A small-molecule organic acid modified hyaluronic acid, and a preparation method and application thereof

Abstract

The application discloses a kind of small molecule organic acid modified hyaluronic acid and its preparation method and application, belong to the field of polymer synthesis.The method first hyaluronic acid is modified with amino-terminated silane coupling agent and is reacted, and the hyaluronic acid derivative with amino-terminated is generated, then grafting reaction is carried out with small molecule organic acid, and small molecule organic acid modified hyaluronic acid is prepared.The application first combines hyaluronic acid with amino-terminated silane coupling agent, and the hyaluronic acid derivative with amino-terminated is generated.Subsequently, using this intermediate, efficient grafting reaction is carried out with small molecule organic acid, and small molecule organic acid modified hyaluronic acid is successfully prepared.Small molecule organic acid modified hyaluronic acid prepared by the application has good water solubility and high molecular weight, and has important application value in the field of skin care, medical cosmetology and the like.

Description

Technical Field

[0001] This invention belongs to the field of polymer synthesis, specifically relating to a method for preparing and applying hyaluronic acid modified with small molecule organic acids. Background Technology

[0002] Hyaluronic acid (HA), as a natural polymer compound, has wide applications in pharmaceutical research, cosmetics manufacturing, and biomaterials due to its outstanding biocompatibility, excellent water-retention properties, and diverse bioactivities. Especially in the field of functional skincare, it occupies a core position in skincare formulations due to its superior moisturizing, nourishing, repairing, sun protection, lubrication, and film-forming effects. However, the functions of pure hyaluronic acid are relatively limited, thus restricting its application areas. In recent years, with the advancement of skincare technology, many small-molecule organic acids have emerged with their unique efficacy and have been cleverly integrated into skincare systems to work synergistically with hyaluronic acid, aiming to explore a new era in skincare.

[0003] For example, commonly used small-molecule organic acids such as sialic acid, salicylic acid, azelaic acid, decanoic acid, and hydroxyproline have different effects in skincare products. Sialic acid can repair skin cells, nourish the dermis, improve skin firmness, and help slow down skin aging. Salicylic acid has certain antifungal effects and anti-inflammatory properties, relieving symptoms such as redness and stinging, and can reduce sebum secretion, preventing clogged pores and acne. Azelaic acid can inhibit melanin production, reduce the formation of age spots and melasma, achieving a whitening effect, and has a good therapeutic effect on acne, blackheads, and pimples. Decanoic acid has anti-free radical effects, helping to delay the aging process and protect the skin from environmental pollution and other harmful substances. Hydroxyproline improves skin elasticity and reduces wrinkles by promoting collagen synthesis. As one of the main components of the stratum corneum, it helps maintain skin integrity and moisture balance, promotes epidermal cell renewal, and is suitable for repairing and maintaining damaged or sensitive skin.

[0004] However, direct physical mixing is often limited by the solubility characteristics and release rate of small molecule organic acids, which affects the full realization of its overall effectiveness. Summary of the Invention

[0005] This invention proposes a general technical route for modifying hyaluronic acid with small-molecule organic acids, addressing the aforementioned problems. The technique first combines hyaluronic acid with a silane coupling agent containing an amino group to generate a hyaluronic acid derivative with an amino group. Subsequently, this intermediate is used to undergo a highly efficient grafting reaction with a small-molecule organic acid, successfully preparing hyaluronic acid modified with small-molecule organic acids. The method used in this invention has a certain degree of versatility; almost any small molecule containing a carboxyl group can be directly grafted onto hyaluronic acid to modify it. Furthermore, the hyaluronic acid modified with small-molecule organic acids prepared by this invention exhibits good water solubility and high molecular weight, making it valuable for applications in skincare products, medical aesthetics, and other fields.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A method for preparing hyaluronic acid modified with small molecule organic acids is disclosed. The method first involves reacting hyaluronic acid with a silane coupling agent with an amino group to generate a hyaluronic acid derivative with an amino group, and then subjecting it to a grafting reaction with a small molecule organic acid to prepare hyaluronic acid modified with small molecule organic acids.

[0008] In the technical solution of this invention: the small molecule organic acid is any one or a combination of several of sialic acid, salicylic acid, azelaic acid, decanoic acid, and hydroxyproline.

[0009] In the technical solution of this invention, the molecular weight of the hyaluronic acid is 10kDa to 3000kDa.

[0010] In the technical solution of this invention: the silane coupling agent with terminal amino group is any one or a combination of several of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyl(diethoxy)methylsilane, and 3-aminopropyldimethoxymethylsilane.

[0011] In the technical solution of this invention: the mass ratio of hyaluronic acid to amino-terminated silane coupling agent is 1:1-10;

[0012] The solvent for the modification reaction is toluene, the reaction temperature is 50-90℃, and the reaction time is 12-24h.

[0013] In the technical solution of this invention: the mass ratio of the hyaluronic acid derivative with terminal amino group to the small molecule organic acid is 1:0.5-1:5; the reaction solvent for the grafting reaction is water, the reaction temperature is 50-75℃, the reaction time is 12-24h; the reaction is carried out in an acidic environment.

[0014] The catalyst used in the reaction is EDC HCl and NHS in a mass ratio of 5-10:3-8, and the mass ratio of small molecule organic acid to catalyst is 100:80-150.

[0015] A small molecule organic acid modified hyaluronic acid material is prepared by the above-described preparation method.

[0016] The present invention relates to the application of small molecule organic acid-modified hyaluronic acid prepared by the method in medical aesthetics.

[0017] Further optimization: the application of hyaluronic acid modified with small molecule organic acids as a skin care product or skin care product excipient.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention first combines hyaluronic acid with a silane coupling agent containing an amino group to generate a hyaluronic acid derivative with an amino group. Subsequently, using this intermediate, a highly efficient grafting reaction is carried out with a small-molecule organic acid to successfully prepare hyaluronic acid modified with a small-molecule organic acid. This technology has a certain degree of versatility for modifying hyaluronic acid, the preparation process is simple, the raw materials are readily available, and it is easy to scale up production. The small-molecule organic acid-modified hyaluronic acid obtained by this invention has good water solubility and can combine the functions of both hyaluronic acid and specific small-molecule organic acids, making it suitable for use in skincare and medical aesthetic products. Attached Figure Description

[0020] Figure 1 NMR spectrum of sialic acid-modified hyaluronic acid (deuterated water as solvent).

[0021] Figure 2 NMR spectrum of hyaluronic acid modified with decanoic acid (deuterated water as solvent). Detailed Implementation

[0022] To make the present invention easier to understand, specific embodiments of the present invention will be further described below.

[0023] The present invention will be further described below with reference to embodiments and accompanying drawings, the purpose of which is to help to better understand the content of the present invention, but the scope of protection of the present invention is not limited thereto:

[0024] Comparative Example 1: Physical mixing of hyaluronic acid and sialic acid.

[0025] Dissolve 1g of hyaluronic acid (10kDa) in a 1% aqueous solution, mix it with 100mg of sialic acid, observe its dissolution state, and test its small molecule sustained-release function.

[0026] Comparative Example 2: Physical mixing of hyaluronic acid and salicylic acid.

[0027] Dissolve 1g of hyaluronic acid (100kDa) in a 1% aqueous solution, mix it with 200mg of salicylic acid, observe its dissolution state, and test its small molecule sustained-release function.

[0028] Comparative Example 3: Physically mixed hyaluronic acid and azelaic acid.

[0029] Dissolve 1g of hyaluronic acid (1000kDa) in a 1% aqueous solution, mix it with 300mg of azelaic acid, observe its dissolution state, and test its small molecule sustained-release function.

[0030] Comparative Example 4: Physically mixed hyaluronic acid and decanoic acid.

[0031] Dissolve 1g of hyaluronic acid (2500kDa) in a 1% aqueous solution and mix it with 200mg of decanoic acid. Observe its dissolution state and test the small molecule sustained-release function.

[0032] Example 1: Preparation of sialic acid-modified hyaluronic acid

[0033] Hyaluronic acid particles (1g, 10kDa) were added to 50mL of toluene and stirred to disperse them evenly. Then, 3-aminopropyltrimethoxysilane (2g) was added dropwise to the above solution. The reaction was carried out at 60℃ for 12h. After centrifugation, the precipitate was collected and washed three times with toluene to obtain hyaluronic acid with terminal amino groups.

[0034] 100 mg of amino-terminated hyaluronic acid was dissolved in 50 mL of water and set aside. Then, 100 mg of sialic acid was dissolved in 50 mL of water, and 74 mg of EDC HCl and 45 mg of NHS were added. The mixture was stirred for 30 min at 60 °C and pH 5.0-6.0. The amino-terminated hyaluronic acid solution was then added dropwise, and the reaction was continued for 24 h. Ethanol was used for precipitation, and the resulting white solid was centrifuged, dried, and its structure was analyzed using NMR. Figure 1 As shown.

[0035] Example 2: Preparation of salicylic acid-modified hyaluronic acid

[0036] Hyaluronic acid particles (1g, 100kDa) were added to 50mL of toluene and stirred to disperse them evenly. Then, 3-aminopropyltriethoxysilane (4g) was added dropwise to the above solution. The reaction was carried out at 60℃ for 15h. After centrifugation, the precipitate was collected and washed three times with toluene to obtain hyaluronic acid with terminal amino groups.

[0037] 100 mg of amino-terminated hyaluronic acid was dissolved in 50 mL of water and set aside. Then, 200 mg of salicylic acid was dissolved in 50 mL of ethanol, and 134 mg of EDC HCl and 115 mg of NHS were added. The mixture was stirred at 60 °C and pH 5.0-6.0 for 30 min. The amino-terminated hyaluronic acid solution was then added dropwise, and the reaction continued for 24 h. Ethanol was used to precipitate the solid, which was then centrifuged and dried to obtain salicylic acid-modified hyaluronic acid.

[0038] Example 3: Preparation of azelaic acid-modified hyaluronic acid

[0039] Hyaluronic acid particles (1g, 1000kDa) were added to 50mL of toluene and stirred to disperse them evenly. Then, 3-aminopropyl(diethoxy)methylsilane (6g) was added dropwise to the above solution. The reaction was carried out at 60℃ for 24h. After centrifugation, the precipitate was collected and washed three times with toluene to obtain hyaluronic acid with terminal amino groups.

[0040] 100 mg of amino-terminated hyaluronic acid was dissolved in 50 mL of water and set aside. Then, 300 mg of azelaic acid was dissolved in 50 mL of ethanol, and 134 mg of EDC HCl and 115 mg of NHS were added. The mixture was stirred at 60 °C and pH 5.0-6.0 for 30 min. Subsequently, the amino-terminated hyaluronic acid solution was added dropwise, and the reaction was continued for 24 h. Ethanol was used to precipitate the solid, and the resulting white solid was centrifuged and dried to obtain azelaic acid-modified hyaluronic acid.

[0041] Example 4: Preparation of decanoic acid-modified hyaluronic acid

[0042] Hyaluronic acid particles (1g, 2500kDa) were added to 50mL of toluene and stirred to disperse them evenly. Then, 3-aminopropyldimethoxymethylsilane (8g) was added dropwise to the above solution. The reaction was carried out at 60℃ for 18h. After centrifugation, the precipitate was collected and washed three times with toluene to obtain hyaluronic acid with terminal amino groups.

[0043] 100 mg of amino-terminated hyaluronic acid was dissolved in 50 mL of water and set aside. Then, 200 mg of decanoic acid was dissolved in 50 mL of ethanol, and 134 mg of EDC HCl and 115 mg of NHS were added. The mixture was stirred at 60 °C and pH 5.0-6.0 for 30 min. Subsequently, the amino-terminated hyaluronic acid solution was added dropwise, and the reaction was continued for 16 h. Precipitation was performed using ethanol, and the resulting white solid was centrifuged, dried, and the decanoic acid-modified hyaluronic acid was obtained. Its structure was analyzed using NMR. Figure 2 As shown.

[0044] Example 5 tested samples from Comparative Examples 1-4 and Examples 1-4.

[0045] Take 1 ml of each of the comparative examples 1-4 for subsequent tests.

[0046] Take 1g of the solid from Examples 1-4 and dissolve it in 100ml of water. Take 1ml of the solution for subsequent tests.

[0047] The solutions of Comparative Examples 1-4 and Examples 1-4 were visually inspected for turbidity and precipitation. The test results are shown in Table 1.

[0048] The solutions of Comparative Examples 1-4 and Examples 1-4 were placed in dialysis bags with a molecular weight cutoff of 3000 and then added to 100 ml of ethanol-water solution (50:50 mass ratio) to test the acidity and alkalinity of the ethanol-water solution. The test results are shown in Table 1.

[0049] Table 1. Detection of sample appearance and acidity / alkalinity.

[0050] Appearance acidity and alkalinity Comparative Example 1 Clarity and transparency acidic Comparative Example 2 There is sediment acidic Comparative Example 3 The solution was turbid. acidic Comparative Example 4 There is sediment acidic Example 1 Clarity and transparency neutral Example 2 Clarity and transparency neutral Example 3 Clarity and transparency neutral Example 4 Clarity and transparency neutral

[0051] The results show that the grafted small molecules dissolve well in water without causing turbidity or precipitation. Since the small molecules are grafted onto the high molecular weight hyaluronic acid, they also cannot pass through dialysis bags, effectively reducing the irritation caused by the small molecules.

[0052] Toxicological experiments:

[0053] Two toxicological tests were conducted on the comparative solutions 1-4 and the solutions from Examples 1-4. These tests were conducted according to Part II of the 2002 edition of the "Disinfection Technical Specifications" for skin irritation and skin allergic reactions. The results are shown in the table below:

[0054] Table 2 Results of skin irritation test

[0055] Stimulus-response integral Sample group data Control group data Comparative Example 1 3.2 0 Comparative Example 2 3.4 0 Comparative Example 3 3.1 0 Comparative Example 4 3.6 0 Example 1 1.4 0 Example 2 1.5 0 Example 3 1.3 0 Example 4 1.2 0

[0056] Conclusion: As shown in Table 2, the small molecule organic acid modified hyaluronic acid prepared by this invention is mildly irritating, and the simple mixture of the two is moderately irritating. This invention improves the irritation of small molecule acids.

[0057] Table 3 Skin Allergy Tests

[0058] Sensitization rate (%) Sample group data Positive control group data Negative control group data Comparative Example 1 50 87 0 Comparative Example 2 45 87 0 Comparative Example 3 32 87 0 Comparative Example 4 56 87 0 Example 1 0 87 0 Example 2 0 87 0 Example 3 0 87 0 Example 4 0 87 0

[0059] Conclusion: As shown in Table 3, the sensitization rate of the small molecule organic acid-modified hyaluronic acid prepared in this invention is 0. It exhibits no sensitization, a significant improvement over the direct use of small molecule hyaluronic acid.

[0060] Example 6: Preparation of salicylic acid modified hyaluronic acid facial mask

[0061] 4g of propylene glycol, 6g of glycerin, 1g of hydroxyethyl cellulose, and 0.5g of Tween-40 were added to 60mL of water and heated to 60℃ with stirring until homogeneous to obtain mixture A. Mixture A was cooled to 40℃, and 1g of salicylic acid-modified hyaluronic acid (as described in Example 2) was added. After stirring until homogeneous, 4g of sea buckthorn oil and 0.5g of PEG-40 hydrogenated castor oil were added, and stirring continued until homogeneous to obtain mixture B. Mixture B was further cooled to 30℃, and 8g of modified fish collagen, 5g of aloe vera extract, 4g of palmitoyl tripeptide-5, 5g of gentian root extract, and 0.05g of p-hydroxyacetophenone were added. After homogeneous mixing, the mixture was filtered through a 200-mesh filter to obtain the mask liquid. The mask liquid was poured into packaging containing the mask base fabric and sealed. The mask liquid prepared by this method showed no salicylic acid precipitation and could be stored stably for a long time. The stability data are shown in Table 2.

[0062] Table 4 Sample Stability Test

[0063] sample temperature Storage conditions Experimental results Example 6 50℃ Return to room temperature after 10 days Clear, transparent and free of sediment Example 6 50℃ Return to room temperature after 50 days Clear, transparent and free of sediment Example 6 25℃ Return to room temperature after 10 days Clear, transparent and free of sediment Example 6 25℃ Return to room temperature after 50 days Clear, transparent and free of sediment Example 6 -5℃ Return to room temperature after 10 days Clear, transparent and free of sediment Example 6 -5℃ Return to room temperature after 50 days Clear, transparent and free of sediment

Claims

1. A method for preparing hyaluronic acid modified with small molecule organic acid, characterized in that: This method first modifies hyaluronic acid with a silane coupling agent with an amino group to generate a hyaluronic acid derivative with an amino group, and then performs a grafting reaction with a small molecule organic acid to prepare hyaluronic acid modified with a small molecule organic acid. The small molecule organic acid is any one or a combination of several of sialic acid, salicylic acid, azelaic acid, and decanoic acid; The silane coupling agent with the terminal amino group is any one or a combination of several of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyl(diethoxy)methylsilane, and 3-aminopropyldimethoxymethylsilane. The mass ratio of amine-terminated hyaluronic acid derivatives to small molecule organic acids is 1:0.5-1:5; the reaction solvent for the grafting reaction is water, the reaction temperature is 50-75℃, and the reaction time is 12-24h; the reaction is carried out in an acidic environment. The catalyst used in the reaction is EDC HCl and NHS in a mass ratio of 5~10:3~8, and the mass ratio of small molecule organic acid to catalyst is 100:80~150.

2. The method for preparing small molecule organic acid-modified hyaluronic acid according to claim 1, characterized in that: The molecular weight of the hyaluronic acid is 10kDa to 3000kDa.

3. The method for preparing small molecule organic acid-modified hyaluronic acid according to claim 1, characterized in that: The mass ratio of hyaluronic acid to amino-terminated silane coupling agent is 1:1-10; The solvent for the modification reaction is toluene, the temperature of the modification reaction is 50-90℃, and the reaction time is 12-24h.

4. A small molecule organic acid-modified hyaluronic acid material, characterized in that, It is prepared by any one of the preparation methods described in 1 to 3.

5. The application of the small molecule organic acid-modified hyaluronic acid obtained by the method of claim 1 in the preparation of skin care products or skin care product excipients.

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