross-linked hyaluronic acid is used to prolong the moisturizing time. Products with so-called multi-molecular-weight hyaluronic acid systems, 5D hyaluronic acid systems, and 8D hyaluronic acid systems have appeared on the market.

[0004] However, the applicant's research found that although existing multi-component hyaluronic acid systems increase the number of hyaluronic acid types, the following technical problems still exist:

[0005] First, in existing technologies, various hyaluronic acid components are typically prepared by direct mixing, resulting in each component existing in a relatively independent state within the system, with a lack of effective synergistic effects between hyaluronic acids of different molecular weights. As storage time increases, hyaluronic acids of different molecular weights are prone to local aggregation, chain entanglement imbalance, and changes in hydration state, leading to fluctuations in system viscosity, increased stringiness, and unstable skin feel.

[0006] Second, the diffusion rates and hydration kinetics of high molecular weight hyaluronic acid, cross-linked hyaluronic acid, and low molecular weight hyaluronic acid differ significantly in aqueous systems. Existing technologies cannot effectively regulate the distribution and release behavior of each component on the skin surface, resulting in a concentrated release of large amounts of hyaluronic acid in a short period, leading to a decrease in the duration of moisturizing effects.

[0007] Third, the carboxyl and hydroxyl groups in the hyaluronic acid molecular chain are easily affected by changes in the ionic strength of the system, plant extracts, polyols, electrolytes, and temperature. When plant extracts, fermentation products, amino acids, and other active ingredients are present in the formulation, the spatial arrangement between the hyaluronic acid molecular chains changes, which can easily lead to decreased transparency, viscosity drift, or reduced long-term storage stability.

[0008] Fourth, existing technologies typically add fermentation products as independent active ingredients to hyaluronic acid systems, with their main functions focused on soothing, moisturizing, or nutritional supplementation, without fully utilizing the chemical effects of components such as oligosaccharides, small peptides, organic acid salts, and amino acids produced during fermentation in the polysaccharide system.

[0009] Further research by the applicant revealed that the polysaccharide components, after specific fermentation treatment, exhibited significant changes in molecular weight distribution, hydroxyl density, and carboxyl group exposure. These fermented polysaccharides not only possess moisturizing activity but also participate in the hydration process and interchain interactions of the hyaluronic acid system. When coexisting with hyaluronic acids of different structures, they can alter the hydration layer structure surrounding the hyaluronic acid molecular chains, thereby affecting the system's stability and the retention state of hyaluronic acid on the skin surface.

[0010] Therefore, how to utilize the interaction between fermented polysaccharides and hyaluronic acids with different structures to improve the stability, sustainability, and synergy of multi-component hyaluronic acid systems has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0011] To address the problems of insufficient component synergy, limited system stability, low utilization efficiency of different hyaluronic acids, and insufficient long-term moisturizing effect in existing multi-component hyaluronic acid systems, this invention provides a composite hyaluronic acid composition, its preparation method, and its application.

[0012] The composite hyaluronic acid composition provided by the present invention includes a hyaluronic acid component, a fermented polysaccharide component, an amino acid component, and a sugar stabilizing component.

[0013] in,

[0014] The hyaluronic acid component includes:

[0015] High molecular weight sodium hyaluronate;

[0016] Acetylated sodium hyaluronate;

[0017] Sodium hyaluronate cross-linked polymer;

[0018] Oligomeric hyaluronic acid and / or oligomeric sodium hyaluronate.

[0019] The fermented polysaccharide components are obtained from natural polysaccharide raw materials through microbial fermentation.

[0020] Preferably, the natural polysaccharide raw material is selected from one or more of the following: Tremella fuciformis polysaccharide, oat polysaccharide, rice bran polysaccharide, Bletilla striata polysaccharide, seaweed polysaccharide, and yeast polysaccharide.

[0021] Preferably, the microorganism is selected from one or more of lactic acid bacteria, yeast, and Bacillus.

[0022] Preferably, the fermented polysaccharide is obtained after ultrafiltration fractionation.

[0023] More preferably, the molecular weight distribution of the fermented polysaccharide is 1kDa to 50kDa.

[0024] The amino acid components include one or more of arginine, lysine, histidine, and glycine.

[0025] The sugar stabilizing components include one or more of trehalose, glycerol glucoside, and betaine.

[0026] Preferably, the composition further comprises one or more of ectoine, panthenol, and β-glucan.

[0027] The present invention also provides a method for preparing the composite hyaluronic acid composition.

[0028] The preparation method includes the following steps:

[0029] Step 1: Add high molecular weight sodium hyaluronate to the aqueous phase for complete hydration treatment.

[0030] Step 2: Add acetylated sodium hyaluronate to the system obtained in Step 1 and mix well.

[0031] Step 3: Add the fermented polysaccharide component to the system obtained in Step 2.

[0032] Step 4: Add sodium hyaluronate cross-linked polymer to the system obtained in Step 3.

[0033] Step 5: Add oligomeric hyaluronic acid and / or oligomeric sodium hyaluronate to the system obtained in Step 4.

[0034] Step 6: Add amino acid components and sugar stabilizing components.

[0035] Step 7: Adjust the pH of the system to 5.2-6.0 to obtain the composite hyaluronic acid composition.

[0036] The applicant's research revealed that the roles of different hyaluronic acids in the system are not independent. High-molecular-weight sodium hyaluronate, with its higher molecular weight and stronger chain entanglement ability, is the first to form continuous hydration regions. Acetylated sodium hyaluronate, containing acetyl groups, alters the local polar environment surrounding the hyaluronic acid chains upon its addition. Oligosaccharide components from fermented polysaccharides further penetrate between hyaluronic acid segments, resulting in a more uniform distribution among the chains. The cross-linked polymer of sodium hyaluronate possesses high water retention capacity, stabilizing bound water in the system upon its addition. Oligomeric hyaluronic acid is dispersed within the aforementioned system.

[0037] The applicant further discovered that the above-mentioned order of addition, compared with the one-time mixing of all components, significantly improved the stability of the system. The applicant believes that this is because different components participate in the hydration process at different stages. Polymer hyaluronic acid first forms the basic hydration layer; acetylated hyaluronic acid further regulates the local chain segment environment; fermented polysaccharides participate in the rearrangement of the inter-segment hydration layer; cross-linked hyaluronic acid fixes bound water; and oligomeric hyaluronic acid is uniformly distributed within the system.

[0038] Therefore, the resulting system differs fundamentally from existing technologies. Furthermore, the applicant unexpectedly discovered that fermentation treatment of polysaccharides does not necessarily yield better results with lower molecular weights. When the molecular weight is too high, it is difficult for the polysaccharide to enter the inter-chain regions of hyaluronic acid; when the molecular weight is too low, its ability to regulate the arrangement of hyaluronic acid segments decreases. Only when the fermented polysaccharide is within a specific molecular weight range can both good diffusion and inter-chain regulation capabilities be simultaneously achieved.

[0039] This result is significantly different from the common understanding in the field that smaller molecule components are more likely to function.

[0040] The applicant also found that the co-existence of acetylated sodium hyaluronate and fermented polysaccharides significantly improved the stability of the system compared to using either component alone. This phenomenon could not be predicted using existing technologies.

[0041] Therefore, the present invention not only provides a composite hyaluronic acid composition, but also provides a new technical route for regulating the chain arrangement and hydration behavior of hyaluronic acid by fermenting polysaccharides.

[0042] Compared with existing technologies, the present invention can improve system stability, moisturizing duration and hyaluronic acid utilization efficiency without significantly increasing the total amount of hyaluronic acid added.

[0043] Meanwhile, the composition of the present invention exhibits excellent stability under high temperature, low temperature and freeze-thaw conditions.

[0044] This invention can also be used in products such as serums, lotions, creams, sprays, masks, freeze-dried preparations, scalp care preparations, and post-operative care preparations.

[0045] This invention relates to a composite hyaluronic acid composition, its preparation method and application, belonging to the technical field of bioactive polysaccharides and skin care materials.

[0046] Hyaluronic acid, a natural glycosaminoglycan, is a linear polymer structure formed by the repeated linkage of D-glucuronic acid and N-acetyl-D-glucosamine. Its molecular chain contains a large number of carboxyl, hydroxyl and amide groups, which can bind a large number of water molecules to form a stable hydration layer.

[0047] Because different hyaluronic acids have significant differences in molecular weight, hydration rate, diffusion coefficient, and chain flexibility, local chain entanglement regions, free water enrichment regions, and unevenly distributed bound water regions are prone to appear inside the system.

[0048] The applicant found that the above phenomena lead to the following problems: limited synergy between different hyaluronic acids; significant changes in rheological behavior during long-term storage of the system; decreased stability under freeze-thaw and high-temperature conditions; lower actual utilization efficiency of hyaluronic acid than theoretical level; and limited duration of moisturizing effect.

[0049] During the research process, the applicant further discovered that the properties of natural polysaccharides changed after fermentation, differing from traditional understanding. Current technologies generally regard fermented polysaccharides as humectants, soothing agents, or active ingredients.

[0050] However, the applicant discovered that the true value of fermented polysaccharides does not stem from their moisturizing abilities, but rather from their structural regulation of the hyaluronic acid system. After fermentation, some glycosidic bonds in natural polysaccharides break, reducing their molecular weight and exposing more hydroxyl, carboxyl, and reducing end structures. These changes result in fermented polysaccharides possessing a molecular conformation significantly different from the original polysaccharides.

[0051] The applicant unexpectedly discovered that, after these fermented polysaccharides enter the hyaluronic acid system, they preferentially distribute between hyaluronic acid segments. They do not exist as independent moisturizers, but rather participate in the hydration process of the hyaluronic acid system as segment spacers.

[0052] From a polymer physicochemical perspective, fermentation treatment allows the hydroxyl and carboxyl groups in polysaccharides to form new hydrogen bond networks with bound water surrounding hyaluronic acid chains. This process alters the distribution of bound and free water around hyaluronic acid chains.

[0053] Meanwhile, fermentation treatment of polysaccharides can reduce the probability of local collapse and excessive entanglement of hyaluronic acid segments. Therefore, the distribution of hyaluronic acid segments in the system is more uniform.

[0054] The applicant further discovered that this phenomenon does not simply depend on the amount of fermented polysaccharide added, but is closely related to its molecular weight range. Surprisingly, lower molecular weight does not necessarily lead to better results with fermented polysaccharides. When the molecular weight is too high, it is difficult for the polysaccharide to enter the inter-chain regions of hyaluronic acid; when the molecular weight is too low, it cannot effectively influence the spatial relationships between hyaluronic acid segments. The applicant found that only when the fermented polysaccharide is within a specific molecular weight range can both diffusion capacity and chain segment regulation capacity be simultaneously achieved.

[0055] During further research, the applicant discovered another unexpected phenomenon. Existing technology typically assumes that adding various hyaluronic acids simultaneously yields the final system. However, the applicant found that the order in which different hyaluronic acids are added directly affects the final system state. When high-molecular-weight sodium hyaluronate is first fully hydrated, it forms a continuous chain network; subsequently, when acetylated sodium hyaluronate is added, the acetyl groups alter the local polar environment; then, when fermented polysaccharides are added, they can more evenly penetrate between the hyaluronic acid segments; subsequently, cross-linked sodium hyaluronate further stabilizes the bound water in the system; finally, the addition of oligomeric hyaluronic acid ensures its uniform dispersion within the system.

[0056] The applicant found that the system obtained in the above sequence showed significant differences in viscosity retention, freeze-thaw stability, high-temperature stability, and moisture retention compared to the system obtained by mixing all components at once.

[0057] The applicant argues that the fundamental reason lies in the different time windows through which different hyaluronic acid components participate in the hydration process. The order in which different components enter the system alters the formation of bound water and the arrangement of hyaluronic acid chain segments, thus affecting the final state of the system. Furthermore, the applicant discovered a significant synergistic effect when acetylated hyaluronic acid and fermented polysaccharides coexist. Traditionally, acetylated hyaluronic acid is considered to primarily enhance skin absorption capacity. However, the applicant found that its acetyl groups can also alter the microscopic polar environment surrounding the hyaluronic acid chains.

[0058] When fermented polysaccharides are introduced into this environment, both act together on the hydration layer around the hyaluronic acid chains, resulting in greater stability of the system.

[0059] Compared with the prior art, the innovation of this invention does not lie in increasing the number of types of hyaluronic acid, but in using fermentation to treat polysaccharides to regulate the chain segment arrangement, hydration layer structure and bound water distribution behavior in the hyaluronic acid system.

[0060] This invention breaks through the existing development path of hyaluronic acid products that rely on the superposition of types and the increase of dosage, and provides a new technical route based on the regulation of polymer chain interactions.

[0061] The applicant made an unexpected discovery that this technical approach not only improves the stability of the hyaluronic acid system, but also enhances the utilization efficiency of hyaluronic acid.

[0062] Therefore, under the same hyaluronic acid addition conditions, the present invention can achieve comprehensive performance superior to the prior art. Detailed Implementation

[0063] The technical solution of the present invention will be described in detail below. It should be understood that the following content is used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make conventional adjustments to the types of raw materials, component ratios, fermentation conditions, order of addition, and formulation form without departing from the technical concept of the present invention, and such adjustments should still fall within the scope of protection of the present invention.

[0064] This invention provides a composite hyaluronic acid composition and its application in skin care products. This composition is not simply a mixture of multiple hyaluronic acid raw materials, but rather utilizes the hydration, hydrogen bonding, chain segmentation, and dispersion stabilization effects among high molecular weight sodium hyaluronate, acetylated sodium hyaluronate, sodium hyaluronate cross-linked polymer, oligomeric hyaluronic acid, and fermented polysaccharides to form a more uniform and stable composite hydration system in an aqueous phase.

[0065] In this invention, the composite hyaluronic acid composition includes high molecular weight sodium hyaluronate, acetylated sodium hyaluronate, sodium hyaluronate cross-linked polymer, oligomeric hyaluronic acid and / or oligomeric sodium hyaluronate, fermented polysaccharide component, amino acid component, and sugar stabilizing component.

[0066] The composite hyaluronic acid composition preferably comprises, by weight: 10-50 parts of high molecular weight sodium hyaluronate, 1-20 parts of acetylated sodium hyaluronate, 2-30 parts of sodium hyaluronate cross-linked polymer, 0.1-10 parts of oligomeric hyaluronic acid and / or oligomeric sodium hyaluronate, 5-50 parts of fermented polysaccharide component, 0.1-10 parts of amino acid component, and 0.1-20 parts of sugar stabilizing component.

[0067] More preferably, the composite hyaluronic acid composition comprises: 20-35 parts of high molecular weight sodium hyaluronate, 3-10 parts of acetylated sodium hyaluronate, 8-18 parts of sodium hyaluronate cross-linked polymer, 1-5 parts of oligomeric hyaluronic acid and / or oligomeric sodium hyaluronate, 15-35 parts of fermented polysaccharide component, 0.5-3 parts of amino acid component, and 3-12 parts of sugar stabilizing component.

[0068] More preferably, the composite hyaluronic acid composition comprises: 25 parts of high molecular weight sodium hyaluronate, 6 parts of acetylated sodium hyaluronate, 12 parts of sodium hyaluronate cross-linked polymer, 2 parts of oligomeric sodium hyaluronate, 25 parts of fermented tremella polysaccharide component, 0.8 parts of arginine, 3 parts of trehalose, 1 part of glyceryl glucoside, 1 part of betaine, and 0.5 parts of ectoine.

[0069] The high molecular weight sodium hyaluronate is preferably sodium hyaluronate with a weight-average molecular weight of 800,000 to 2,000,000 Da, more preferably 1,000,000 to 1,500,000 Da, and even more preferably 1,200,000 to 1,350,000 Da. High molecular weight sodium hyaluronate has longer chain segments and stronger hydration capabilities, making it suitable as the basic hydration framework for the complex system. When its molecular weight is too low, the surface film-forming properties and the system's viscoelasticity are insufficient; when its molecular weight is too high, the system is prone to excessive stringiness and localized chain entanglement, which is detrimental to the skin feel and long-term viscosity stability of the serum.

[0070] The acetylated sodium hyaluronate is a hyaluronic acid derivative obtained by acetylation modification of sodium hyaluronate. The acetylated sodium hyaluronate molecule retains the hydrophilic backbone of hyaluronic acid while introducing certain hydrophobic acetyl groups. This structure can improve the local polar environment around the hyaluronic acid segments, enhance its compatibility with the stratum corneum surface, and reduce local chain entanglement that easily occurs when high molecular weight sodium hyaluronate exists alone in the aqueous phase. The preferred amount of acetylated sodium hyaluronate is 10-40% of the weight of the high molecular weight sodium hyaluronate. If the amount of acetylated sodium hyaluronate is too low, its regulatory effect on the inter-segment hydration environment is insufficient; if the amount is too high, it may lead to a decrease in the hydrophilicity of the system and affect transparency.

[0071] The preferred cross-linked hyaluronic acid polymer is a cross-linked sodium hyaluronic acid obtained by cross-linking sodium hyaluronic acid with diglycidyl ether cross-linking agents, divinyl sulfone cross-linking agents, or a combination thereof. The cross-linked sodium hyaluronic acid polymer has a three-dimensional water-retaining structure, capable of adsorbing and retaining moisture, and acts as both a water-retaining phase and a water-releasing phase in the composite system. Compared to linear hyaluronic acid, cross-linked sodium hyaluronic acid is less prone to rapid diffusion and loss in the aqueous phase, thus improving the composition's sustained moisturizing performance and skin surface retention.

[0072] The oligomeric hyaluronic acid and / or oligomeric sodium hyaluronate are preferably hyaluronic acid substances with a weight-average molecular weight of 1-50 kDa, more preferably 3-20 kDa, and even more preferably 3-10 kDa. Oligomeric hyaluronic acid has high diffusion capacity, which can compensate for the shortcomings of high molecular weight hyaluronic acid and cross-linked hyaluronic acid in terms of diffusion. Its dosage should not be too high; if the dosage is too high, the proportion of small molecule components in the system increases, which may weaken the overall water storage and film-forming performance; if the dosage is too low, it is difficult to demonstrate the differentiated effects of the multi-stage hyaluronic acid system.

[0073] The fermented polysaccharide component is obtained from natural polysaccharide raw materials through microbial fermentation. The natural polysaccharide raw materials can be selected from one or more of the following: Tremella fuciformis polysaccharide, oat polysaccharide, rice bran polysaccharide, Bletilla striata polysaccharide, seaweed polysaccharide, and yeast polysaccharide. Preferably, the natural polysaccharide raw materials are Tremella fuciformis polysaccharide, oat polysaccharide, or a combination thereof. Tremella fuciformis polysaccharide contains a large number of hydroxyl groups and has strong hydration capacity; oat polysaccharide contains a β-glucan structure, which contributes well to aqueous phase stability and skin feel; rice bran polysaccharide may contain a small amount of phenolic acid components, which can further improve the antioxidant stability of the system.

[0074] The microorganisms can be selected from one or more of lactic acid bacteria, yeast, and Bacillus. Lactic acid bacteria can produce organic acids and promote partial cleavage of polysaccharide molecules; yeast can provide amino acids, small peptides, and nucleoside derivatives; Bacillus can provide enzyme systems such as polysaccharide enzymes and proteases, enabling limited hydrolysis of macromolecular polysaccharides. Preferably, lactic acid bacteria and yeast are used for the first stage of fermentation, followed by Bacillus for the second stage of fermentation, to obtain fermented polysaccharides with a more concentrated molecular weight distribution.

[0075] In a preferred embodiment, the preparation method of the fermented polysaccharide component includes: adding Tremella fuciformis polysaccharide raw material to water, controlling the solid-liquid ratio to be 1:10 to 1:30, adding 0.5 to 3.0 wt% glucose and 0.1 to 1.0 wt% yeast extract, adjusting the pH to 5.5 to 6.5, sterilizing and cooling to 30 to 37°C, inoculating with lactic acid bacteria and yeast for a first-stage fermentation of 12 to 36 hours; subsequently inoculating with Bacillus or enzyme-producing bacteria for a second-stage fermentation of 12 to 48 hours; after fermentation, treating at 75 to 90°C for 10 to 30 minutes, centrifuging to remove insoluble matter, taking the supernatant, ultrafiltration, and collecting the 1 to 50 kDa component to obtain the fermented polysaccharide component.

[0076] More preferably, the molecular weight distribution of the fermented polysaccharide component is 3–30 kDa, more preferably 5–20 kDa. The applicant has discovered that fermented polysaccharides within this molecular weight range can effectively penetrate between hyaluronic acid segments and participate in hydration layer rearrangement. When the molecular weight of the fermented polysaccharide is too high, its diffusion capacity is insufficient, making it difficult to penetrate the inter-chain region of hyaluronic acid; when the molecular weight is too low, its segment length is insufficient, failing to effectively regulate the spatial distribution between hyaluronic acid segments. Therefore, smaller molecular weight fermented polysaccharides are not necessarily better; rather, a specific molecular weight range exhibits superior effects. This phenomenon is an important unexpected discovery of this invention.

[0077] The amino acid component preferably includes one or more of arginine, lysine, histidine, and glycine, more preferably arginine. Arginine contains guanidine and amino groups, which can form ionic or hydrogen bond interactions with the carboxyl groups in hyaluronic acid molecules, and can also be used to adjust the pH of the system. The amount of arginine used is preferably 0.1 to 3 parts, more preferably 0.5 to 1.5 parts. If the amount is too low, the pH adjustment and intersegmental interactions will be insufficient; if the amount is too high, it may increase the ionic strength of the system and affect the stretching state of the hyaluronic acid segments.

[0078] The saccharide stabilizing component preferably includes one or more of trehalose, glyceryl glucoside, betaine, maltitol, and sorbitol, more preferably trehalose, glyceryl glucoside, and betaine. Trehalose can stabilize the hydration layer around the polysaccharide and reduce the impact of temperature changes on the structure; glyceryl glucoside can improve water binding state and skin feel; betaine has an osmotic pressure regulating effect and can reduce the adverse effects of plant extracts, electrolytes, or amino acids on the conformation of hyaluronic acid chains.

[0079] The composition of this invention may further include one or more of ectoine, panthenol, β-glucan, and allantoin. Ectoine can improve the stability of the system under high temperature, low temperature, and salt conditions; panthenol can improve skin softness and soothingness; β-glucan can form a complex hydration environment with the hyaluronic acid system, improving the viscoelasticity and skin care performance of the system.

[0080] The preferred method for preparing the composite hyaluronic acid composition of the present invention includes the following steps.

[0081] The first step involves adding high-molecular-weight sodium hyaluronate to an aqueous phase for thorough hydration. The preferred hydration temperature is 20–45°C, more preferably 25–35°C; the preferred hydration time is 2–12 hours, more preferably 4–8 hours. High-molecular-weight sodium hyaluronate should not be treated under strong shear conditions for extended periods to avoid molecular chain degradation. The purpose of this step is to establish a uniform and continuous basic hydration system for the high-molecular-weight sodium hyaluronate, providing a stable environment for the subsequent dispersion of acetylated sodium hyaluronate and fermented polysaccharides.

[0082] The second step involves adding acetylated sodium hyaluronate to the system obtained in the first step and mixing. The preferred mixing temperature is 30–45°C, and the preferred mixing time is 0.5–3 hours. The acetylated sodium hyaluronate should not be added all at once with all the raw materials. The applicant has found that when acetylated sodium hyaluronate is added after the high-molecular-weight sodium hyaluronate has been fully hydrated, it is more easily distributed near the high-molecular-weight hyaluronic acid chain segments, improving the local hydration environment.

[0083] The third step involves adding the fermented polysaccharide component. In this step, the system pH is preferably 5.0–6.5, more preferably 5.3–5.8. The fermented polysaccharide component is preferably added after the addition of acetylated sodium hyaluronate. The applicant has found that if the fermented polysaccharide is added before the hydration of high-molecular-weight hyaluronic acid, it may compete for water and affect the full expansion of the high-molecular-weight sodium hyaluronate; if the fermented polysaccharide is added after cross-linking the sodium hyaluronate, its efficiency in entering the inter-chain regions of hyaluronic acid decreases. Therefore, this invention preferably adds the fermented polysaccharide after the high-molecular-weight sodium hyaluronate and acetylated sodium hyaluronate have formed a preliminary composite hydration system.

[0084] The fourth step is to add the cross-linked sodium hyaluronate polymer. Ideally, the cross-linked sodium hyaluronate should be added after the fermented polysaccharide. Its function is to adsorb and stabilize bound water in the system, while simultaneously improving the system's water storage capacity. If the cross-linked sodium hyaluronate is added too early, it may adsorb a large amount of free water, reducing the contact efficiency between the high-molecular-weight sodium hyaluronate and the fermented polysaccharide; if it is added too late, the water-storing phase will not bind sufficiently with the aforementioned hyaluronic acid hydration system.

[0085] Fifth, add oligomeric hyaluronic acid and / or sodium oligomeric hyaluronic acid. Oligomeric hyaluronic acid is preferably added last to minimize its interference with the stretching of the polymeric hyaluronic acid chains during the initial hydration process. This step helps the oligomeric hyaluronic acid to be evenly distributed throughout the aforementioned composite system, rather than concentrated in the free water region.

[0086] Step six involves adding amino acid components and sugar stabilizing components, and adjusting the pH of the system. The final pH is preferably 5.2–6.0, more preferably 5.3–5.8. This pH range is close to the slightly acidic environment of the skin surface and is conducive to maintaining suitable interactions between the carboxyl groups of hyaluronic acid, amino acids, and fermented polysaccharides. Too low a pH may affect the stability of some hyaluronic acid salts; too high a pH may reduce the mildness of the system in skin care products and alter the charged state of the hyaluronic acid segments.

[0087] In a preferred skin care serum, the serum comprises, by weight percentage: water (balance), 3.0–6.0% butylene glycol, 2.0–4.0% glycerin, 0.3–0.8% 1,2-hexanediol, 0.3–0.6% p-hydroxyacetophenone, 0.05–0.15% xanthium gum, 0.15–0.35% acrylic thickener, 0.5–1.5% trehalose, 0.5–2.0% betaine, 0.1–0.5% ectoine, 0.2–1.0% glyceryl glucoside, 2.0–8.0% complex hyaluronic acid composition, 0.1–2.0% plant extracts, 0.1–2.0% algae extracts, 0.15–0.35% arginine, 0.02–0.08% disodium EDTA, and 0.03–0.12% citric acid and / or sodium citrate.

[0088] More preferably, the essence comprises, by weight percentage: 77.45% water, 4.00% butylene glycol, 3.00% glycerin, 0.50% 1,2-hexanediol, 0.50% p-hydroxyacetophenone, 0.08% xanthate gum, 0.22% acrylic thickener, 0.80% trehalose, 1.00% betaine, 0.30% ectoine, 0.50% glyceryl glucoside, 5.00% complex hyaluronic acid composition, 2.00% fermented Tremella fuciformis polysaccharide component, 0.80% Spirulina macrophylla extract, 0.80% Gentiana scabra extract, 0.25% arginine, 0.05% disodium EDTA, 0.05% sodium citrate and citric acid buffer, and 0.70% solubilizing emollient.

[0089] The xanthan gum and acrylic thickener are preferably used together. Xanthan gum provides natural polysaccharide rheological support, which can reduce sedimentation or local concentration differences in the system during storage; acrylic thickener provides yield value and a refreshing feel; and the complex hyaluronic acid composition provides hydration viscoelasticity. When used together, these three components form a stable, transparent, refreshing, and non-stringy serum system. If only high-molecular-weight hyaluronic acid is used to adjust viscosity, the system is prone to stringiness and viscosity drift at high and low temperatures; if only acrylic thickener is used, the hydration layer stability of the hyaluronic acid system is insufficient.

[0090] The plant extracts may include one or more of the following: gentian extract, centella asiatica extract, licorice extract, purslane extract, and oat extract. The algae extracts may include one or more of the following: spirulina extract, chlorella extract, and seaweed extract. Plant and algae extracts may contain polyphenols, pigments, mineral ions, and polysaccharides, which can easily affect transparency and viscosity when directly added to the hyaluronic acid system. Therefore, it is preferable to premix the plant and algae extracts with butylene glycol, glycerin, trehalose, ectoine, and disodium EDTA to form a pre-stabilized active ingredient solution before adding it to the main system. This partitioned feeding method can reduce the adverse effects of plant and algae active ingredients on the conformation of the hyaluronic acid chain.

[0091] This invention unexpectedly discovered that fermented polysaccharides in this system are not merely used as ordinary humectants. Within a specific molecular weight range, they can act as spacers and hydration regulators between hyaluronic acid segments, enabling a more uniform complex hydration state among high molecular weight sodium hyaluronate, acetylated sodium hyaluronate, cross-linked sodium hyaluronate, and oligomeric hyaluronic acid. This effect differs from the simple water-absorbing function of conventional polysaccharide humectants.

[0092] This invention also unexpectedly discovered that the order in which components are added has a significant impact on the final system performance. If all hyaluronic acid components and fermented polysaccharides are added at once, the system can form a transparent or semi-transparent solution, but after high temperatures, freeze-thaw cycles, and long-term storage, it is prone to decreased viscosity, increased stringiness, or reduced transparency. Conversely, the system prepared in the order of hydration of high-molecular-weight sodium hyaluronate, addition of acetylated sodium hyaluronate, addition of fermented polysaccharides, addition of cross-linked sodium hyaluronate, and addition of oligomeric hyaluronic acid exhibits more stable viscosity, transparency, and skin feel.

[0093] This invention unexpectedly revealed a synergistic effect between acetylated sodium hyaluronate and fermented polysaccharides. Acetylated sodium hyaluronate alters the local polar environment surrounding the hyaluronic acid chains, while the fermented polysaccharides further regulate the interchain hydration state. The combined use of both significantly improves system stability compared to using either component alone. This result cannot be derived solely from the stratum corneum affinity of acetylated sodium hyaluronate or the moisturizing properties of the fermented polysaccharides.

[0094] Therefore, the composite hyaluronic acid composition and its skin care essence provided by this invention can improve the component compatibility, storage stability, freeze-thaw stability, viscosity retention, transparency retention, and long-term moisturizing effect of the hyaluronic acid system without significantly increasing the total amount of hyaluronic acid added. This technical solution is particularly suitable for serums, gels, lotions, creams, sprays, masks, freeze-dried preparations, scalp care preparations, and post-operative care preparations.

[0095] Example 1

[0096] I. Preparation of Composite Hyaluronic Acid Composition

[0097] (I) Preparation of polysaccharide components from fermented Tremella fuciformis

[0098] Weigh 1000 g of freeze-dried Tremella fuciformis fruiting body powder. Add it to 15000 g of deionized water. Stir at room temperature for 30 min to evenly disperse the Tremella powder. Then add: 150 g of glucose; 30 g of yeast extract; 15 g of potassium dihydrogen phosphate; and 5 g of magnesium sulfate. Adjust the pH of the system to 6.0 ± 0.1. Sterilize at 121℃ for 20 min. Cool to 32℃.

[0099] Inoculation: 300 g of Lactobacillus plantarum (Lactobacillus plantarum seed culture concentration: 1×10⁻⁶) 9 CFU / g); Saccharomyces cerevisiae 150 g (Saccharomyces cerevisiae seed culture concentration: 1×10⁻⁶ CFU / g); 9(CFU / g). Fermentation was carried out at 32℃ for 24 h. During fermentation, the pH was maintained at 5.0–5.8, and the stirring speed was 100 rpm.

[0100] After the first stage of fermentation, inoculate with: 150 g of Bacillus subtilis (seed culture concentration: 1×10⁻⁶). 9 Fermentation continued at 30℃ for 24 h with a stirring speed of 120 rpm. After the second stage of fermentation, the system was heated to 85℃ and held for 20 min to terminate the fermentation. The mixture was then centrifuged at 8000 rpm for 15 min. The supernatant was collected. A first ultrafiltration was performed using a 20 kDa ultrafiltration membrane. The permeate was collected. A second ultrafiltration was performed using a 5 kDa ultrafiltration membrane. The retentate was collected. The retentate was concentrated under reduced pressure to a solid content of approximately 10 wt%. It was then freeze-dried to obtain the fermented Tremella fuciformis polysaccharide fraction.

[0101] The obtained fermented Tremella polysaccharide components have the following characteristics: average molecular weight 11.6 kDa; molecular weight distribution 5–20 kDa; moisture content ≤5 wt%.

[0102] (II) Preparation of composite hyaluronic acid composition

[0103] Weigh 450.0 g of deionized water and add it to the mixing tank. Start the anchor stirrer. Control the speed at 150 rpm. Slowly add 25.0 g of high molecular weight sodium hyaluronate. The high molecular weight sodium hyaluronate has a weight average molecular weight of 1.2 million Da. After addition, continue stirring at 30°C for 6 h to allow the hyaluronic acid molecular chains to fully expand and form a homogeneous hydration system. Then add 6.0 g of acetylated sodium hyaluronate. Maintain the system temperature at 35°C. Continue stirring for 2 h. Then add 25.0 g of fermented Tremella fuciformis polysaccharide. Control the system temperature at 35°C. Continue stirring for 2 h. Then add 12.0 g of sodium hyaluronate cross-linked polymer. Continue stirring for 1 h. The sodium hyaluronate cross-linked polymer is BDDE cross-linked sodium hyaluronate with a cross-linking degree of approximately 3% and an average particle size of approximately 150 nm. Then add 2.0 g of oligomeric sodium hyaluronate with a weight average molecular weight of approximately 5 kDa. Continue stirring for 30 min. Then, add the following ingredients in sequence: 0.8 g arginine; 3.0 g trehalose; 1.0 g glyceryl glucoside; 1.0 g betaine; and 0.5 g ectoine. Stir for 30 minutes. Adjust the pH of the system to 5.5 ± 0.1. Filter. Obtain the composite hyaluronic acid composition.

[0105] II. Preparation of the serum

[0106] (a) Composition of the serum

[0107] By weight percentage: 5.00% composite hyaluronic acid composition; 4.00% butylene glycol; 3.00% glycerin; 0.50% 1,2-hexanediol; 0.50% p-hydroxyacetophenone; 0.08% xanthate gum; 0.22% ammonium acryloyldimethyl taurate / VP copolymer (U20); 0.80% trehalose; 1.00% betaine; 0.30% ectoine; 0.50% glyceryl glucoside; 0.80% gentian extract; 0.80% spirulina extract; 0.05% EDTA-2Na; 0.05% sodium citrate / citric acid buffer system; balance deionized water.

[0108] (II) Preparation method of essence

[0109] Phase A: Add the following to the mixing tank: deionized water, EDTA-2Na, glycerol, and butylene glycol. Stir until homogeneous. Then add: xanthan gum. Disperse at high speed (3000 rpm) for 20 minutes. Then add: U20 thickener. Continue dispersing for 15 minutes. A homogeneous gel system is formed. Then add: the composite hyaluronic acid composition. Stir for 30 minutes. The main system is formed.

[0110] Phase B:

[0111] Mix the following ingredients: gentian extract; spirulina extract; trehalose; ectoine; glyceryl glucoside. Stir at 25°C for 20 min to obtain an active pre-stabilized solution.

[0112] Slowly add phase B to phase A. Stir for 20 min. Then add: 1,2-hexanediol; p-hydroxyacetophenone. Continue stirring for 15 min. Adjust the final pH to 5.5±0.1 using arginine. Filter through a 100-mesh filter. Let stand for 12 h to remove bubbles. Fill into vials. Obtain the compound hyaluronic acid repair essence.

[0113] Example 2

[0114] The only difference from Example 1 is that in the composite hyaluronic acid composition, 25 parts of fermented Tremella fuciformis polysaccharide (20-50 kDa) replace the 5-20 kDa component in Example 1. All other components are identical.

[0115] Example 3

[0116] The only difference from Example 1 is that the fermented Tremella polysaccharide in the complex hyaluronic acid composition contains 3-10 kDa.

[0117] 25 copies were identical to the rest.

[0118] Comparative Example 1

[0119] The only difference from Example 1 is that the fermented Tremella polysaccharide in the composite hyaluronic acid composition is replaced with 25 parts of unfermented Tremella polysaccharide. All other components are the same.

[0120] Comparative Example 2

[0121] The only difference from Example 1 is that the fermented tremella polysaccharide is replaced with 25 parts of fermented tremella polysaccharide with a content of >100kDa.

[0122] Comparative Example 3

[0123] The only difference from Example 1 is that the fermented tremella polysaccharide is replaced with 25 parts of fermented tremella polysaccharide with a content of <1kDa.

[0124] Comparative Example 4

[0125] The only difference from Example 1 is the deletion of 6 parts of acetylated sodium hyaluronate. Everything else is the same.

[0126] Comparative Example 5

[0127] The only difference from Example 1 is the deletion of 25 parts of fermented Tremella polysaccharide. Everything else is the same.

[0128] Comparative Example 6

[0129] The only difference from Example 1 is the deletion of 12 parts of sodium hyaluronate cross-linked polymer. Everything else is the same.

[0130] Comparative Example 7

[0131] The only difference from Example 1 is the deletion of 2 parts of oligomeric sodium hyaluronate. Everything else is the same.

[0132] Comparative Example 8

[0133] The only difference from Example 1 is that, during the preparation of the composite hyaluronic acid composition, all hyaluronic acid components and fermented Tremella fuciformis polysaccharide are added at once. Everything else is the same.

[0134] Comparative Example 9

[0135] The composite hyaluronic acid composition in this comparative example has the same components as in Example 1, and by weight includes:

[0136] 25.0 parts of high molecular weight sodium hyaluronate; 6.0 parts of acetylated sodium hyaluronate; 12.0 parts of cross-linked sodium hyaluronate; 2.0 parts of oligomeric sodium hyaluronate; 25.0 parts of fermented Tremella fuciformis polysaccharide; 0.8 parts of arginine; 3.0 parts of trehalose; 1.0 part of glyceryl glucoside; 1.0 part of betaine; 0.5 parts of ectoine; balance of deionized water.

[0137] The high molecular weight sodium hyaluronate has a weight-average molecular weight of 1.2 million Da; the oligomeric sodium hyaluronate has a weight-average molecular weight of 5 kDa; and the fermented Tremella polysaccharide is the 5-20 kDa fermented Tremella polysaccharide prepared in Example 1.

[0138] II. Preparation method of composite hyaluronic acid composition

[0139] The difference between this comparative example and Example 1 is that fermented Tremella polysaccharide was added first, followed by sodium hyaluronate.

[0140] The specific preparation method is as follows:

[0141] Weigh 450.0g of deionized water and add it to the mixing tank. Start the anchor stirrer, controlling the speed at 150rpm and the system temperature at 30℃. Add 25.0g of fermented Tremella fuciformis polysaccharide to the deionized water and stir for 2 hours to allow the fermented Tremella fuciformis polysaccharide to form a hydrated dispersion. Then, slowly add 25.0g of high molecular weight sodium hyaluronate to the above system and continue stirring at 30℃ for 6 hours to hydrate the high molecular weight sodium hyaluronate. Then, add 6.0g of acetylated sodium hyaluronate, raise the system temperature to 35℃, and stir for 2 hours. Then, add 12.0g of sodium hyaluronate crosspolymer and continue stirring for 1 hour. Next, add 2.0g of oligomeric sodium hyaluronate and stir for 30 minutes. Finally, add 0.8g of arginine, 3.0g of trehalose, 1.0g of glyceryl glucoside, 1.0g of betaine, and 0.5g of ectoine, and continue stirring for 30 minutes. The pH of the system was adjusted to 5.5±0.1, and the mixture was filtered through a 100-mesh filter to obtain the composite hyaluronic acid composition of Comparative Example 9.

[0142] III. Ingredients of the Serum

[0143] The essence was prepared using the composite hyaluronic acid composition obtained in Comparative Example 9.

[0144] By weight percentage, the serum comprises: 5.00% of the comparative example 9 complex hyaluronic acid composition; 4.00% of butylene glycol; 3.00% of glycerin; 0.50% of 1,2-hexanediol; 0.50% of p-hydroxyacetophenone; 0.08% of xanthan gum; 0.22% of ammonium acryloyldimethyl taurate / VP copolymer; 0.80% of trehalose; 1.00% of betaine; 0.30% of ectoine; 0.50% of glyceryl glucoside; 0.80% of gentian extract; 0.80% of Spirulina macrophylla extract; 0.05% of EDTA-2Na; 0.05% of sodium citrate / citric acid buffer system; and the balance being deionized water.

[0145] IV. Preparation Method of Serum

[0146] Deionized water, EDTA-2Na, glycerol, and butylene glycol were added to a mixing tank and stirred at room temperature for 10 min. Xanthan gum was added and dispersed at 3000 rpm for 20 min. Acryloyldimethyl taurate ammonium / VP copolymer was added and dispersion continued for 15 min to form a basic gel system. Then, the comparative example 9 composite hyaluronic acid composition was added and stirred for 30 min. In a separate container, gentian extract, Spirulina macrophylla extract, trehalose, ectoine, and glyceryl glucoside were mixed and stirred at 25°C for 20 min to form an active pre-stabilized solution. This active pre-stabilized solution was slowly added to the basic gel system and stirred for 20 min. Then, 1,2-hexanediol and p-hydroxyacetophenone were added and stirring continued for 15 min. The final pH was adjusted to 5.5 ± 0.1, filtered through a 100-mesh filter, allowed to stand for 12 h to degas, and then filled into vials to obtain the comparative example 9 essence.

[0147] Comparative Example 10

[0148] I. Components of the Composite Hyaluronic Acid Composition

[0149] The composite hyaluronic acid composition in this comparative example has the same components as in Example 1, and by weight includes:

[0150] 25.0 parts of high molecular weight sodium hyaluronate; 6.0 parts of acetylated sodium hyaluronate; 12.0 parts of cross-linked sodium hyaluronate; 2.0 parts of oligomeric sodium hyaluronate; 25.0 parts of fermented Tremella fuciformis polysaccharide; 0.8 parts of arginine; 3.0 parts of trehalose; 1.0 part of glyceryl glucoside; 1.0 part of betaine; 0.5 parts of ectoine; balance of deionized water.

[0151] The high molecular weight sodium hyaluronate has a weight-average molecular weight of 1.2 million Da; the oligomeric sodium hyaluronate has a weight-average molecular weight of 5 kDa; and the fermented Tremella polysaccharide is the 5-20 kDa fermented Tremella polysaccharide prepared in Example 1.

[0152] II. Preparation method of composite hyaluronic acid composition

[0153] The difference between this comparative example and Example 1 is that acetylated sodium hyaluronate was added last.

[0154] The specific preparation method is as follows: Weigh 450.0g of deionized water and add it to the mixing tank. Start the anchor stirrer, control the speed at 150rpm, and control the system temperature at 30℃. Slowly add 25.0g of high molecular weight sodium hyaluronate to the deionized water and stir at 30℃ for 6 hours to hydrate the high molecular weight sodium hyaluronate. Then add 25.0g of fermented Tremella fuciformis polysaccharide and continue stirring for 2 hours. Next, add 12.0g of sodium hyaluronate cross-linked polymer and stir for 1 hour. Add 2.0g of oligomeric sodium hyaluronate and stir for 30 minutes. Then add 0.8g of arginine, 3.0g of trehalose, 1.0g of glyceryl glucoside, 1.0g of betaine, and 0.5g of ectoine and continue stirring for 30 minutes. Finally, add 6.0g of acetylated sodium hyaluronate, raise the system temperature to 35℃, and stir for 2 hours. The pH of the system was adjusted to 5.5±0.1, and the mixture was filtered through a 100-mesh filter to obtain the composite hyaluronic acid composition of Comparative Example 10.

[0155] III. Ingredients of the Serum

[0156] The serum was prepared using the composite hyaluronic acid composition obtained in Comparative Example 10. By weight percentage, the serum comprised: 5.00% of the composite hyaluronic acid composition from Comparative Example 10; 4.00% butylene glycol; 3.00% glycerin; 0.50% 1,2-hexanediol; 0.50% p-hydroxyacetophenone; 0.08% xanthate gum; 0.22% ammonium acryloyldimethyl taurate / VP copolymer; 0.80% trehalose; 1.00% betaine; 0.30% ectoine; 0.50% glyceryl glucoside; 0.80% gentian extract; 0.80% spirulina extract; 0.05% EDTA-2Na; 0.05% sodium citrate / citric acid buffer system; and the balance being deionized water.

[0157] IV. Preparation Method of Serum

[0158] Deionized water, EDTA-2Na, glycerol, and butylene glycol were added to a mixing tank and stirred at room temperature for 10 min. Xanthan gum was added and dispersed at 3000 rpm for 20 min. Acryloyldimethyl taurate ammonium / VP copolymer was added and dispersion continued for 15 min to form a basic gel system. Then, the Comparative Example 10 composite hyaluronic acid composition was added and stirred for 30 min. In a separate container, gentian extract, Spirulina macrophylla extract, trehalose, ectoine, and glyceryl glucoside were mixed and stirred at 25°C for 20 min to form an active pre-stabilized solution. This active pre-stabilized solution was slowly added to the basic gel system and stirred for 20 min. Then, 1,2-hexanediol and p-hydroxyacetophenone were added and stirring continued for 15 min. The final pH was adjusted to 5.5 ± 0.1, filtered through a 100-mesh filter, allowed to stand for 12 h to degas, and then filled into vials to obtain the Comparative Example 10 essence.

[0159] Performance testing

[0160] The following test samples are the serums prepared in Examples 1-3 and Comparative Examples 1-10. All samples were equilibrated for 24 hours at 25°C and 50% relative humidity before testing. Unless otherwise stated, each group of samples was tested in triplicate, and the average value was taken.

[0161] Appearance and transparency stability test

[0162] Take 50g of each of the essences from Examples 1-3 and Comparative Examples 1-10, and place them into 100mL transparent glass bottles, then seal them. Store them at 25℃, 45℃, and -5℃ for 30 days, respectively.

[0163] Samples were taken on days 0, 7, 14, and 30 to observe the appearance and record whether turbidity, flocculation, precipitation, stratification, darkening of color, or bubble aggregation occurred.

[0164] Transparency was measured using a UV-Vis spectrophotometer. 2.0 g of sample was taken, 8.0 g of deionized water was added, and the mixture was stirred until homogeneous. After standing for 30 min, the mixture was placed in a 1 cm quartz cuvette. Using deionized water as a blank, the transmittance T was measured at 600 nm.

[0165] Transparency retention rate is calculated using the following formula:

[0166] Transparency retention rate (%) = Tt / T0 × 100%

[0167] Where T0 is the transmittance of the sample on day 0, and Tt is the transmittance of the sample after storage.

[0168] Centrifugal stability test

[0169] Take 10.0g of each of the essences from Examples 1-3 and Comparative Examples 1-10 and place them in a 15mL centrifuge tube.

[0170] Place the centrifuge tubes in a high-speed centrifuge and centrifuge at 4000 rpm for 30 minutes at 25°C.

[0171] After centrifugation, observe whether the sample shows stratification, precipitation, flocculation, or water separation.

[0172] The evaluation criteria are as follows: no stratification, no sedimentation, and no water separation are recorded as stable; slight flocculation but no obvious stratification is recorded as basically stable; obvious stratification, sedimentation, or water separation is recorded as unstable.

[0173] Freeze-thaw cycle stability test

[0174] Take 30.0g of each of the essence from Examples 1-3 and Comparative Examples 1-10, put them into a 50mL glass bottle, and seal it.

[0175] The samples were placed in a -5°C freezer for 12 hours, then transferred to a 45°C incubator for another 12 hours, constituting one freeze-thaw cycle. Five freeze-thaw cycles were performed consecutively.

[0176] After the freeze-thaw cycle, the sample was placed at 25°C for 4 hours to equilibrate. The appearance changes were observed, and the viscosity, pH, and transparency were measured.

[0177] Viscosity retention after freeze-thaw is calculated using the following formula:

[0178] Viscosity retention rate (%) = V5 / V0 × 100%

[0179] Where V0 is the sample viscosity before freeze-thaw cycles, and V5 is the sample viscosity after 5 freeze-thaw cycles.

[0180] High-temperature accelerated stability test

[0181] Take 50.0g of each of the essence from Examples 1-3 and Comparative Examples 1-10, place them in a 100mL glass bottle, and seal it.

[0182] The samples were stored in a 45°C incubator for 30 days. Samples were taken on days 0, 7, 14, and 30 to test changes in appearance, pH, viscosity, transparency, and odor. pH was measured using a calibrated pH meter at 25°C. 10.0g of sample was taken each time, directly inserted into the pH electrode, and recorded after the reading stabilized.

[0183] pH change value is calculated using the following formula:

[0184] ΔpH = |pHt - pH0|

[0185] Where pH0 is the pH on day 0, and pHt is the pH after storage.

[0186] Low temperature stability test

[0187] Take 50.0g of each of the essence from Examples 1-3 and Comparative Examples 1-10, place them in a 100mL glass bottle, and seal it.

[0188] The samples were stored at -5°C for 30 days. Samples were taken on days 0, 7, 14, and 30 to observe for crystallization, flocculation, water separation, stratification, or abnormal increase in viscosity. The viscosity and transparency were measured after the samples were brought back to 25°C.

[0189] Viscosity and viscosity retention test

[0190] The viscosity of the samples was tested using a Brookfield rotational viscometer.

[0191] The test conditions are as follows: Rotor: LV-3 rotor; Rotation speed: 12 rpm; Test temperature: 25℃; Sample amount: approximately 100g; Balancing time: 30min; Reading time: Read after the rotor has been running for 60s.

[0192] The viscosity of the samples was tested on day 0, after 30 days of storage at 45℃, and after 5 freeze-thaw cycles.

[0193] Viscosity retention rate is calculated using the following formula:

[0194] Viscosity retention rate (%) = Vt / V0 × 100%

[0195] Where V0 is the viscosity on day 0, and Vt is the viscosity after treatment.

[0196] Rheological performance testing

[0197] The rheological properties of the samples were tested using a rotational rheometer.

[0198] The test conditions are as follows: Fixture: 40mm parallel plate; Gap: 1.0mm; Test temperature: 25℃; Shear rate range: 0.1~100s. -1 Test mode: Steady-state shear mode.

[0199] Record the sample at 0.1s. -1 1s -1 10s -1 100s -1 The apparent viscosity was measured. Further tests were conducted using an oscillating mode to determine the storage modulus G' and loss modulus G''.

[0200] The oscillation test conditions were as follows: frequency range: 0.1–10 Hz; strain: 1%; temperature: 25 °C. The stability of the hydration network and the skin feel of the samples were evaluated by comparing G', G'', and shear thinning behavior.

[0201] Particle size and particle size stability test

[0202] The particle size of the complex hydration aggregates in the sample was determined using a dynamic light scattering particle size analyzer.

[0203] Take 1.0g of the essence, add 99.0g of deionized water, dilute 100 times, gently invert and mix 10 times, avoiding vigorous shaking.

[0204] Place the diluted solution at 25°C for 30 minutes to equilibrate.

[0205] The test conditions are as follows: test temperature: 25℃; scattering angle: 173°; equilibration time: 120s; each sample is tested 3 times.

[0206] Record the average particle size (Z-average) and polydispersity index (PDI). Analyze samples from day 0 and samples stored at 45°C for 30 days.

[0207] The particle size growth rate is calculated using the following formula: Particle size growth rate (%) = (Dt - D0) / D0 × 100%

[0208] Where D0 is the average particle size on day 0, and Dt is the average particle size after storage.

[0209] In vitro moisturizing performance test

[0210] The hygroscopic and moisture-retaining capacities of the samples were evaluated using a weighing method. A dry weighing bottle was filled with 0.500 g of the sample and placed in a desiccator with a relative humidity of 75%. The constant humidity environment was maintained by a saturated sodium chloride solution at a temperature of 25°C. The samples were weighed at 0 h, 2 h, 4 h, 8 h, and 24 h.

[0211] The moisture absorption rate is calculated using the following formula:

[0212] Moisture absorption rate (%) = (Mt - M0) / M0 × 100%

[0213] Where M0 is the initial sample mass and Mt is the sample mass after t hours of storage.

[0214] During the moisturizing performance test, 0.500g of sample was placed in a weighing bottle and first placed in an environment with a relative humidity of 75% for 24 hours to equilibrate. Then it was transferred to an environment with a relative humidity of 43% and a temperature of 25℃.

[0215] Weigh the items at 0h, 4h, 8h, 24h, and 48h respectively.

[0216] Moisturizing rate is calculated using the following formula: Moisturizing rate (%) = Mt / M0 × 100%

[0217] Where M0 is the sample mass when transferred to a 43% humidity environment, and Mt is the sample mass after t hours of storage.

[0218] Human stratum corneum moisture content test

[0219] Twenty healthy volunteers, aged 20-45, were selected. They were not allowed to use skin care products on the inside of their forearms for 24 hours before the test.

[0220] The test environment was 22±2℃ and 50±5% relative humidity. Subjects sat quietly for 30 minutes after entering the test environment.

[0221] Divide the inner forearm into 3cm×3cm areas, apply 20mg of sample to each area, and spread evenly for 30s.

[0222] The stratum corneum moisture content was determined using a Corneometer skin moisture meter. Test time points were before application, and 0.5h, 2h, 8h, 24h, and 48h after application.

[0223] The rate of increase in stratum corneum moisture content is calculated using the following formula:

[0224] Moisture content increase rate (%) = (Wt - W0) / W0 × 100%

[0225] Where W0 is the moisture content of the stratum corneum before application, and Wt is the moisture content of the stratum corneum at the corresponding time point after application.

[0226] Transdermal water loss (TEWL) test

[0227] Transdermal water loss (TEWL) was determined using a Tewameter transdermal water loss meter. The test area, sample amount, and environmental conditions were the same as for human stratum corneum moisture content testing. Test time points were before application, and 2h, 8h, 24h, and 48h after application.

[0228] The TEWL reduction rate is calculated using the following formula:

[0229] TEWL reduction rate (%) = (TEWL0 - TEWLt) / TEWL0 × 100%

[0230] Wherein, TEWL0 is the transdermal water dispersion loss before coating, and TEWLt is the transdermal water dispersion loss at the corresponding time point after coating.

[0231] pH response release test

[0232] This test is used to evaluate the release behavior of hyaluronic acid under different pH conditions.

[0233] Take 2.0 g of sample and place it in a dialysis bag. The molecular weight cutoff of the dialysis bag is 3.5 kDa.

[0234] Place the dialysis bag in 100 mL of pH 5.5 phosphate buffer and pH 6.8 phosphate buffer, respectively.

[0235] The test temperature was 32℃ and the oscillation speed was 100rpm.

[0236] Take 2.0 mL of the external solution at 0.5 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h respectively, and add an equal amount of fresh buffer solution at the same time.

[0237] The hyaluronic acid content in the release solution was determined by HPLC.

[0238] The cumulative release rate is calculated using the following formula:

[0239] Cumulative release rate (%) = Mt / Mtotal × 100%

[0240] Where Mt is the cumulative amount of hyaluronic acid released at time t, and Mtotal is the total amount of hyaluronic acid in the sample.

[0241] Mild patch test

[0242] Thirty healthy volunteers were selected. 0.02g of sample was placed in a patch applicator and applied to the skin on the back of each subject. The patch was left in place for 24 hours. Skin reactions were observed at 0.5h, 24h, and 48h after removing the patch applicator.

[0243] Evaluation will be conducted according to the following criteria:

[0244] 0 points: No response; 1 point: Mild erythema; 2 points: Significant erythema; 3 points: Erythema with mild edema; 4 points: Significant edema or other strong response. Record the average stimulus score for each sample.

[0245] Table 1. Viscosity retention rate after 30 days of storage at 45℃

[0246] sample Initial viscosity (mPa·s) Viscosity (mPa·s) after 30 days Viscosity retention rate (%) Example 1 8250 7960 96.5 Example 2 8180 7640 93.4 Example 3 8220 7830 95.3 Comparative Example 1 8110 6680 82.4 Comparative Example 2 8160 6920 84.8 Comparative Example 3 8080 6520 80.7 Comparative Example 4 7850 6030 76.8 Comparative Example 5 7920 6250 78.9 Comparative Example 6 7480 5480 73.3 Comparative Example 7 8030 6980 86.9 Comparative Example 8 8140 6390 78.5 Comparative Example 9 8090 6620 81.8 Comparative Example 10 8110 6730 83

[0247] Table 2. Transparency retention rate after 5 freeze-thaw cycles

[0248] sample Initial transmittance (%) Light transmittance after freeze-thaw cycle (%) Retention rate (%) Example 1 98.6 96.1 97.5 Example 2 98.2 94.3 96 Example 3 98.4 95.1 96.6 Comparative Example 1 97.9 87.8 89.7 Comparative Example 2 98 88.6 90.4 Comparative Example 3 97.8 86.4 88.3 Comparative Example 4 97.2 84.7 87.1 Comparative Example 5 97.5 85.8 88 Comparative Example 6 96.8 82.9 85.6 Comparative Example 7 97.4 89.1 91.5 Comparative Example 8 97.7 84.5 86.5 Comparative Example 9 97.6 85.7 87.8 Comparative Example 10 97.8 86.2 88.1

[0249] Table 3 DLS particle size stability results

[0250] sample Initial particle size (nm) After 30 days at 45℃ (nm) growth rate(%) Example 1 218 236 8.3 Example 2 226 254 12.4 Example 3 221 243 10 Comparative Example 1 238 396 66.4 Comparative Example 2 245 418 70.6 Comparative Example 3 241 428 77.6 Comparative Example 4 256 475 85.5 Comparative Example 5 248 451 81.9 Comparative Example 6 262 503 92 Comparative Example 7 236 354 50 Comparative Example 8 231 518 124.2 Comparative Example 9 233 476 104.3 Comparative Example 10 229 468 104.4

[0251] Table 4. Increase rate of stratum corneum moisture content over 48 hours

[0252] sample Moisture content increase rate (%) Example 1 36.8 Example 2 31.5 Example 3 34.7 Comparative Example 1 19.4 Comparative Example 2 21.6 Comparative Example 3 18.9 Comparative Example 4 24.8 Comparative Example 5 23.5 Comparative Example 6 22.1 Comparative Example 7 28.4 Comparative Example 8 21.5 Comparative Example 9 22.7 Comparative Example 10 23.3

[0253] Table 5 TEWL reduction rate (48h)

[0254] sample TEWL reduction rate (%) Example 1 29.4 Example 2 24.8 Example 3 27.8 Comparative Example 1 13.5 Comparative Example 2 15.2 Comparative Example 3 14.1 Comparative Example 4 18.7 Comparative Example 5 17.9 Comparative Example 6 16.8 Comparative Example 7 21.3 Comparative Example 8 15.6 Comparative Example 9 16.9 Comparative Example 10 17.5

[0255] Table 6. Results of appearance and transparency stability tests (45℃, 30 days)

[0256] sample Initial transmittance (%) 30-day transmittance (%) Retention rate (%) Appearance changes Example 1 98.6 96.1 97.5 No change Example 2 98.2 94.3 96 No change Example 3 98.4 95.1 96.6 No change Comparative Example 1 97.9 87.8 89.7 Slightly cloudy Comparative Example 2 98 88.6 90.4 Slightly cloudy Comparative Example 3 97.8 86.4 88.3 turbid Comparative Example 4 97.2 84.7 87.1 turbid Comparative Example 5 97.5 85.8 88 turbid Comparative Example 6 96.8 82.9 85.6 Turbid, slight separation of water Comparative Example 7 97.4 89.1 91.5 Slightly cloudy Comparative Example 8 97.7 84.5 86.5 Noticeably turbid Comparative Example 9 97.6 85.7 87.8 Noticeably turbid Comparative Example 10 97.8 86.2 88.1 Noticeably turbid

[0257] Table 7. Centrifugation stability test results (4000 rpm, 30 min)

[0258] sample Layering precipitation water analysis evaluate Example 1 none none none Stablize Example 2 none none none Stablize Example 3 none none none Stablize Comparative Example 1 none trace amounts none Basically stable Comparative Example 2 none trace amounts none Basically stable Comparative Example 3 none small amount none Basically stable Comparative Example 4 none small amount none Basically stable Comparative Example 5 none small amount none Basically stable Comparative Example 6 slight none slight Unstable Comparative Example 7 none trace amounts none Basically stable Comparative Example 8 obvious small amount obvious Unstable Comparative Example 9 obvious small amount obvious Unstable Comparative Example 10 obvious small amount slight Unstable

[0259] Table 8. Results of accelerated stability test at high temperature (45℃, 30 days)

[0260] sample ΔpH Viscosity retention rate (%) evaluate Example 1 0.08 96.5 excellent Example 2 0.1 93.4 excellent Example 3 0.09 95.3 excellent Comparative Example 1 0.25 82.4 generally Comparative Example 2 0.22 84.8 generally Comparative Example 3 0.28 80.7 Poor Comparative Example 4 0.31 76.8 Poor Comparative Example 5 0.29 78.9 Poor Comparative Example 6 0.35 73.3 Poor Comparative Example 7 0.18 86.9 generally Comparative Example 8 0.3 78.5 Poor Comparative Example 9 0.27 81.8 Poor Comparative Example 10 0.24 83 generally

[0261] Table 9. Low temperature stability test results (-5℃, 30 days)

[0262] sample crystallization Layering Transparency changes evaluate Example 1 none none No significant changes excellent Example 2 none none No significant changes excellent Example 3 none none No significant changes excellent Comparative Example 1 none none Slight decline generally Comparative Example 2 none none Slight decline generally Comparative Example 3 trace amounts none Significant decline Poor Comparative Example 4 trace amounts none Significant decline Poor Comparative Example 5 trace amounts none Significant decline Poor Comparative Example 6 trace amounts slight Significant decline Poor Comparative Example 7 none none Slight decline generally Comparative Example 8 trace amounts slight Significant decline Poor Comparative Example 9 trace amounts slight Significant decline Poor Comparative Example 10 trace amounts none Significant decline Poor

[0263] Table 10 Rheological property test results (25℃)

[0264] sample G' (Pa) G'' (Pa) G' / G'' Example 1 156 74 2.11 Example 2 142 73 1.95 Example 3 148 72 2.06 Comparative Example 1 96 81 1.19 Comparative Example 2 102 82 1.24 Comparative Example 3 88 79 1.11 Comparative Example 4 105 84 1.25 Comparative Example 5 108 85 1.27 Comparative Example 6 82 80 1.03 Comparative Example 7 121 79 1.53 Comparative Example 8 84 86 0.98 Comparative Example 9 92 85 1.08 Comparative Example 10 95 84 1.13

[0265] Table 11 Results of in vitro moisturizing performance test (48h moisturizing rate)

[0266] sample Moisturizing rate (%) Example 1 81.2 Example 2 75.4 Example 3 78.8 Comparative Example 1 59.6 Comparative Example 2 62.4 Comparative Example 3 58.8 Comparative Example 4 65.3 Comparative Example 5 64.7 Comparative Example 6 61.9 Comparative Example 7 70.5 Comparative Example 8 60.2 Comparative Example 9 62 Comparative Example 10 63.4

[0267] Table 12 pH response release test results (cumulative release rate over 48 hours)

[0268] sample pH 5.5 (%) pH 6.8 (%) Example 1 36.5 58.8 Example 2 38.1 54.3 Example 3 37.4 56.9 Comparative Example 1 42.6 46.8 Comparative Example 2 40.8 48.5 Comparative Example 3 44.2 47.1 Comparative Example 8 45.6 49.4 Comparative Example 9 44.8 48.7 Comparative Example 10 43.9 48.2

[0269] Table 13 Results of the mild patch test (30 subjects)

[0270] sample Average Stimulus Rating Example 1 0.05 Example 2 0.06 Example 3 0.05 Comparative Example 1 0.08 Comparative Example 2 0.07 Comparative Example 3 0.09 Comparative Example 4 0.12 Comparative Example 5 0.1 Comparative Example 6 0.11 Comparative Example 7 0.08 Comparative Example 8 0.09 Comparative Example 9 0.1 Comparative Example 10 0.09

[0271] Evaluation criteria: 0-0.25: no irritation, 0.25-0.50: slight irritation, 0.50-1.00: mild irritation, >1.00: moderate or higher irritation; all samples were classified as no irritation.

[0272] The results showed that Example 1 exhibited the best performance in terms of viscosity retention, transparency retention, particle size stability, stratum corneum water content improvement rate, and TEWL reduction rate.

[0273] In particular, studies on the molecular weight of polysaccharides treated by fermentation revealed that lower molecular weight does not necessarily lead to better results. The system exhibited optimal performance when the molecular weight of the fermented polysaccharide was in the range of 5–20 kDa; however, when the molecular weight was higher than 100 kDa or lower than 1 kDa, all indicators decreased significantly.

[0274] The above results indicate that there is a specific effective molecular weight range for fermented polysaccharides. This result is inconsistent with the technical understanding in the field that smaller molecule components are more likely to play a role, and has obvious unpredictability.

[0275] Furthermore, the results of Comparative Examples 8-10 showed that the order in which different components were added had a significant impact on the final system performance. The composite hyaluronic acid composition prepared in the order described in Example 1 exhibited superior stability and moisturizing properties, indicating that there is a specific synergistic relationship between high molecular weight sodium hyaluronate, acetylated sodium hyaluronate, fermented polysaccharide, cross-linked sodium hyaluronate, and oligomeric hyaluronic acid, and that the same effect cannot be obtained simply by mixing them.

[0276] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite hyaluronic acid composition, characterized in that, Includes the following components by weight: (1) 10-50 parts of high molecular weight sodium hyaluronate; (2) 1-20 parts of acetylated sodium hyaluronate; (3) 2-30 parts of sodium hyaluronate cross-linked polymer; (4) 0.1 to 10 parts of oligomeric hyaluronic acid and / or sodium oligomeric hyaluronic acid.

2. The composite hyaluronic acid composition according to claim 1, characterized in that, The weight-average molecular weight of the sodium hyaluronate is 800,000 Da to 2,000,000 Da. The weight-average molecular weight of the oligomeric hyaluronic acid and / or oligomeric sodium hyaluronate is 1 kDa to 50 kDa. The sodium hyaluronate crosslinked polymer is a crosslinked sodium hyaluronate obtained by crosslinking sodium hyaluronate with diglycidyl ether crosslinking agents, divinyl sulfone crosslinking agents, or a combination thereof.

3. The composite hyaluronic acid composition according to claim 1, characterized in that, It also includes 5 to 50 parts of fermented polysaccharide components; the fermented polysaccharide components are selected from one or more of fermented tremella polysaccharide, fermented oat polysaccharide, fermented rice bran polysaccharide, fermented Bletilla striata polysaccharide, fermented seaweed polysaccharide, and fermented yeast polysaccharide.

4. The composite hyaluronic acid composition according to claim 3, characterized in that, It also includes 0.1 to 10 parts of amino acid components; the amino acid components are selected from one or more of arginine, lysine, histidine, and glycine.

5. The composite hyaluronic acid composition according to claim 4, characterized in that, It also includes 0.1 to 20 parts of a carbohydrate stabilizing component; the carbohydrate stabilizing component is selected from one or more of trehalose, glycerol glucoside, betaine, maltitol, and sorbitol.

6. The composite hyaluronic acid composition according to claim 5, characterized in that, The composition further includes one or more of ectoine, panthenol, β-glucan, and allantoin.

7. A method for preparing the composite hyaluronic acid composition according to claim 5 or 6, characterized in that, The steps include the following: (1) Add high molecular weight sodium hyaluronate to the aqueous phase for hydration treatment; (2) Add acetylated sodium hyaluronate and mix well; (3) Add the fermented polysaccharide components and mix well; (4) Add sodium hyaluronate cross-linking polymer and mix well; (5) Add oligomeric hyaluronic acid and / or oligomeric sodium hyaluronate; (6) Add amino acid components and sugar stabilizing components; (7) Adjust the pH of the system to obtain the composite hyaluronic acid composition.

8. A skin care composition, characterized in that, Includes the composite hyaluronic acid composition according to any one of claims 1 to 6 and cosmetically acceptable excipients.

9. The skin care composition according to claim 8, characterized in that, The skin care composition is a serum, gel, lotion, cream, spray, mask, freeze-dried preparation, or scalp care preparation.

10. The use of the composite hyaluronic acid composition according to any one of claims 1 to 6 in the preparation of skin moisturizing products; in the preparation of skin barrier care products; in the preparation of skin soothing products; and in the preparation of postoperative care products.