Small molecular organic acid modified hyaluronic acid as well as preparation method and application thereof

Abstract

The invention discloses small molecular organic acid modified hyaluronic acid as well as a preparation method and application thereof, and belongs to the field of polymer synthesis. The method comprises the following steps: firstly, carrying out modification reaction on hyaluronic acid and an amino-terminated silane coupling agent to generate an amino-terminated hyaluronic acid derivative, and then carrying out grafting reaction on the amino-terminated hyaluronic acid derivative and small-molecular organic acid to prepare the small-molecular organic acid modified hyaluronic acid. The preparation method comprises the following steps: firstly, combining hyaluronic acid with an amino-terminated silane coupling agent to generate an amino-terminated hyaluronic acid derivative; then, the intermediate and small-molecular organic acid are subjected to efficient grafting reaction, and small-molecular organic acid modified hyaluronic acid is successfully prepared. The small molecular organic acid modified hyaluronic acid prepared by the invention has good water solubility and high molecular weight, and has important application value in the fields of skin care products, medical cosmetology and the like.

Description

Technical Field

[0001] The invention belongs to the field of polymer synthesis, and specifically relates to a preparation method and application of small molecule organic acid modified hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA), as a natural polymer compound, has a wide range of applications in pharmaceutical research and development, cosmetics manufacturing, and biomaterials due to its outstanding biocompatibility, excellent water retention, and diverse biological activities. Especially in the field of functional skin care, it occupies a core position in skin care formulas with its excellent moisturizing, nourishing, repairing, sunscreen, lubricating, and film-forming functions. However, the functions of pure hyaluronic acid are relatively limited, and its application areas are therefore also subject to certain restrictions. In recent years, with the advancement of skin care technology, many small molecule organic acids have emerged with their unique efficacy and have been cleverly integrated into the skin care system to work synergistically with hyaluronic acid, aiming to explore a new era of skin care.

[0003] For example, the commonly used small molecule organic acids, sialic acid, salicylic acid, azelaic acid, capric acid, and hydroxyproline have different effects in skin care products. Sialic acid can repair skin cells, nourish the dermis, improve skin firmness, and help slow down skin aging. Salicylic acid has certain antifungal and anti-inflammatory effects, can relieve symptoms such as redness, swelling, and stinging, and can reduce skin oil secretion, prevent pore blockage and acne. Azelaic acid can inhibit the production of melanin, reduce the formation of spots and chloasma, achieve a whitening effect, and has a good effect on the treatment of acne, blackheads, and acne. Capric acid has an anti-free radical effect, helps to delay the aging process, and protects the skin from environmental pollution and other harmful substances. Hydroxyproline promotes collagen synthesis, improves skin elasticity, and reduces the formation of wrinkles. As one of the main components of the stratum corneum, it helps maintain the integrity and moisture balance of the skin and promotes the renewal of epidermal cells. It is suitable for repairing and maintaining damaged or sensitive skin.

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

[0005] The present invention proposes a general technical route for small molecule organic acid modified hyaluronic acid in response to the above-mentioned problems. This technology first combines hyaluronic acid with a silane coupling agent with a terminal amino group to generate a hyaluronic acid derivative with a terminal amino group. Subsequently, this intermediate is used to carry out an efficient grafting reaction with a small molecule organic acid to successfully prepare a small molecule organic acid modified hyaluronic acid. The method used in the present invention has a certain versatility. As long as it is a small molecule containing a carboxyl group, it can almost be directly grafted onto hyaluronic acid through the present invention to modify it. In addition, the small molecule organic acid modified hyaluronic acid prepared by the present invention has good water solubility and high molecular weight, and has important application value in the fields of skin care products, medical cosmetology, etc.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for preparing small molecule organic acid modified hyaluronic acid, the method firstly carries out a modification reaction between hyaluronic acid and a terminal amino group silane coupling agent to generate a hyaluronic acid derivative with a terminal amino group, and then carries out a grafting reaction with a small molecule organic acid to prepare the small molecule organic acid modified hyaluronic acid.

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

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

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

[0011] In the technical solution of the present 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°C, and the reaction time is 12-24h.

[0013] In the technical scheme of the present invention: the mass ratio of the amino-terminated hyaluronic acid derivative to the small molecule organic acid is 1:0.5-1:5; the reaction solvent of the grafting reaction is water, the reaction temperature is 50-75°C, and the reaction time is 12-24h; the reaction is 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 the small molecule organic acid to the catalyst is 100:80-150.

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

[0016] The technology of the present invention is applied to the application of small molecule organic acid modified hyaluronic acid prepared by the method in medical cosmetology.

[0017] Further preferred is the use of small molecule organic acid modified hyaluronic acid as a skin care product or skin care product auxiliary material.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention first combines hyaluronic acid with a terminal amino group silane coupling agent to generate a hyaluronic acid derivative with a terminal amino group. Subsequently, this intermediate is used to carry out an efficient grafting reaction with a small molecule organic acid to successfully prepare a small molecule organic acid-modified hyaluronic acid. This technology has certain versatility for the modification of hyaluronic acid, a simple preparation process, readily available raw materials, and easy large-scale production. The small molecule organic acid-modified hyaluronic acid prepared by the present invention has good water solubility, can have the functionality of both hyaluronic acid and a specific small molecule organic acid, and is used in skin care and medical beauty products. BRIEF DESCRIPTION OF THE DRAWINGS

[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 capric acid (deuterated water as solvent). DETAILED DESCRIPTION

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

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

[0024] Comparative Example 1 Physically mixing hyaluronic acid and sialic acid.

[0025] Take hyaluronic acid (1g, 10kDa) and dissolve it into 1% aqueous solution, mix it with 100mg sialic acid, observe its dissolution state and test the small molecule sustained release function.

[0026] Comparative Example 2 Physically mixed hyaluronic acid and salicylic acid.

[0027] Take hyaluronic acid (1g, 100kDa) and dissolve it into 1% aqueous solution, mix it with 200mg salicylic acid, observe its dissolution state and test the small molecule sustained release function.

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

[0029] Take hyaluronic acid (1g, 1000kDa) and dissolve it into 1% aqueous solution, mix it with 300mg azelaic acid, observe its dissolution state and test the small molecule sustained release function.

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

[0031] Take hyaluronic acid (1g, 2500kDa) and dissolve it into 1% aqueous solution, mix it with 200mg capric 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 (1 g, 10 kDa) were placed in 50 mL of toluene and stirred to disperse them evenly. Subsequently, 3-aminopropyltrimethoxysilane (2 g) was added dropwise to the above solution. The mixture was reacted at 60° C. for 12 h. The precipitate was collected by centrifugation and washed three times with toluene to obtain amino-terminated hyaluronic acid.

[0034] Dissolve the amino-terminated hyaluronic acid (100 mg) in 50 mL of water for later use. Then dissolve sialic acid (100 mg) in 50 mL of water, add 74 mg of EDC HCl and 45 mg of NHS, stir for 30 min at 60°C pH = 5.0-6.0, then drop the amino-terminated hyaluronic acid solution, continue the reaction for 24 h, use ethanol for precipitation, centrifuge the obtained white solid, dry it, and use nuclear magnetic resonance to detect its structure, such as Figure 1 shown.

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

[0036] Hyaluronic acid particles (1 g, 100 kDa) were placed in 50 mL of toluene and stirred to disperse them evenly. Subsequently, 3-aminopropyltriethoxysilane (4 g) was added dropwise to the above solution. The mixture was reacted at 60°C for 15 h, centrifuged, and the precipitate was collected and washed three times with toluene to obtain amino-terminated hyaluronic acid.

[0037] Dissolve the amino-terminated hyaluronic acid (100 mg) in 50 mL of water for later use. Then dissolve salicylic acid (200 mg) in 50 mL of ethanol, add 134 mg of EDC HCl and 115 mg of NHS, stir for 30 min at 60°C pH = 5.0-6.0, then drop the amino-terminated hyaluronic acid solution, continue the reaction for 24 h, use ethanol for precipitation, centrifuge the obtained white solid, and dry it to obtain salicylic acid-modified hyaluronic acid.

[0038] Example 3 Preparation of Azelaic Acid Modified Hyaluronic Acid

[0039] Hyaluronic acid particles (1 g, 1000 kDa) were placed in 50 mL of toluene and stirred to disperse them evenly. Subsequently, 3-aminopropyl(diethoxy)methylsilane (6 g) was added dropwise to the above solution. The mixture was reacted at 60°C for 24 h, centrifuged, and the precipitate was collected and washed three times with toluene to obtain amino-terminated hyaluronic acid.

[0040] Dissolve the amino-terminated hyaluronic acid (100 mg) in 50 mL of water for later use. Then dissolve azelaic acid (300 mg) in 50 mL of ethanol, add 134 mg of EDC HCl and 115 mg of NHS, stir for 30 min at 60°C pH = 5.0-6.0, then drop the amino-terminated hyaluronic acid solution, continue the reaction for 24 h, use ethanol for precipitation, centrifuge the obtained white solid, and dry it to obtain azelaic acid-modified hyaluronic acid.

[0041] Example 4 Preparation of capric acid-modified hyaluronic acid

[0042] Hyaluronic acid particles (1 g, 2500 kDa) were put into 50 mL of toluene and stirred to make them evenly dispersed. Then 3-aminopropyldimethoxymethylsilane (8 g) was added dropwise to the above solution. The mixture was reacted at 60°C for 18 h. The precipitate was collected by centrifugation and washed three times with toluene to obtain amino-terminated hyaluronic acid.

[0043] Dissolve the amino-terminated hyaluronic acid (100 mg) in 50 mL of water for later use. Then dissolve the capric acid (200 mg) in 50 mL of ethanol, add 134 mg of EDC HCl and 115 mg of NHS, stir for 30 min at 60°C pH = 5.0-6.0, then drop the amino-terminated hyaluronic acid solution, continue the reaction for 16 h, precipitate with ethanol, centrifuge the obtained white solid, dry it, and obtain the capric acid-modified hyaluronic acid. Use nuclear magnetic resonance to detect its structure, as shown in FIG. Figure 2 shown.

[0044] Example 5 The samples of Comparative Examples 1-4 and Examples 1-4 were tested.

[0045] 1 ml of the solutions of Comparative Examples 1-4 were taken respectively for subsequent tests.

[0046] 1 g of the solid of Examples 1-4 was respectively dissolved in 100 ml of water, and 1 ml of the solution was taken for subsequent testing.

[0047] Use visual method to observe the appearance of the solution of comparative examples 1-4 and examples 1-4 to see if there is turbidity or precipitation. The test results are shown in Table 1.

[0048] The solutions of Comparative Examples 1-4 and Examples 1-4 to be tested were placed in a dialysis bag with a molecular weight cut-off of 3000, and then placed in 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. Sample appearance and acidity and alkalinity tests.

[0050] Appearance Acidity and alkalinity Comparative Example 1 Clarity and transparency Acidic Comparative Example 2 There is precipitation Acidic Comparative Example 3 Turbid solution Acidic Comparative Example 4 There is precipitation 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] From the above results, it can be seen that the grafted small molecules can be well dissolved in water without turbidity, precipitation, etc. Since the small molecules are grafted onto the high molecular weight hyaluronic acid, they cannot pass through the dialysis bag, which can effectively reduce the irritation of the small molecules.

[0052] Toxicology test:

[0053] Two toxicological tests were conducted on the solutions of comparative examples 1-4 and examples 1-4. The tests were conducted in accordance with the second part of the "Technical Specifications for Disinfection" 2002 edition, skin irritation test and skin allergy test. The results are shown in the following table:

[0054] Table 2 Skin irritation test results

[0055] Stimulus response score Sample set 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 in the present invention is mildly irritating, and a simple mixture of the two is moderately irritating. The present invention improves the irritation of small molecule acids.

[0057] Table 3 Skin allergy test

[0058] Sensitization rate (%) Sample set 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 the present invention is 0. It is non-allergenic, which is greatly improved compared with 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 glycerol, 1g of hydroxyethyl cellulose, and 0.5g of Tween-40 were added to 60mL of water, heated to 60°C and stirred, and mixed evenly to obtain a mixed solution A; after cooling the mixed solution A to 40°C, 1g of salicylic acid-modified hyaluronic acid in Example 2 was added, and after stirring and mixing evenly, 4g of sea buckthorn oil and 0.5g of PEG-40 hydrogenated castor oil were added, and stirring and mixing evenly to obtain a mixed solution B; after the mixed solution B was further cooled to 30°C, 8g of modified fish collagen, 5g of aloe extract, 4g of palmitoyl tripeptide-5, 5g of gentian root extract, and 0.05g of p-hydroxyacetophenone were added, and after mixing evenly, a 200-mesh filter was filtered to obtain a facial mask liquid, and the facial mask liquid was poured into a packaging material containing a facial mask base cloth, and sealed to obtain the facial mask liquid. The facial mask liquid prepared by this method has no salicylic acid precipitation and can 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 and transparent without precipitation Example 6 50℃ Return to room temperature after 50 days Clear and transparent without precipitation Example 6 25℃ Return to room temperature after 10 days Clear and transparent without precipitation Example 6 25℃ Return to room temperature after 50 days Clear and transparent without precipitation Example 6 -5℃ Return to room temperature after 10 days Clear and transparent without precipitation Example 6 -5℃ Return to room temperature after 50 days Clear and transparent without precipitation

Claims

1. A method for preparing small molecule organic acid modified hyaluronic acid, characterized in that: The method firstly performs a modification reaction on hyaluronic acid and a terminal amino group silane coupling agent to generate a terminal amino group hyaluronic acid derivative, and then performs a grafting reaction with a small molecule organic acid to prepare a small molecule organic acid modified hyaluronic acid.

2. The method for preparing small molecule organic acid modified hyaluronic acid according to claim 1, characterized in that: The small molecule organic acid is any one or a combination of sialic acid, salicylic acid, azelaic acid, capric acid, and hydroxyproline.

3. 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 10 kDa to 3000 kDa.

4. The method for preparing small molecule organic acid modified hyaluronic acid according to claim 1, characterized in that: The amino-terminated silane coupling agent is any one of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyl(diethoxy)methylsilane and 3-aminopropyldimethoxymethylsilane or a combination thereof.

5. The amino-terminated hyaluronic acid derivative 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 for the modification reaction is 50-90° C., and the time for the modification reaction is 12-24 hours.

6. According to claim 1, the intermediate is subjected to efficient grafting reaction with a small molecule organic acid, characterized in that: The mass ratio of the amino-terminated hyaluronic acid derivative to the small molecule organic acid is 1:0.5-1:5; the reaction solvent of the grafting reaction is water, the reaction temperature is 50-75°C, and the reaction time is 12-24h; the reaction is 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 the small molecule organic acid to the catalyst is 100:80-150.

7. A small molecule organic acid modified hyaluronic acid material, characterized in that: The compound is prepared by the preparation method described in any one of items 1 to 6.

8. Application of the small molecule organic acid modified hyaluronic acid obtained by the method of claim 1 in medical cosmetology.

9. The use according to claim 8, characterized in that Application of small molecule organic acid modified hyaluronic acid as skin care products or skin care product excipients.

Images