Hyaluronic acid and azelaic acid graft as well as preparation method and application thereof

Abstract

The invention provides a hyaluronic acid and azelaic acid graft as well as a preparation method and application thereof. The hyaluronic acid and azelaic acid graft provided by the invention solves the problem that the azelaic acid is low in solubility and large in usage amount so as to cause irritation, and has an obvious soothing effect compared with azelaic acid, hyaluronic acid and a physical mixture of hyaluronic acid and azelaic acid with the same amount.

Description

Technical Field

[0001] This application belongs to the field of hyaluronic acid derivative technology, specifically, it relates to a hyaluronic acid-azelaic acid graft compound, its preparation method and application. Background Technology

[0002] Azelaic acid, also known as azelaic acid, possesses various properties such as antibacterial, anti-inflammatory, anti-acne, pigmentation improvement, and exfoliation, making it widely used in personal care products. However, azelaic acid is poorly soluble in water and oil, resulting in low bioavailability. Most commercially available products containing azelaic acid are formulated as creams using various emulsification systems, with concentrations typically ranging from 15% to 20% to ensure effective absorption. High concentrations of azelaic acid can easily lead to precipitation, resulting in a gritty feel on the skin, a rough and unstable system, and potential local skin irritation from excessive use, primarily manifesting as erythema, itching, scaling, and burning sensations. These factors contribute to the limited variety of formulations available for azelaic acid-related products.

[0003] To address the water solubility issue of azelaic acid, some methods have been published in the literature, mainly focusing on cosmetic formulation and dosage form design. For example: CN 113248364 A discloses a method for compounding azelaic acid with alkaline substances (such as carnitine, echinocoline, theophylline, etc.) to form salts, which can increase the water solubility of azelaic acid, but the prepared salts have not been shown to enhance the efficacy of azelaic acid or reduce the amount of azelaic acid used; patents CN 110669226 A and CN 115463044B disclose methods for preparing supramolecular systems through solvents and additives, and the prepared compositions have long-lasting high-temperature resistance, but the supramolecular systems are easily damaged by other compound additives; patent CN 114181072A discloses a preparation process for ultrafine azelaic acid, but the solubilization range of azelaic acid is limited, and there is a risk of recrystallization when the formulation or the use environment changes; CN 116270268 A, CN 115501171 A, CN117045523A and CN Patents such as 115919683A disclose preparation methods for different compositions. They improve the solubility of azelaic acid by compounding it with different components, but the use of large amounts of excipients and solvents limits the use of other active substances in its application. Other patents, CN 110974711 A and CN 113520890 B, enhance the solubility of azelaic acid through microcapsules and dispersions, respectively. However, similarly, the use of inorganic calcium carbonate shells, large amounts of solvents, and stabilizers increases the proportion of excipients in the formulation. In summary, these methods improve the water solubility of azelaic acid to some extent, but require a large amount of excipients. Furthermore, the solubilizing effect may be compromised by the addition of other substances, changes in environmental pH, or solvent evaporation during use. Therefore, solutions for improving the solubility and efficiency of azelaic acid in end-product applications are still needed. Summary of the Invention

[0004] To address the issues of low solubility of azelaic acid and the potential for irritation from large-volume use, this application provides a hyaluronic acid-azelaic acid graft, its preparation method, and its application.

[0005] Specifically, this application relates to the following aspects:

[0006] 1. A hyaluronic acid-azelaic acid graft compound having the structure shown in Formula I:

[0007]

[0008] The R1 group in the hyaluronic acid-azelaic acid graft compound includes at least H and / or metal ions, and... The group, that is, hyaluronic acid is replaced by azelaic acid at at least one hydroxyl position;

[0009] At least one of the R2 groups is H and / or a metal ion or a quaternary ammonium salt;

[0010] Preferably, the grafting degree of the hyaluronic acid-azelaic acid graft is 0.01-4.

[0011] 2. The hyaluronic acid-azelaic acid graft compound according to item 1, wherein n is an integer from 1 to 2000.

[0012] 3. The hyaluronic acid-azelaic acid graft compound according to item 1 or 2, wherein the metal ion includes one or more of sodium ions, potassium ions, calcium ions, zinc ions or magnesium ions.

[0013] 4. A method for preparing the hyaluronic acid-azelaic acid graft material according to any one of items 1-3, comprising the following steps:

[0014] (1) Dissolve hyaluronic acid or its salt in an organic solvent, add azelaic anhydride or azelaic acid or its derivative, and react in the presence of an organic base to obtain the target crude product.

[0015] (2) The crude product from step (1) is purified to obtain hyaluronic acid azelaic acid graft.

[0016] 5. The preparation method according to item 4, wherein the organic solvent in step (1) includes one or more of formamide, N,N-dimethylformamide, and dimethyl sulfoxide, preferably formamide.

[0017] 6. The preparation method according to item 4, wherein the organic base in step (1) includes one or more of amine compounds, nitrogen-containing heterocyclic compounds, and alkaloids;

[0018] Preferably, the organic base includes one or more of triethylamine, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine, and N,N-diisopropylethylamine.

[0019] 7. The preparation method according to item 4, wherein the molar ratio of organic base to hyaluronic acid or its salt in step (1) is (0.1-5):1.

[0020] 8. The preparation method according to item 4, wherein in step (1), the molar ratio of azelaic anhydride or azelaic acid or its derivative to hyaluronic acid or its salt is (0.1-10):1.

[0021] 9. The preparation method according to any one of items 4-8, wherein the reaction temperature in step (1) is 10-80°C and the reaction time is 2-48h.

[0022] 10. The application of the hyaluronic acid-azelaic acid graft compound according to any one of items 1-3 in the preparation of cosmetics and pharmaceutical products, preferably, the cosmetics and pharmaceutical products have a soothing effect.

[0023] The hyaluronic acid-azelaic acid grafted compound of this application solves the problems of low solubility of azelaic acid and potential irritation caused by large dosage. Compared with an equal amount of azelaic acid, hyaluronic acid, or a physical mixture of hyaluronic acid and azelaic acid, the hyaluronic acid-azelaic acid grafted compound of this application has a significant soothing effect. Attached Figure Description

[0024] Figure 1 This shows a graft of 40kDa sodium hyaluronate and 40kDa azelaic acid hyaluronate. 1 H-NMR comparison;

[0025] Figure 2 IR comparison of 40kDa sodium hyaluronate and 40kDa azelaic acid-grafted hyaluronate.

[0026] Figure 3 The high-performance liquid chromatogram of azelaic acid is shown.

[0027] Figure 4 The high-performance liquid chromatography-mass spectra of azelaic acid are shown.

[0028] Figure 5 The liquid phase integral area-concentration curve of azelaic acid is shown. Detailed Implementation

[0029] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0030] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0031] This application provides a hyaluronic acid-azelaic acid grafted compound having the structure shown in Formula I:

[0032]

[0033] Wherein, the R1 group includes at least H and / or metal ions, and and The functional group, namely hyaluronic acid, is replaced by azelaic acid at at least one hydroxyl position;

[0034] At least one of the R2 groups is H and / or a metal ion or a quaternary ammonium salt;

[0035] The grafting degree of the hyaluronic acid-azelaic acid graft is 0.01-4, for example, it can be 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1. 35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, and any value between these values.

[0036] In some specific embodiments, the grafting degree of the hyaluronic acid-azelaic acid graft is 0.2-1.5.

[0037] In this application, the grafting degree of the hyaluronic acid-azelaic acid graft can be detected by methods known in the art.

[0038] In some specific embodiments, the grafting degree of the hyaluronic acid-azelaic acid graft can be calculated using the following formula:

[0039]

[0040] Where DS represents the grafting degree; m 壬二酸 M represents the mass of azelaic acid grafted onto the hyaluronic acid-azelaic acid graft; 壬二酸 m is the molar mass of azelaic acid; HA-壬二酸 The mass of the hyaluronic acid-azelaic acid grafted product; M HA The molar mass is the disaccharide structural unit of hyaluronic acid. The mass of azelaic acid grafted onto the hyaluronic acid-azelaic acid graft can be determined by methods known in the art, such as detection by liquid chromatography-mass spectrometry.

[0041] In some specific implementations, n is an integer from 1 to 2000, such as 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, and any value between these values.

[0042] In some specific embodiments, the metal ion is one or more of sodium ions, potassium ions, calcium ions, zinc ions, or magnesium ions.

[0043] In some specific embodiments, the hyaluronic acid-azelaic acid grafted product has the structure shown in Formula I:

[0044]

[0045] Wherein, the R1 group includes at least H and / or metal ions, and The functional group, namely hyaluronic acid, is replaced by azelaic acid at at least one hydroxyl position;

[0046] R2 is H;

[0047] Wherein, the grafting degree of the hyaluronic acid-azelaic acid graft is 0.01-4; n is an integer from 1 to 2000.

[0048] In some specific embodiments, the hyaluronic acid-azelaic acid grafted product has the structure shown in Formula I:

[0049]

[0050] Wherein, the R1 group includes at least H and / or metal ions, and The functional group, namely hyaluronic acid, is replaced by azelaic acid at at least one hydroxyl position;

[0051] R2 is a metal ion, which includes one or more of sodium ions, potassium ions, calcium ions, zinc ions or magnesium ions.

[0052] Wherein, the grafting degree of the hyaluronic acid-azelaic acid graft is 0.01-4; n is an integer from 1 to 2000.

[0053] In some specific embodiments, the hyaluronic acid-azelaic acid grafted product has the structure shown in Formula I:

[0054]

[0055] Wherein, the R1 group includes at least H and / or metal ions, and and The functional group, namely hyaluronic acid, is replaced by azelaic acid at at least one hydroxyl position;

[0056] R2 is H and a metal ion, wherein the metal ion includes one or more of sodium ions, potassium ions, calcium ions, zinc ions or magnesium ions.

[0057] Wherein, the grafting degree of the hyaluronic acid-azelaic acid graft is 0.01-4; n is an integer from 1 to 2000.

[0058] In some specific embodiments, the hyaluronic acid-azelaic acid grafted product has the structure shown in Formula I:

[0059]

[0060] Wherein, the R1 group includes at least H and / or metal ions, and The functional group, namely hyaluronic acid, is replaced by azelaic acid at at least one hydroxyl position;

[0061] R2 is a quaternary ammonium salt;

[0062] Wherein, the grafting degree of the hyaluronic acid-azelaic acid graft is 0.01-4; n is an integer from 1 to 2000.

[0063] This application also provides a method for preparing any of the above-mentioned hyaluronic acid-azelaic acid grafts, comprising the following steps:

[0064] (1) Dissolve hyaluronic acid or its salt in an organic solvent, add azelaic anhydride or azelaic acid or its derivative, and react in the presence of an organic base to obtain the target crude product.

[0065] (2) The crude product from step (1) is purified to obtain hyaluronic acid azelaic acid grafted product;

[0066] The organic solvent includes one or more of formamide, N,N-dimethylformamide, and dimethyl sulfoxide.

[0067] In some specific embodiments, the organic solvent includes formamide.

[0068] The organic base includes one or more of amine compounds, nitrogen-containing heterocyclic compounds, and alkaloids.

[0069] In some specific embodiments, the organic base includes one or more of triethylamine, 4-dimethylaminopyridine, 4-pyrrolylpyridine, and N,N-diisopropylethylamine.

[0070] In some specific embodiments, the molar ratio of the organic base to hyaluronic acid or its salt is (0.1-5):1, for example, it can be 0.1:1, 0.2:1, 0.5:1, 0.8:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, and any value between these values.

[0071] In some specific embodiments, the molar ratio of azelaic anhydride or azelaic acid or its derivative to hyaluronic acid or its salt is (0.1-10):1, for example, it can be 0.1:1, 0.2:1, 0.5:1, 0.8:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, and any value between these values.

[0072] In some specific embodiments, the reaction temperature in step (1) is 10-80℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, and any value between these values.

[0073] In some specific embodiments, the reaction temperature in step (1) is 20-40°C.

[0074] In some specific embodiments, the reaction time in step (1) is 2-48h, for example, it can be 2h, 5h, 8h, 10h, 12h, 15h, 18h, 20h, 22h, 25h, 28h, 30h, 32h, 35h, 38h, 40h, 42h, 45h, 48h, and any value between these values.

[0075] In some specific embodiments, the purification method includes precipitation, dialysis, and tangential flow filtration.

[0076] Furthermore, the precipitant used for precipitation includes one or more of ethanol, methanol, isopropanol, acetone, and methyl tert-butyl ether.

[0077] In some specific embodiments, the precipitant includes ethanol.

[0078] Furthermore, the preparation method of hyaluronic acid-azelaic acid grafts may also include processing steps such as washing, filtering, and drying.

[0079] Furthermore, the detergent includes one or more of ethanol, methanol, isopropanol, acetone, and methyl tert-butyl ether.

[0080] In some specific embodiments, the detergent includes ethanol.

[0081] In some specific embodiments, a method for preparing a hyaluronic acid-azelaic acid graft includes the following steps:

[0082] (1) Dissolve hyaluronic acid or its salt in an organic solvent, add azelaic anhydride, and react in the presence of an organic base to obtain the target crude product.

[0083] The organic solvent includes one or more of formamide, N,N-dimethylformamide, and dimethyl sulfoxide;

[0084] The organic base includes one or more of triethylamine, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine, and N,N-diisopropylethylamine;

[0085] The molar ratio of the organic base to hyaluronic acid or its salt is (0.1-5):1;

[0086] The molar ratio of azelaic anhydride to hyaluronic acid or its salt is (0.1-10):1;

[0087] The reaction temperature is 10-80℃; the reaction time is 2-48h.

[0088] (2) The crude product from step (1) is purified to obtain the hyaluronic acid-azelaic acid graft.

[0089] In some specific embodiments, the purification method includes precipitation, dialysis, and tangential flow filtration.

[0090] Furthermore, the precipitant used for precipitation includes one or more of ethanol, methanol, isopropanol, acetone, and methyl tert-butyl ether.

[0091] In some specific embodiments, the precipitant includes ethanol.

[0092] Furthermore, the preparation method of hyaluronic acid azelaic acid grafts also includes processing steps such as washing, filtering, and drying.

[0093] Furthermore, the detergent includes one or more of ethanol, methanol, isopropanol, acetone, and methyl tert-butyl ether.

[0094] In some specific embodiments, the detergent includes ethanol.

[0095] In some specific embodiments, the method for preparing a hyaluronic acid azelaic acid graft further includes: adding a precipitant to the crude product to precipitate it, and washing, filtering and drying the obtained precipitate to obtain the hyaluronic acid azelaic acid graft.

[0096] This application also provides the application of any of the above-mentioned hyaluronic acid-azelaic acid grafts in the preparation of cosmetics and pharmaceutical products.

[0097] In some specific embodiments, the cosmetics and medical devices have a soothing effect.

[0098] The hyaluronic acid-azelaic acid grafted compound of this application dissolves rapidly at all tested concentrations, solving the problems of low solubility of azelaic acid and potential irritation from large dosages. Furthermore, compared to equal amounts of azelaic acid, hyaluronic acid, or a physical mixture of hyaluronic acid and azelaic acid, the hyaluronic acid-azelaic acid grafted compound of this application exhibits significant soothing effects, thereby expanding the application of azelaic acid in the cosmetics field.

[0099] Example

[0100] Example 1

[0101] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 40kDa and dissolve it in 600mL of formamide. Heat the solution in an oil bath at 100℃ and cool it to room temperature. Add 2.758g of 4-pyrrolidinylpyridine and stir at room temperature for 30min. Then add 10.55g of azelaic anhydride solid and react at 30℃ for 12h to obtain the crude product.

[0102] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0103] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol. The liquid phase analysis showed no residual azelaic acid molecular weight (see the graft test section for liquid phase conditions). Finally, it was washed with 300 mL of methyl tert-butyl ether and rinsed twice with a small amount of methyl tert-butyl ether. After vacuum drying, 3.7 g of white powder was obtained with a grafting degree of 0.6.

[0104] The structure and grafting degree of the obtained grafts were tested using the following methods:

[0105] 1. Proton NMR spectroscopy

[0106] The 1H NMR spectra of sodium hyaluronate (40 kDa) and the aforementioned white powder were measured using a nuclear magnetic resonance spectroscopy (NMR) instrument to obtain the 1H NMR spectra of the hyaluronic acid-azelaic acid grafted product, as shown below. Figure 1 As shown in the figure, compared with the 1H NMR spectrum of hyaluronic acid, the 1H NMR spectrum of the hyaluronic acid-azelaic acid graft shows new absorption peaks at 1.0–1.5 ppm, which are absorption peaks of a series of hydrogen atoms on the -CH2 group in the azelaic acid structure, and new absorption peaks at 2.0–2.5 ppm, which are absorption peaks of hydrogen atoms on the carbon atoms adjacent to the carboxyl or ester groups in the azelaic acid structure, indicating that the azelaic acid graft was successful.

[0107] 2. Fourier transform infrared spectroscopy test

[0108] The infrared spectra of sodium hyaluronate and hyaluronic acid-azelaic acid grafts were detected by Fourier transform infrared spectroscopy using the potassium bromide pellet method. Figure 2 As shown. Compared with the infrared spectrum of sodium hyaluronate, at 1730 cm⁻¹... -1 The appearance of a new absorption peak at this point is the absorption peak of the C=O stretching vibration of the ester bond, indicating that azelaic acid and hyaluronic acid are connected by an ester bond.

[0109] 3. Grafting degree test

[0110] Liquid phase conditions: mobile phase: water (0.1% formic acid): methanol (0.1% formic acid) = 40:60; column temperature: 40℃; injection volume: 10μL; flow rate: 0.2mL / min; isocratic elution for 8min.

[0111] Mass spectrometry conditions: -MRM mode

[0112] Q1 Q3 Time DP EP CE CXP 187 125 1000 -80 -5 -20 -20

[0113] CEP CUR CAD IS TEM GS1 GS2 -20 15 12 -4500 400 50 50

[0114] Standard curve: Accurately weigh 1 mg of azelaic acid and dissolve and dilute it with the mobile phase (water (0.1% formic acid): methanol (0.1% formic acid) = 40:60) to prepare a series of concentrations of 62.5, 125, 250, 500, and 1000 ng / mL. Perform liquid chromatography-mass spectrometry (LC-MS) analysis to create a standard curve. The high-performance liquid chromatography (HPLC) of azelaic acid reference standard is as follows: Figure 3 As shown, the peak at 4.35 min is azelaic acid.

[0115] The mass spectrum of azelaic acid reference standard is as follows: Figure 4As shown, m / z = 187 is the relative molecular mass of azelaic acid in negative mode. The standard curve is shown below. Figure 5 As shown, the equation for calculating the integral area-concentration of azelaic acid in the liquid phase is: y = 43.1x + 220, R 2 =0.999, indicating good linearity.

[0116] Accurately weigh 1 mg of hyaluronic acid-azelaic acid graft and dissolve it in 1 mL (V1) of 1 mol / L sodium hydroxide solution. Hydrolyze the solution at room temperature for 24 h. Then, accurately measure 0.02 mL (V2) of the reaction solution and add it dropwise to 1.98 mL (V3) of ethanol to precipitate hyaluronic acid. Centrifuge at 10000 rpm for 5 min. Accurately measure 0.2 mL (V4) of the supernatant and dilute it with 0.8 mL (V5) of methanol (0.1% formic acid). Perform a liquid chromatography-mass spectrometry (LC-MS). Calculate the concentration of azelaic acid (x ng / mL) based on the peak area integral (y) and substitute it into the standard curve equation above. Calculate the grafting degree using the following formula.

[0117]

[0118] Where, m 壬二酸 The mass of azelaic acid grafted onto the hyaluronic acid-azelaic acid graft is expressed in mg.

[0119]

[0120] Where DS represents the grafting degree;

[0121] m 壬二酸 The mass of azelaic acid grafted onto the hyaluronic acid-azelaic acid graft is measured, in mg.

[0122] M 壬二酸 This represents the molar mass of azelaic acid, which is 188.2 mg / mmol.

[0123] m HA-壬二酸 The mass of the hyaluronic acid-azelaic acid grafted compound weighed is in mg;

[0124] M HA The molar mass of the disaccharide structural unit of hyaluronic acid is 401.3 mg / mmol.

[0125] Furthermore, the above calculation formula is also used when preparing a physical mixture of hyaluronic acid and azelaic acid. For example, in preparing a 10 mL physical mixture of hyaluronic acid and azelaic acid at a concentration of 2 mg / mL (0.2%), similar to Example 1, the amount of azelaic acid used is calculated using the above formula as follows:

[0126]

[0127] Calculations show that m 壬二酸It is 4.4 mg, m HA It is 15.6 mg.

[0128] Example 2

[0129] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 9kDa and dissolve it in 600mL of formamide. Place it in an oil bath at 100℃ and heat it to dissolve. Cool it to room temperature, add 2.758g of 4-pyrrolidinylpyridine and stir at room temperature for 30min. Then add 10.55g of azelaic anhydride solid and react at 30℃ for 12h to obtain the crude product.

[0130] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0131] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol, and then washed with 300 mL of methyl tert-butyl ether. After washing with a small amount of methyl tert-butyl ether twice, the precipitate was dried under vacuum to obtain 3.5 g of white powder with a grafting degree of 0.91.

[0132] Example 3

[0133] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 500kDa and dissolve it in 600mL of formamide. Heat the solution in an oil bath at 100℃ and cool it to room temperature. Add 2.758g of 4-pyrrolidinylpyridine and stir at room temperature for 30min. Then add 10.55g of azelaic anhydride solid and react at 30℃ for 12h to obtain the crude product.

[0134] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0135] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol, and then washed with 300 mL of methyl tert-butyl ether. After washing with a small amount of methyl tert-butyl ether twice, the precipitate was dried under vacuum to obtain 4.5 g of white powder with a grafting degree of 0.42.

[0136] Example 4

[0137] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 1kDa and dissolve it in 600mL of formamide. Place it in an oil bath at 100℃ and heat to dissolve. Cool to room temperature, add 2.758g of 4-pyrrolidinylpyridine and stir at room temperature for 30min. Then add 10.55g of azelaic anhydride solid and react at 30℃ for 12h to obtain crude product.

[0138] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0139] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol, and then washed with 300 mL of methyl tert-butyl ether. After washing with a small amount of methyl tert-butyl ether twice, the precipitate was dried under vacuum to obtain 2.8 g of white powder with a grafting degree of 1.25.

[0140] Example 5

[0141] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 40kDa and dissolve it in 600mL of formamide. Heat the solution in an oil bath at 100℃ and cool it to room temperature. Add 0.36g of 4-pyrrolidinylpyridine and stir at room temperature for 30min. Then add 10.55g of azelaic anhydride solid and react at 30℃ for 12h to obtain the crude product.

[0142] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0143] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol, and then washed with 300 mL of methyl tert-butyl ether. After washing with a small amount of methyl tert-butyl ether twice, the precipitate was dried under vacuum to obtain 4.5 g of white powder with a grafting degree of 0.12.

[0144] Example 6

[0145] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 40kDa and dissolve it in 600mL of formamide. Place it in an oil bath at 100℃ and heat it to dissolve. Cool it to room temperature, add 9.19g of 4-pyrrolidinylpyridine and stir at room temperature for 30min. Then add 10.55g of azelaic anhydride solid and react at 30℃ for 12h to obtain the crude product.

[0146] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0147] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol, and then washed with 300 mL of methyl tert-butyl ether. After washing with a small amount of methyl tert-butyl ether twice, the precipitate was dried under vacuum to obtain 4.4 g of white powder with a grafting degree of 0.83.

[0148] Example 7

[0149] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 40kDa and dissolve it in 600mL of formamide. Heat the solution in an oil bath at 100℃ and cool it to room temperature. Add 2.758g of 4-pyrrolidinylpyridine and stir at room temperature for 30min. Then add 0.42g of azelaic anhydride solid and react at 30℃ for 12h to obtain the crude product.

[0150] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0151] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol, and then washed with 300 mL of methyl tert-butyl ether. After washing with a small amount of methyl tert-butyl ether twice, the precipitate was dried under vacuum to obtain 4.1 g of white powder with a grafting degree of 0.14.

[0152] Example 8

[0153] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 40kDa and dissolve it in 600mL of formamide. Heat the solution in an oil bath at 100℃ and cool it to room temperature. Add 2.758g of 4-pyrrolidinylpyridine and stir at room temperature for 30min. Then add 21.1g of azelaic anhydride solid and react at 30℃ for 12h to obtain the crude product.

[0154] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0155] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol, and then washed with 300 mL of methyl tert-butyl ether. After washing with a small amount of methyl tert-butyl ether twice, the precipitate was dried under vacuum to obtain 4.3 g of white powder with a grafting degree of 0.75.

[0156] Example 9

[0157] (1) Weigh 7.7g of tetrabutylammonium hyaluronic acid with a molecular weight of 62kDa and dissolve it in 600mL of N,N-dimethylformamide. Stir and dissolve at room temperature, add 2.758g of 4-pyrrolidinylpyridine and stir at room temperature for 30min, then add 10.55g of azelaic anhydride solid and react at 30℃ for 12h to obtain crude product.

[0158] (2) After cooling the crude product to room temperature, slowly add 600 mL of 1% sodium chloride aqueous solution to the reaction solution and stir at room temperature for 2 h. Pour the mixture into 2 L of anhydrous ethanol to precipitate the product, stir for 10 min, and centrifuge to collect the precipitate.

[0159] (3) Wash the above precipitate three times with 300 mL of 85% ethanol, then wash it twice with 300 mL of anhydrous ethanol, and vacuum dry to obtain 4.0 g of white powder with a grafting degree of 0.65.

[0160] Example 10

[0161] (1) Weigh 5g of sodium hyaluronate with a molecular weight of 40kDa and dissolve it in 600mL of formamide. Heat the solution in an oil bath at 100℃ and cool it to room temperature. Add 2.274g of 4-dimethylaminopyridine and stir at room temperature for 30min. Then add 10.55g of azelaic anhydride solid and react at 30℃ for 12h to obtain the crude product.

[0162] (2) Pour the crude product into 2L of anhydrous ethanol to precipitate the product, stir for 10min, and centrifuge to collect the precipitate.

[0163] (3) The above precipitate was washed three times with 300 mL of anhydrous ethanol, and then washed with 300 mL of methyl tert-butyl ether. After washing with a small amount of methyl tert-butyl ether twice, the precipitate was dried under vacuum to obtain 3.7 g of white powder. The grafting degree was determined to be 0.72.

[0164] The main conditions and grafting results of the above embodiments are shown in Table 1.

[0165] Table 1. Reaction conditions and grafting degree data for the examples.

[0166]

[0167]

[0168] Note: The molar amount of hyaluronic acid refers to the molar amount of disaccharide structural units in the hyaluronic acid molecule.

[0169] Experiment Example 1: Solubility Test

[0170] Experimental methods

[0171] Weigh azelaic acid, sodium hyaluronate with a molecular weight of 40 kDa, and the hyaluronic acid-azelaic acid grafts from Examples 1-8, add 5 mL of purified water to each, and dissolve by vigorous shaking at 20°C. Observe the dissolution after standing for 30 min and 12 h.

[0172] Solubility increase factor = (Azelaic acid concentration in the example at 5% concentration - 0.22%) / 0.22%

[0173] The experimental results are shown in Table 2.

[0174] Table 2 Solubility Test Data

[0175]

[0176]

[0177] Experiment Example 2: Soothing Efficacy Test

[0178] 1. Testing System

[0179] Macrophages RAW264.7.

[0180] 2. Main reagents

[0181] High glucose DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (VivaCell), MTT (Sigma), DMSO (Sinopharm), trypsin (Gibco), LPS (Sigma), dexamethasone (Sigma), Mouse TNF-α ELISA Kit (Shanghai Enzyme-Linked Biotechnology), Mouse IL-6 ELISA Kit (Shanghai Enzyme-Linked Biotechnology).

[0182] 3. Main Equipment

[0183] CO2 incubator (Thermo, 160i), biosafety cabinet (Sujing Antai, BSC-1604ⅡA2), inverted fluorescence microscope (Keyence BZ-X810), microplate reader (Tecan, Spark).

[0184] 4. Sample Information

[0185] Sample information is shown in Table 3.

[0186] Table 3

[0187] Sample Name Storage conditions Sodium hyaluronate (HA, MW = 40kDa) 4℃, protected from light azelaic acid 4℃, protected from light Hyaluronic acid-azelaic acid grafted product of Example 1 4℃, protected from light Sodium hyaluronate and azelaic acid were physically mixed according to the amounts used in Example 1. 4℃, protected from light

[0188] 5. Testing Methods

[0189] The specific experimental group settings are shown in Table 4.

[0190] Table 4

[0191]

[0192] 1) Cell seeding: at 1×10 5 Cells were seeded at a density of cells / well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0193] 2) Solution preparation: Prepare working solutions of different concentrations of the test substance according to the experimental design table, using PBS solution (0.15M, pH=7.2) as the solvent.

[0194] 3) Induction and drug administration: When the cell deposition rate in the 24-well plate reached 40%–60%, 100 μL of 10×LPS working solution was added to each well according to the experimental design. The wells were shaken left and right to mix the drug. Samples were added simultaneously. The final LPS concentration was 1 μg / mL. Each group had 3 replicates. After drug administration, the wells were placed in an incubator (37℃, 5% CO2) and cultured for another 24 h.

[0195] 4) Sample collection: After incubation, collect the cell culture supernatant into EP tubes (Note: Determine the amount of sample to be collected according to the detection indicators). After collection, freeze the sample in a -80℃ freezer.

[0196] 5) TNF-α content detection: The detection was performed according to the instructions of the Mouse TNF-α ELISA kit.

[0197] 6. TNF-α content test results

[0198] Cell supernatant was collected and TNF-α content was detected. The results are shown in Table 5.

[0199] Table 5 Summary of TNF-α Data

[0200] serial number Group Average concentration (pg / mL) 1 Blank control (BC) 105.33 2 Negative control (NC) 692.00### 3 Positive control (PC) 226.28*** 4 Sodium hyaluronate - 0.200% 493.34** 5 Azelaic acid - 0.200% 477.14** 6 Hyaluronic Acid Azelaic Acid Graft-0.200% 421.12 7 Sodium hyaluronate and azelaic acid physically mixed - 0.200% 450.12**

[0201] Note: When performing statistical analysis using the two-tailed t-test, compared with the BC group, a p-value < 0.05 is represented as #, a p-value < 0.01 as ##, and a p-value < 0.001 as ###.

[0202] Significance between the sample group, the PC group, and the 0.2% hyaluronic acid azelaic acid graft group is indicated by *, p-value < 0.05 is indicated by *, p-value < 0.01 is indicated by **, and p-value < 0.001 is indicated by ***.

[0203] Compared with the BC group, the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages in the NC group was significantly increased (p<0.001), indicating that LPS stimulation modeling was successful in this experiment. Compared with the NC group, the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages in the PC group was significantly decreased at a drug concentration of dexamethasone 100 μg / mL (p<0.001), indicating that the positive control test was effective.

[0204] Based on the LPS-stimulated RAW264.7 macrophage model, the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages was significantly reduced when the samples of sodium hyaluronate, azelaic acid, and a physical mixture of sodium hyaluronate and azelaic acid were at a concentration of 0.200% (m / V). These results showed statistically significant differences compared to the hyaluronic acid-azelaic acid graft group (p<0.01). This indicates that the hyaluronic acid-azelaic acid graft, azelaic acid, sodium hyaluronate, and azelaic acid physical mixture can inhibit the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages, exhibiting a soothing effect, especially the hyaluronic acid-azelaic acid graft group, which showed a significant soothing effect.

[0205] In summary, grafting sodium hyaluronate and azelaic acid can reduce the amount of azelaic acid used, decrease its irritation, and increase its solubility. At the same time, compared with physical mixtures, the grafted material can better exert its soothing effect of inhibiting the secretion of the inflammatory factor TNF-α.

Claims

1. A hyaluronic acid-azelaic acid graft compound having the structure shown in Formula I: in, The R1 group in the hyaluronic acid-azelaic acid graft contains at least H and / or metal ions, and... The group, that is, hyaluronic acid is replaced by azelaic acid at at least one hydroxyl position; At least one of the R2 groups is H and / or a metal ion or a quaternary ammonium salt; Preferably, the grafting degree of the hyaluronic acid-azelaic acid graft is 0.01-4.

2. The hyaluronic acid-azelaic acid graft compound according to claim 1, wherein n is an integer from 1 to 2000.

3. The hyaluronic acid-azelaic acid graft compound according to claim 1 or 2, wherein the metal ion includes one or more of sodium ions, potassium ions, calcium ions, zinc ions or magnesium ions.

4. A method for preparing the hyaluronic acid-azelaic acid graft compound according to any one of claims 1-3, comprising the following steps: (1) Dissolve hyaluronic acid or its salt in an organic solvent, add azelaic anhydride or azelaic acid or its derivative, and react in the presence of an organic base to obtain the target crude product. (2) The crude product from step (1) is purified to obtain hyaluronic acid azelaic acid graft.

5. The preparation method according to claim 4, wherein the organic solvent in step (1) includes one or more of formamide, N,N-dimethylformamide, and dimethyl sulfoxide, preferably formamide.

6. The preparation method according to claim 4, wherein the organic base in step (1) includes one or more of amine compounds, nitrogen-containing heterocyclic compounds, and alkaloids; Preferably, the organic base includes one or more of triethylamine, 4-dimethylaminopyridine, 4-pyrrolidinylpyridine, and N,N-diisopropylethylamine.

7. The preparation method according to claim 4, wherein the molar ratio of organic base to hyaluronic acid or its salt in step (1) is (0.1-5):

1.

8. The preparation method according to claim 4, wherein in step (1), the molar ratio of azelaic anhydride or azelaic acid or its derivative to hyaluronic acid or its salt is (0.1-10):

1.

9. The preparation method according to any one of claims 4-8, wherein the reaction temperature in step (1) is 10-80℃ and the reaction time is 2-48h.

10. The application of the hyaluronic acid-azelaic acid graft compound according to any one of claims 1-3 in the preparation of cosmetics and pharmaceutical products, preferably, the cosmetics and pharmaceutical products have a soothing effect.

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