Functional hyaluronic acid derivative as well as preparation method and application thereof

Abstract

The invention discloses a functional hyaluronic acid derivative as well as a preparation method and application thereof, and belongs to the field of daily chemicals and medicines. The preparation method of the functional hyaluronic acid derivative comprises the following steps: (1) reacting ascorbate and alkyl dihalide in a molar ratio of (1-8): 1 to obtain a mixed solution containing ascorbic acid ether; (2) hyaluronic acid quaternary ammonium salt is added into the mixed solution for a reaction, the molar ratio of ascorbate to hyaluronic acid quaternary ammonium salt is (2-10): 1, and the functional hyaluronic acid derivative is obtained. The functional hyaluronic acid derivative prepared by the method is of a cross-linked structure and is wider in application scene.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on October 11, 2024, with application number "202411417850.1" and invention name "A functional hyaluronic acid derivative, its preparation method and application", the entire contents of which are incorporated by reference in this application. Technical Field

[0002] The present application relates to a functional hyaluronic acid derivative and a preparation method and application thereof, belonging to the fields of daily chemicals and medicine. Background Art

[0003] An intact skin barrier can reduce damage from external mechanical, physical, chemical, and microbial stimuli, prevent the loss of nutrients and moisture in the skin, and has irritation tolerance, water-locking ability, and immune function. However, as we age, the skin barrier is constantly damaged and aged due to external and internal reasons, resulting in reduced elasticity, thinning, fragility, sagging, and dryness. Therefore, functional cosmetic raw materials have become the main direction of research and development.

[0004] Hyaluronic acid (HA) is a linear polysaccharide with a high molecular weight chain. Its molecular structure is a repeating structure of disaccharide units composed of D-glucuronic acid and N-acetylglucosamine. The molecular weight is between 100,000 and 8 million. Unlike other mucopolysaccharides, it does not contain sulfur. Each disaccharide unit in the hyaluronic acid molecule contains a carboxyl group, which can be dissociated into negative ions under physiological conditions. Negative ions at equal spatial distances repel each other, making the hyaluronic acid molecules in a loose state in the aqueous solution. It can bind 500 to 1000 times more water than itself, preventing the loss of skin moisture through the epidermis, making the skin smooth and elastic, and is widely used in beauty and skin care products.

[0005] The moisturizing function of hyaluronic acid is to moisturize the skin by improving the hydration of the skin. Large molecular hyaluronic acid reduces the loss of epidermal water, and small molecular hyaluronic acid reduces the barrier function of the skin by changing the structure of keratin. Both high molecular hyaluronic acid and low molecular hyaluronic acid have moisturizing effects. In addition, hyaluronic acid also has anti-aging functions by promoting the proliferation and differentiation of epidermal cells and scavenging oxygen free radicals, and can promote skin regeneration in injured areas. Its mechanism of action is different from that of the UV absorbers commonly used in sunscreens. Because of its synergistic effect, it can simultaneously reduce UV transmission and repair skin damage caused by UV rays, achieving double protection. When the skin is mildly burned, applying hyaluronic acid-containing aqueous cosmetics to the epidermis can relieve pain and accelerate the healing of the injured area.

[0006] Vitamin C (VC) has the function of preventing and treating scurvy, so it is also called ascorbic acid. It is a water-soluble vitamin. Its molecular formula is C6H8O6. It is a strong reducing agent. The two enol hydroxyl groups on the 2nd and 3rd carbon atoms in the molecule are easily dissociated to release H + , thereby oxidizing to form dehydrogenated vitamin C. Vitamin C and dehydrogenated vitamin C form a reversible redox system, exerting multiple effects such as anti-oxidation. However, the pH of vitamin C in skin care products is generally not more than 3.5, which can easily cause skin irritation. In addition, the structure of vitamin C is unstable and easily oxidized and deteriorated. Factors affecting the degradation rate of vitamin C include: solution pH, temperature, light, dissolved oxygen, and metal ions, which are factors that are difficult to avoid during research, use, and storage. The degradation of vitamin C is also accompanied by its color turning yellow and dark, which greatly affects the appearance and use effect of the product. At present, measures to prevent degradation include: isolating oxygen and light during formulation, packaging and storage, lowering the pH value of the formula, minimizing the use of water and adding other antioxidants, and not using iron and copper utensils. Even so, its stability cannot be effectively guaranteed, and the cost and difficulty of the formulation are increased.

[0007] Patent CN116410346A discloses a reducing hyaluronic acid ester and a preparation method thereof, wherein sodium ascorbate and hyaluronic acid are bonded to obtain reducing hyaluronic acid ester by the action of halogenated alkyl alcohol, and the reducing hyaluronic acid ester has the moisturizing effect, anti-inflammatory effect, cell growth promotion effect of hyaluronic acid, and the effects of vitamin C such as skin brightening, collagen formation promotion, and melanin inhibition, and the substance improves the stability of vitamin C and improves the penetration of vitamin C in the skin. However, the reducing hyaluronic acid ester is a linear product with poor enzymolysis resistance, which makes it easy to produce opalescence when preparing a solution formula system, and the reducing hyaluronic acid ester has poor stability and a short storage period. Summary of the invention

[0008] In order to solve the above problems, a functional hyaluronic acid derivative and a preparation method and application thereof are provided. The functional hyaluronic acid derivative prepared by the method has a cross-linked structure and can solve the above technical problems.

[0009] According to one aspect of the present application, a functional hyaluronic acid derivative is provided, and the structural formula of the functional hyaluronic acid derivative is as follows:

[0010]

[0011] Where n≥4, the R is selected from one of the following four structures (a), (b), (c), (d), and in the same long-chain molecule, there is no situation where all of them are a single structure of (a), (b), (c), or (d),

[0012] (a): (b): metal ion or ammonium ion, (c): H, (d): alkyl group;

[0013] (a) represents the junction site;

[0014] When R is selected from (d) an alkyl group, the alkyl group is connected as a connection point to a carboxyl group on another molecular chain containing a hyaluronic acid disaccharide structural unit through an ester bond.

[0015] The functional hyaluronic acid derivative is connected to another molecular chain containing a hyaluronic acid unit through an (d) alkyl group, thereby obtaining a cross-linked hyaluronic acid derivative. The derivative can overcome the shortcomings of hyaluronic acid esters bonded with linear hyaluronic acid-VC and is more conducive to exerting the effects of VC and hyaluronic acid.

[0016] Optionally, the molecular weight of the hyaluronic acid is 1 kDa-600 kDa.

[0017] For example, the molecular weight of the hyaluronic acid is 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa.

[0018] Preferably, the molecular weight of the hyaluronic acid is 10 kDa-200 kDa.

[0019] More preferably, the molecular weight of the hyaluronic acid is 10 kDa-100 kDa.

[0020] Optionally, the cross-linking degree of the functional hyaluronic acid derivative is 0.1%-25.0%.

[0021] For example, the degree of cross-linking of the functional hyaluronic acid derivative can be 0.1%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0% and any percentage in between.

[0022] Preferably, the cross-linking degree of the functional hyaluronic acid derivative is 5.0%-20.0%.

[0023] Optionally, the intrinsic viscosity of the functional hyaluronic acid derivative is less than or equal to 5.0 dL / g.

[0024] For example, the intrinsic viscosity of the functional hyaluronic acid derivative can be 0.1 dL / g, 0.5 dL / g, 0.9 dL / g, 1.0 dL / g, 1.5 dL / g, 2.0 dL / g, 2.5 dL / g, 3.0 dL / g, 3.5 dL / g, 4.0 dL / g, 4.5 dL / g, 5.0 dL / g and any value therebetween.

[0025] Preferably, the intrinsic viscosity of the functional hyaluronic acid derivative is less than or equal to 4.0 dL / g.

[0026] Optionally, in the functional hyaluronic acid derivative, the total substitution degree of the structure R being (a) in (a), (b), (c), and (d) is 1.0%-40.0%. For example, in the functional hyaluronic acid derivative, the total substitution degree of the structure R being (a) in (a), (b), (c), and (d) can be 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 11.0%, 12.0%, 13.0%, 14.0%, 15%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 30.0%, 31.0%, 32.0%, 33.0%, 34.0%, 35.0%, 36.0%, 37.0%, 38.0%, 39.0%, 40.0%, 41.0%, 42.0%, 43.0%, 44.0%, 45.0%, 46.0%, 47.0%, 48.0%, 49.0%, 50.0%, 51.0%, 52.0%, 53.0%, 54.0%, 55.0%, 56.0%, 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72. 8.0%, 19.0%, 20%, 21.0%, 22.0%, 23.0%, 24.0%, 25%, 26.0%, 27.0%, 28.0%, 29.0%, 30%, 31.0%, 32.0%, 33.0%, 34.0%, 35%, 36.0%, 37.0%, 38.0%, 39.0%, 40% and any percentage in between.

[0027] Optionally, in the functional hyaluronic acid derivative, the total substitution degree of structure (a) where R accounts for (a), (b), (c), and (d) is preferably 5.0%-20.0%.

[0028] According to another aspect of the present application, a method for preparing the functional hyaluronic acid derivative described in any one of the above items is provided, comprising the following steps:

[0029] (1) reacting an ascorbate salt with a dihaloalkane in a molar ratio of 1-8:1 to obtain a mixed solution containing ascorbic acid ether;

[0030] (2) adding quaternary ammonium salt of hyaluronic acid to the mixed solution for reaction, wherein the molar ratio of ascorbate to tetrabutylammonium salt of hyaluronic acid is 2-10:1, thereby obtaining the functional hyaluronic acid derivative.

[0031] In the method, the crosslinking degree of the hyaluronic acid derivative is adjusted by the molar ratio of ascorbate to dihaloalkane and the molar ratio of ascorbate to hyaluronic acid tetrabutylammonium salt, and the substitution degree of the hyaluronic acid derivative is adjusted by the molar ratio of ascorbate to hyaluronic acid tetrabutylammonium salt.

[0032] For example, the molar ratio of ascorbate to dihaloalkane can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 and any ratio therebetween.

[0033] Optionally, the molar ratio of the ascorbate to the dihaloalkane is 2-6:1.

[0034] Optionally, the reaction temperature of step (1) is 20°C-60°C, preferably 20°C-40°C.

[0035] For example, the reaction temperature of step (1) can be 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C and any temperature therebetween.

[0036] Optionally, the reaction time of step (1) is 5 h-30 h, preferably the reaction time is at least 20 h-30 h.

[0037] For example, the reaction time of step (1) can be 5 h, 10 h, 15 h, 20 h, 25 h, 30 h or any time therebetween.

[0038] Optionally, the molar ratio of ascorbate to tetrabutylammonium hyaluronic acid is 2-10:1, preferably 2-6:1.

[0039] For example, the molar ratio of ascorbate to tetrabutylammonium hyaluronate can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and any ratio therebetween.

[0040] Optionally, the reaction temperature of step (2) is 25°C-60°C, preferably 35°C-55°C.

[0041] For example, the reaction temperature of step (2) can be 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C and any temperature therebetween.

[0042] Optionally, the reaction time of step (2) is 10h-30h, preferably 20h-30h.

[0043] For example, the reaction time of step (2) can be 10 h, 15 h, 20 h, 25 h, 30 h or any time therebetween.

[0044] Optionally, the ascorbate salt includes at least one of sodium ascorbate, potassium ascorbate, magnesium ascorbate, calcium ascorbate, zinc ascorbate, and ammonium ascorbate;

[0045] The dihaloalkane includes dihalo C 2-10 The alkyl group preferably includes at least one of 1,2-dichloroethane, 1,2-dichloropropane, 1,3-dichloropropane, 1,2-dichlorobutane, 1,3-dichlorobutane, 1,4-dichlorobutane, 1,2-dibromoethane, 1,2-dibromopropane, 1,3-dibromopropane, 1,2-dibromobutane, 1,3-dibromobutane, 1,4-dibromobutane, 1-bromo-2-chloroethane, 1-bromo-2-chloropropane, 1-bromo-3-chloropropane, 1-bromo-2-chlorobutane, 1-bromo-3-chlorobutane, and 1-bromo-4-chlorobutane.

[0046] The dihalogenated C 2-10 The alkyl group is a dihaloalkane group having 2 to 10 carbon atoms.

[0047] According to another aspect of the present application, a composition is provided, comprising any of the functional hyaluronate derivatives described above.

[0048] According to another aspect of the present application, provided is the use of the functional hyaluronic acid derivative described in any one of the above or the functional hyaluronic acid derivative prepared by the preparation method of the functional hyaluronic acid derivative described in any one of the above in the preparation of cosmetics and medical devices.

[0049] According to another aspect of the present application, provided is the use of the functional hyaluronic acid derivative described in any one of the above or the functional hyaluronic acid derivative prepared by the preparation method of the functional hyaluronic acid derivative described in any one of the above in repairing the skin barrier after acid damage.

[0050] The beneficial effects of this application include but are not limited to:

[0051] 1. The functional hyaluronic acid derivatives of the present application solve the shortcomings of vitamin C such as instability, strong irritation, easy yellowing, easy inactivation and short action time, so that the activity of vitamin C can be maintained, showing effective antioxidant and wrinkle improvement effects, while having the moisturizing, antioxidant and skin protection, and barrier repair effects of hyaluronic acid after acid damage.

[0052] 2. Compared with the current linear reduced hyaluronic acid esters, the functional hyaluronic acid derivatives of the present application have strong resistance to enzymatic hydrolysis, strong stability, and their solutions are not prone to opalescence and have a long storage period. At the same time, they reduce the stickiness of the product, are easy to use in formulations, and have a wider range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0054] Figure 1 This is the ultraviolet spectra of the three substances involved in Example 3 of the present application: functional hyaluronic acid derivatives, vitamin C and sodium hyaluronate.

[0055] Figure 2 The functional hyaluronic acid derivative, vitamin C (VC) and sodium hyaluronate involved in Example 3 of the present application are 13 C-NMR spectrum. DETAILED DESCRIPTION

[0056] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0057] Unless otherwise specified, the raw materials in the examples and comparative examples of the present application were purchased through commercial channels.

[0058] Unless otherwise specified, the methods used in the examples and comparative examples of the present application are conventional methods in the prior art.

[0059] Example 1

[0060] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 40 kDa as a raw material and is prepared by the method disclosed in Chinese patent CN107459590B.

[0061] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0062] (1) dissolving 1.0 mmol sodium ascorbate and 1.0 mmol ethylene dichloride in DMSO, reacting at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0063] (2) Add 0.25 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0064] Example 2

[0065] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 40 kDa as a raw material and is prepared by the method disclosed in Chinese patent CN107459590B.

[0066] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0067] (1) dissolving 2.0 mmol sodium ascorbate and 1.0 mmol dichloroethane in DMSO, reacting at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0068] (2) Add 0.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0069] Example 3

[0070] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 30 kDa as a raw material and is prepared by the method disclosed in Chinese patent CN107459590B.

[0071] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0072] (1) 6.0 mmol sodium ascorbate and 1.0 mmol dichloroethane were dissolved in DMSO and reacted at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0073] (2) Add 1.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0074] Example 4

[0075] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 40 kDa as a raw material and is prepared by the method disclosed in Chinese patent CN107459590B.

[0076] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0077] (1) 8.0 mmol sodium ascorbate and 1.0 mmol dichloroethane were dissolved in DMSO and reacted at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0078] (2) Add 2.0 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0079] Example 5

[0080] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 40 kDa as a raw material and is prepared by the method disclosed in Chinese patent CN107459590B.

[0081] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0082] (1) dissolving 6.0 mmol sodium ascorbate and 1.0 mmol ethylene dichloride in DMSO, reacting at 60° C. for 25 hours to obtain a mixed solution containing ascorbic acid ether;

[0083] (2) Add 1.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0084] Example 6

[0085] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 40 kDa as a raw material and is prepared by the method disclosed in Chinese patent CN107459590B.

[0086] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0087] (1) 6.0 mmol sodium ascorbate and 1.0 mmol dichloroethane were dissolved in DMSO and reacted at 25° C. for 5 h to obtain a mixed solution containing ascorbic acid ether;

[0088] (2) Add 1.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0089] Example 7

[0090] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 10 kDa as a raw material and using the method disclosed in Chinese patent CN107459590B.

[0091] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0092] (1) 6.0 mmol sodium ascorbate and 1.0 mmol dichloroethane were dissolved in DMSO and reacted at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0093] (2) Add 1.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0094] Example 8

[0095] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 200 kDa as a raw material and using the method disclosed in Chinese patent CN107459590B.

[0096] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0097] (1) 6.0 mmol sodium ascorbate and 1.0 mmol dichloroethane were dissolved in DMSO and reacted at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0098] (2) Add 1.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0099] Example 9

[0100] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 600 kDa as a raw material and using the method disclosed in Chinese patent CN107459590B.

[0101] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0102] (1) 6.0 mmol sodium ascorbate and 1.0 mmol dichloroethane were dissolved in DMSO and reacted at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0103] (2) Add 1.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0104] Example 10

[0105] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 40 kDa as a raw material and is prepared by the method disclosed in Chinese patent CN107459590B.

[0106] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0107] (1) 6.0 mmol potassium ascorbate and 1.0 mmol dichloroethane were dissolved in DMSO and reacted at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0108] (2) Add 1.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0109] Embodiment 11

[0110] In this embodiment, the preparation of tetrabutylammonium salt of hyaluronic acid is carried out using hyaluronic acid with a molecular weight of 40 kDa as a raw material and is prepared by the method disclosed in Chinese patent CN107459590B.

[0111] The method for preparing the functional hyaluronic acid derivative in this embodiment comprises the following steps:

[0112] (1) 6.0 mmol of tetrabutylammonium ascorbate and 1.0 mmol of dichloroethane were dissolved in DMSO and reacted at 25° C. for 25 h to obtain a mixed solution containing ascorbic acid ether;

[0113] (2) Add 1.5 mmol of tetrabutylammonium hyaluronic acid to the mixed solution and react at 45° C. for 25 h. After the reaction is completed, add ethanol to precipitate and wash the reaction product, and then vacuum dry the filtered precipitate to obtain a functional hyaluronic acid derivative powder.

[0114] Comparative Example 1

[0115] The preparation method of the linear hyaluronic acid graft copolymer powder, namely vitamin C propyl hyaluronate, is as follows.

[0116] 4.0mmol sodium ascorbate and 1.0mmol 3-bromopropanol were dissolved in water and reacted at 60°C for 8h. 2.0mmol sodium hyaluronate (molecular weight 100kDa), 3.6mmol DMAP and 4.0mmol EDC were added to the reaction solution and reacted for 10h. After the reaction, ethanol was added to precipitate the reaction product, and the filtered precipitate was vacuum dried to obtain a linear hyaluronic acid graft copolymer powder, namely vitamin C propyl hyaluronate.

[0117] After testing, the content of the obtained vitamin C propyl hyaluronate was 93.4%, the degree of substitution was 11.2%, the yield was 73.5%, and the intrinsic viscosity was 1.3 dL / g.

[0118] Test Example 1

[0119] The hyaluronic acid derivative prepared in Example 3 was subjected to full-wavelength UV scanning and NMR testing, wherein the UV test was performed using UV-1900i, and the sample test concentration was 0.02%; the NMR test was performed using a Varian INOVA-600 NMR spectrometer under the following test conditions: solvent D2O.

[0120] UV test results are as follows Figure 1 As shown, Figure 1 The figure shows the UV absorption spectra of three substances: vitamin C, functional hyaluronic acid derivatives, and sodium hyaluronate (HA). Sodium hyaluronate has no UV absorption at 230nm-400nm, vitamin C has maximum UV absorption at 265nm, and the functional hyaluronic acid derivative in the figure has maximum UV absorption at 244nm. Therefore, it can be inferred that vitamin C is grafted onto hyaluronic acid, causing the UV absorption of vitamin C to shift, thus proving that a substitution reaction has occurred.

[0121] Hyaluronic acid derivatives 13 C-NMR spectrum is shown in Figure 2 , Figure 2 HA refers to sodium hyaluronate 13 C-NMR spectrum. The functional hyaluronic acid derivative is prepared by reacting the carboxyl group of hyaluronic acid with the hydroxyl group on the carbon 3 of vitamin C (VC) through an organic linking group. 13 In the C-NMR spectrum, the chemical shift of the carboxyl carbon of hyaluronic acid decreases, the chemical shift of the 3rd carbon of vitamin C decreases, and the chemical shift of the 2nd carbon of vitamin C increases.

[0122] Test Example 2

[0123] The functional hyaluronic acid derivatives prepared above were tested for degree of substitution, degree of cross-linking, intrinsic viscosity, purity and yield. The test results are shown in Table 1. The specific test method is as follows:

[0124] (I) Crosslinking degree test:

[0125] 1. Instruments and equipment: gas chromatograph (Agilent 7890B), electronic balance (Mettler Toledo xse105), volumetric flask.

[0126] 2. Solution preparation

[0127] 1) Test solution: weigh W s 0.1 g of the test sample was dissolved in 10 ml of 0.2 M sodium hydroxide solution and subjected to alkaline hydrolysis at 80 °C for 4 h.

[0128] 2) Dilution of test solution: Measure 1 ml of sample into a 10 ml volumetric flask, make up to volume with ethanol, filter and prepare for injection.

[0129] 3) Reference solution: weigh W i 0.1g of 1,3-propylene glycol was placed in a 100ml volumetric flask and fixed to volume with ethanol to obtain a stock solution. When injecting samples, the stock solution was gradually diluted to 100ppm as the reference solution.

[0130] 3. The amount of 1,3-propylene glycol in the test solution is:

[0131]

[0132] Where N1:W s The amount of 1,3-propanediol in the test sample after alkaline hydrolysis, mol;

[0133] A i : Peak area of ​​100ppm 1,3-propylene glycol reference solution;

[0134] A s : Peak area after dilution of the test solution;

[0135] M4: 1,3-propylene glycol molar mass 76 g / mol;

[0136] V: volume of the test solution after dilution;

[0137] 100ppm: The concentration of the reference substance 1,3-propylene glycol solution after dilution.

[0138] 4. Crosslinking degree C is:

[0139]

[0140] Where N1:W s The amount of 1,3-propanediol in the test sample after alkaline hydrolysis, mol;

[0141] W s Weigh the sample for the test article;

[0142] M5 is uronic acid with a molar mass of 194 g / mol;

[0143] G is the uronic acid content;

[0144] h is the drying loss of the test sample.

[0145] (II) Degree of substitution test

[0146] 1. Instruments and equipment: UV spectrophotometer (UV-1900i), electronic balance (Mettler Toledo ME204E), volumetric flask

[0147] 2. Preparation of stock solution

[0148] Weigh 0.01 g of vitamin C ethyl ether, place it in a 50 ml volumetric flask, dissolve it to the mark with purified water, shake well, and use it as the stock solution.

[0149] 3. Preparation of standard solution

[0150] Take 1ml, 2ml, 3ml and 4ml of the stock solution and place them in 50ml volumetric flasks respectively, dilute to the scale with purified water, shake well and use as standard solutions.

[0151] 4. Preparation of test solution

[0152] Weigh about W of the test sample g 0.01 g was placed in a 50 ml volumetric flask, dissolved to the mark with purified water, and shaken well to serve as the test solution.

[0153] 5. Determination of standard curve

[0154] After adjusting to zero with purified water, each standard solution was tested with an ultraviolet spectrophotometer at 244 nm, and linear regression was performed to draw a standard curve and obtain a linear equation.

[0155] 6. Determination

[0156] Use an ultraviolet spectrophotometer to test the test solution, obtain the absorbance value, substitute it into the linear equation, and obtain the corresponding vitamin C ethyl ether concentration.

[0157] 7. Calculation of degree of substitution

[0158]

[0159] in

[0160] Where C (mol / L) is the corresponding vitamin C ethyl ether concentration of the test solution;

[0161] M1 is the molar mass of the modified disaccharide unit of hyaluronic acid, 595 g / mol;

[0162] M2 is the molar mass of the unmodified disaccharide unit of hyaluronic acid, 401 g / mol;

[0163] M3 is a hyaluronic acid cross-linked structural unit with a molar mass of 798 g / mol;

[0164] N2 is the amount of propyl substance in the cross-linked structural unit of hyaluronic acid (equal to the amount of 1,3-propylene glycol substance tested after alkaline hydrolysis);

[0165] h is the drying loss of the test sample;

[0166] 0.05 is the volume of the test sample dissolved in the volumetric flask, 0.05 L;

[0167] N1:W sThe amount of 1,3-propanediol in the test sample after alkaline hydrolysis, mol;

[0168] W g is the sample weight of the test product, g;

[0169] W s This is the sample weight for alkaline hydrolysis test of 1,3-propylene glycol content, g.

[0170] (III) Intrinsic viscosity:

[0171] 1. Instruments and equipment: Ubbelohde viscometer, electronic balance (Mettler Toledo ME204E), volumetric flask

[0172] 2. Reagents

[0173] 0.2mol / L sodium chloride solution: weigh 11.688g of sodium chloride and dissolve it in water to 1000ml.

[0174] 3. Test solution

[0175] Accurately weigh about 0.500g (W1) of the test sample (Note: t1 / t0 should be between 1.3 and 1.5, otherwise the concentration of the test solution should be adjusted before measurement) into a 100ml volumetric flask, add an appropriate amount of 0.2mol / L sodium chloride solution, place on an oscillator to dissolve, then dilute to the scale, shake well, and use as the test solution.

[0176] 4. Determination of solvent outflow time (t0):

[0177] Repeat the measurement at least twice, the two measured values ​​should be the same, and t0 should be greater than 100 seconds.

[0178] 5. Determination of test solution outflow time (t1):

[0179] Take an appropriate amount of the test solution filtered through a No. 3 vertical fritted glass funnel and measure it according to the method for measuring the solvent effluent time. Repeat the measurement at least twice. The difference between the two measured values ​​should not exceed 0.2 seconds. The average of the two values ​​is the effluent time (t1) of the test solution.

[0180] 6. Calculation:

[0181] Calculate the test solution concentration C (g / dL) according to formula (1):

[0182]

[0183] Where: h (%) = loss on drying of the test sample

[0184] ρ 25 =1.000g / ml (density of the test solution at 25°C)

[0185] The intrinsic viscosity is calculated according to formula (2):

[0186]

[0187] Where: t1 is the average outflow time of the test solution, seconds

[0188] t0 is the solvent elution time, seconds

[0189] C is the concentration of the test solution (calculated on the basis of the dry product), g / dL.

[0190] (IV) Content:

[0191] 1. Instruments and equipment: UV spectrophotometer (UV-1900i), electronic balance (Mettler Toledo ME204E), induction cooker

[0192] 2. Reagents

[0193] 0.025mol / L borax-sulfuric acid solution: Weigh 4.77g borax, dissolve it in 500ml concentrated sulfuric acid (super pure), and shake well.

[0194] Carbazole test solution: weigh 0.125g of carbazole and add 100ml of anhydrous ethanol, shake to dissolve it, and the solution should be placed in a brown glass bottle, tightly plugged, and stored in a dark place.

[0195] 3. Reference substances

[0196] D-Glucuronic acid

[0197] 4. Preparation of reference stock solution

[0198] Accurately weigh about 0.100 g of glucuronic acid reference substance (W) which has been dried at 105°C in vacuum with phosphorus pentoxide as a desiccant to a constant weight. s ), put it in a 100ml volumetric flask, add water to dissolve, dilute to scale, shake well, and use it as the reference stock solution. Accurately measure 5.0ml of the stock solution, put it in a 100ml volumetric flask, add water to make a solution containing 50μg per 1ml, shake well, and calculate the concentration of the glucuronic acid reference solution according to the following formula.

[0199]

[0200] Where W s is the sample weight of the glucuronic acid reference substance, in g; Z is the content of the glucuronic acid reference substance.

[0201] 5. Preparation of test solution

[0202] Weigh about 0.10g (W1) of the product (weigh at the same time as the drying loss) accurately and place it in a 100ml volumetric flask, add appropriate amount of water to swell the sample, add water to the scale after it is completely dissolved, and shake well. Then weigh about 4.0g (W2) (the density of the solution is calculated as 1.000g / ml, which is equivalent to 4.00ml) and place it in a 50ml volumetric flask, add water to dilute to the scale, shake well, and use it as the test solution.

[0203] 6. Preparation of standard curve

[0204] Accurately measure 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the reference solution, place them in stoppered test tubes respectively, add water to 1.0 ml each, mix well, cool to below 4°C, slowly drop 5.0 ml of 0.025 mol / L borax sulfuric acid solution, mix well, heat in a boiling water bath for 10 minutes, and cool in ice water. Accurately add 0.20 ml of carbazole test solution, shake well, heat in a boiling water bath for 15 minutes, and cool in ice water. According to the operating procedures of the UV-visible spectrophotometer, measure the absorbance at a wavelength of 530 nm, and use the concentration of glucuronic acid (μg / ml) and the absorbance to display the regression equation and correlation coefficient (R required) on the instrument. 2 >0.990).

[0205] 7. Sample determination

[0206] Accurately measure 1.0 ml of the test solution and prepare two test tubes in parallel. Place them in stoppered test tubes and operate in the same way as under the standard curve starting from "cool to below 4°C". The concentration of glucuronic acid (Ci) (μg / ml) is obtained by the regression equation.

[0207] A standard curve was prepared using the concentration of glucuronic acid in each tube of the control solution and the corresponding absorbance. The glucuronic acid concentration Cg (μg / g) of the sample solution was obtained by the regression equation based on the absorbance of the sample solution.

[0208] 8. Calculation

[0209]

[0210] Where Ci is the glucuronic acid concentration obtained by the regression equation, μg / ml;

[0211] W1 is the amount of test sample weighed, g;

[0212] W2 is the amount of sample weighed for the second dilution, g;

[0213] h (%) is the drying loss of the test sample;

[0214] 9. Content calculation

[0215]

[0216] Wherein G (%) is the glucuronic acid content;

[0217] M1 is the molar mass of the modified disaccharide unit of hyaluronic acid, 595 g / mol;

[0218] M2 is the molar mass of the unmodified disaccharide unit of hyaluronic acid, 401 g / mol;

[0219] M3 is a hyaluronic acid cross-linked structural unit with a molar mass of 798 g / mol;

[0220] N2 is the amount of propyl substance in the cross-linked structural unit of hyaluronic acid (equal to the amount of 1,3-propylene glycol substance tested after alkaline hydrolysis);

[0221] D is the degree of substitution;

[0222] M is the molar mass of glucuronic acid, 194 g / mol.

[0223]

[0224] m1 is the mass of hyaluronic acid derivative powder after drying;

[0225] m2 is the mass of tetrabutylammonium hyaluronic acid salt.

[0226] Table 1

[0227]

[0228]

[0229] Test Example 3 Moisturizing Efficacy Evaluation

[0230] Principle: The moisturizing effect of the skin moisturizing system from the inside out can be summarized as: water capture, water lock, water activation and water storage. Water storage: The hyaluronic acid in the dermis (synthesized by hyaluronan synthase (HAS)) can absorb a large amount of water, like a water reservoir for the skin, providing reserves for the moisture of the entire skin, and also filling the dermis to make the dermis structure more complete. Activated water: Aquaporins (AQP) transport small molecules such as water and glycerol across the membrane and participate in epidermal hydration. This process is called activated water. Water lock: Tight junction proteins connect adjacent keratinocytes like a zipper, closing the gaps between cells and thus preventing excessive evaporation of water through the cell bypass. Keratin, a marker protein of cornification terminal, and filaggrin (FLG) together form the strength and shape of keratinocytes, and bind to lipids through covalent bonds, jointly promoting a tighter and more complete morphology of the stratum corneum and preventing excessive loss of water to the outside world. Water capture: FLG eventually forms a natural moisturizing factor to help cells capture water from the outside. Therefore, this test case evaluates whether the sample has moisturizing effect by detecting the relative expression of the AQP3 gene. The specific operation is as follows:

[0231] 1. Cells were seeded into 6-well plates (6×10 5 cells / well), with 1 mL of solution per well, and incubated at 37°C, 5% CO2 for 24 h.

[0232] 2. Administration: The normal control group was replaced with fresh complete culture medium, and the complete culture medium containing the sample (using Example 3 as the experimental sample) was added to the sample wells, 1 mL per well, and incubated at 37° C., 5% CO 2 for 24 h.

[0233] 3. Extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and target genes.

[0234] 4. Use β-actin as an internal reference for gene expression and calculate the relative RNA expression of the target gene.

[0235] Relative RNA expression = 2 ΔΔC(t)

[0236]

[0237] ΔC(t)=C(t) 目的基因 -C(t) β-actin

[0238] The test results are shown in Table 2:

[0239] Table 2

[0240]

[0241] Test Example 4 Evaluation of Antioxidant Efficacy

[0242] Principle: UVA+UVB is used to damage human epidermal keratinocytes HaCaT, simulating the damage caused by sunlight to the epidermis and stimulating the production of reactive oxygen free radicals (ROS). The cells are contacted with samples before and after irradiation, and the ROS in the cells are labeled with the fluorescent probe DCFH-DA. The content of ROS in the cells is detected by flow cytometry to examine whether the samples have the ability to inhibit the production of ROS or eliminate ROS.

[0243] 1. Contact with samples after UV damage (repair)

[0244] 1.1 Plating: HaCaT cells in the logarithmic growth phase were plated at 1×10 5 The cells were inoculated at a density of 100 cells / mL in a 12-well culture plate, with 1 mL of cell suspension in each well, and cultured in a carbon dioxide incubator at 37°C and 5% CO2 for 24 hours.

[0245] 1.2 Establishment of injury model: The irradiation group was covered with plastic wrap and irradiated with UVA at an intensity of 2000 μW / cm2 for 1 h (the irradiation time was adjusted according to the cell state), and UVB at an intensity of 700 μW / cm 2 The mice were irradiated with an intensity of 100 nm for 3 min, and the normal control group was covered with aluminum foil and not irradiated.

[0246] 1.3 Drug addition detection: After irradiation, discard the old culture medium, add the sample solution (using Example 3 as the experimental sample) to the experimental group, and add serum-free culture medium to the normal control group, 1 mL per well, continue to culture for 24 hours, discard all the culture medium, and wash twice with PBS. Add 1.5 mL DCFH-DA (dichlorofluorescein diacetate, prepared as: diluted at a ratio of 0.375 uL DCFH-DA: 1 mL PBS) to each well, put it in a cell culture incubator and continue to incubate for 30 minutes, mix every 5 minutes to allow the probe to fully bind. Discard the probe, wash twice with preheated serum-free culture medium, add 1 ml serum-free culture medium to each well and incubate at 37 ° C for 10 minutes. After washing once with PBS, trypsinize the cells, wash twice with PBS, resuspend in 300 μL PBS, and detect with a flow cytometer (cells need to be filtered before being put on the machine), FITC-A channel, and collect 10,000 cells for each sample.

[0247] 2. Contact with samples before UV damage (protection)

[0248] 2.1 Plating: HaCaT cells in the logarithmic growth phase were plated at 1×10 5 The cells were inoculated at a density of 100 cells / mL in a 12-well culture plate, with 1 mL of cell suspension in each well, and cultured in a carbon dioxide incubator at 37°C and 5% CO2 for 24 hours.

[0249] 2.2 Sample addition treatment: discard the old culture medium, add the sample solution to the experimental group (using Example 3 as the experimental sample), and add serum-free culture medium to the normal control group, 1 mL per well. Continue to culture for 24 hours and then irradiate. The normal control group is covered with aluminum foil and not irradiated.

[0250] 2.3 Establishment and detection of injury model: The irradiation group was covered with plastic wrap, and UVA was irradiated with an intensity of 2000μW / cm2 for 1h (the irradiation time was adjusted according to the cell state), and UVB was irradiated with an intensity of 700μW / cm2 for 3min. The normal control group was not irradiated. The culture medium was discarded and washed twice with PBS. 1.5mL DCFH-DA was added to each well and placed in a cell culture incubator for further incubation for 30min. Mix every 5min to allow the probe to fully bind. The probe was discarded, washed twice with preheated serum-free culture medium, and 1ml serum-free culture medium was added to each well and incubated at 37℃ for 10min. After washing once with PBS, the cells were digested with trypsin, washed twice with PBS, resuspended with 300μL PBS, and detected by flow cytometry (cells need to be filtered before loading), FL1-H channel, and 10,000 cells were collected for each sample.

[0251] The test results are shown in Table 3:

[0252] Table 3

[0253]

[0254] Test Example 5 Evaluation of Skin Protection Efficacy

[0255] Principle: This experiment uses mouse mononuclear macrophages (RAW264.7). After the samples are applied to the cells, the relative expression levels of IL-6, IL-1β, and TNF-α genes are detected to evaluate whether the samples have an inhibitory effect on the three factors.

[0256] 1. Inoculate cells into 6-well plates (6×10 5 / well) and incubated at 37°C, 5% CO2 for 24 h.

[0257] 2. Modeling: After the incubation, the culture medium was removed, and the cells were washed 1-2 times with D-Hanks. The normal control group was replaced with fresh culture medium, the model control group was replaced with fresh culture medium containing the modeling agent (LPS, 0.5 μg / mL), and the sample group was replaced with fresh culture medium containing the sample (using Example 3 as the experimental sample) and the modeling agent (LPS, 0.5 μg / mL). The cells were incubated at 37°C, 5% CO2 for 24 h.

[0258] 3. Extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and target genes.

[0259] 4. Use β-actin as an internal reference for gene expression and calculate the relative RNA expression of the target gene.

[0260] Relative RNA expression = 2 ΔΔC(t)

[0261]

[0262] The test results of the samples of Example 3 are shown in Table 4:

[0263] Table 4

[0264]

[0265]

[0266] Test Example 6: Stability Evaluation

[0267] Principle: 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable long-lived free radical. Its ethanol solution is dark purple and has strong absorption near 517nm. When there is a free radical scavenger, the light absorption of DPPH ethanol solution is weakened due to its single electron pairing. The degree of fading of DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the antioxidant activity.

[0268] Experimental methods and procedures:

[0269] DPPH ethanol solution: accurately weigh 12 mg of DPPH into a 100 mL volumetric flask, add an appropriate amount of anhydrous ethanol to dissolve it, make up to volume, shake well, and set aside;

[0270] Test solution: Accurately weigh 0.64 g of the sample from Example 3 into a 50 mL beaker, add 20 mL of purified water, and stir to dissolve to obtain a 32 mg / mL sample solution;

[0271] Test solution: Accurately weigh 0.64 g of the sample of comparative example 1 into a 50 mL beaker, add 20 mL of purified water, stir to dissolve, and obtain a 32 mg / mL sample solution;

[0272] Test solution: Accurately weigh 0.0364 g of vitamin C sample into a 50 mL beaker, add 20 mL of purified water, stir to dissolve, and obtain a 1.82 mg / mL (converted according to the vitamin C content in Example 3) sample solution;

[0273] Use 10mL test tubes to set up sample tube (T), sample background (T0), DPPH tube (C) and solvent background (C0). Add 1mL of sample solution of the same concentration to each of the sample tube (T) and sample background (T0); add solvent to all test tubes (T, T0, C, C0), and use water for water-soluble samples to make up to 3mL, and mix well; add 1mL of DPPH ethanol solution to the sample tube (T) and DPPH tube (C), replace the sample background (T0) and solvent background (C0) with 95% ethanol, shake gently, and let stand at room temperature for 5 minutes. Transfer each reaction solution into a cuvette and measure the absorbance at 517nm.

[0274] Table 5 Sample addition requirements

[0275]

[0276] Calculation of DPPH clearance:

[0277]

[0278] Wherein T is the absorbance of the sample tube, i.e., the absorbance of the solution after the sample reacts with DPPH; T is the background absorbance of the sample;

[0279] TComparative Example 1 is the absorbance value of the sample tube, i.e., the absorbance value of the solution after the sample reacts with DPPH;

[0280] T0 comparative example 1 is the background absorbance value of the sample;

[0281] T Vitamin C is the absorbance value of the sample tube, that is, the absorbance value of the solution after the sample reacts with DPPH;

[0282] T0 vitamin C is the background absorbance of the sample;

[0283] C is the average of three absorbance values ​​of the DPPH tube, that is, the absorbance value of the DPPH solution when no sample is added;

[0284] C0 is the background absorbance of the solvent;

[0285] Table 6

[0286]

[0287] Note: ***, compared with comparative example 1, p<0.001.

[0288] Test Example 7

[0289] 1. Valid subjects: 15 people in total, including 3 males and 12 females, aged 22 to 59 years old, with an average age of 36.27±2.77 years old, who met the voluntary inclusion criteria.

[0290] Subject inclusion / exclusion criteria

[0291] 18-60 years old, both men and women;

[0292] Those who have not used antihistamines in the past week or immunosuppressants in the past month;

[0293] Those who have not used any anti-inflammatory drugs in the tested area in the past two months;

[0294] Patients with non-insulin-dependent diabetes mellitus;

[0295] Women who are not breastfeeding or pregnant;

[0296] Those whose test sites have not participated in other clinical trials now or in the past three months;

[0297] The skin to be tested has no scars, moles or other marks that may affect the test results;

[0298] Volunteer to participate in this trial and be able to complete the required content according to the trial requirements.

[0299] 2. Materials and Equipment:

[0300] Instruments and equipment: TM Hex.

[0301] Test samples:

[0302] Table 7

[0303]

[0304] Note: The molecular weight of low molecular weight sodium hyaluronate (HA-TLM for short) sample is similar to that of VCHA.

[0305] 3. Testing environment: temperature 20℃~22℃, humidity 40~60%.

[0306] 4. Inspection method:

[0307] 1) After acid brushing repair set:

[0308] Step 1: Acid brushing and modeling

[0309] Subjects aged 18-60 years old and meeting the project requirements were selected. The subjects used 15% citric acid for modeling, specifically applied to the inner side of the forearm, 4 to 5 times a day, 0.1 ml each time, for 7 consecutive days, and tested the transepidermal water loss value (TEWL value). The modeling was considered successful when the transepidermal water loss was ≥5g / m2 / h.

[0310] Step 2: Apply the sample

[0311] After successful modeling, the subjects applied Essence 1 and Essence 2 to the inner side of their forearms, respectively, and used the products for 7 consecutive days. The TEWL values ​​at 0d, 2h, 2d, and 7d after using the samples were monitored.

[0312] 2) Gentle Skin Renewal and Repair Kit

[0313] Step 1: Acid brushing and modeling

[0314] Subjects aged 18-60 years old and meeting the project requirements were selected (see Appendix 2 for specific standards). The subjects were modeled with 15% citric acid, which was applied to the inner side of the forearm 4 to 5 times a day, 0.1 ml each time, for 7 consecutive days, and the TEWL value was tested. The model was considered successful when the transepidermal water loss was ≥5g / m2 / h.

[0315] Step 2: Apply the sample

[0316] After successful modeling, the subjects applied Essence 3, Essence 4, Essence 5 and Essence 6 to the inner side of their forearms, respectively, and used the products for 7 consecutive days. The TEWL values ​​at 0d, 2h, 2d, and 7d after using the samples were monitored.

[0317] 5. Statistical methods:

[0318] EXCEL software was used to perform descriptive statistics on each measurement value, including quantity, mean, minimum and maximum values.

[0319] SPSS analysis software was used to compare the measured values ​​at different time points in the same test area with the baseline value, and the measured values ​​at the same time points in different test areas were compared. The Shapiro-Wilk Test was used to test the significance of the normal distribution of the difference. If Sig. (bilateral)>0.05, it was normally distributed, and a paired t test was performed, and the significant difference level α was 0.05. If Sig. (bilateral)<0.05, it was non-normally distributed, and a Wilcoxon signed rank test was performed, and the significant difference level α was 0.05.

[0320] 6. Test results:

[0321] The test results of the transepidermal water loss values ​​of the subjects' skin for each essence are shown in Table 8.

[0322] Table 8 Results of transepidermal water loss values ​​of each essence

[0323]

[0324] Note: The smaller the transepidermal water loss value, the better the sample effect, and vice versa.

[0325] * indicates the comparison with the initial value, P < 0.05, ** indicates the comparison with the initial value, P < 0.01, *** indicates the comparison with the initial value, P < 0.001;

[0326] # indicates P<0.05 compared with essence 1, ## indicates P<0.01 compared with essence 1, ### indicates P<0.001 compared with essence 1;

[0327] & indicates P<0.05 compared with essence 4, && indicates P<0.01 compared with essence 4, &&& indicates P<0.001 compared with essence 4;

[0328] Result analysis:

[0329] After the subjects used the test samples for 2 days, the transepidermal water loss value of essence 1 (placebo group) decreased by 17.64% compared with before the use of the samples, and the transepidermal water loss value of essence 2 (VCHA group) decreased by 30.04% compared with before the use of the samples. Compared with essence 1 and essence 2, the difference in the transepidermal water loss value of essence 2 after 2 days of use and before use was lower than that of essence 1 (P < 0.001); after the subjects used the test samples for 7 days, the transepidermal water loss value of essence 1 decreased by 28.76% compared with before the use of the samples, and the transepidermal water loss value of essence 2 decreased by 44.32% compared with before the use of the samples. Compared with essence 1 and essence 2, the difference in the transepidermal water loss value of essence 2 after 7 days of use and before use was lower than that of essence 1 (P < 0.001).

[0330] After the subjects used the test samples for 2 hours, the transepidermal water loss value of Essence 3 (citric acid control group) decreased by 5.12% compared with that before the samples were used, the transepidermal water loss value of Essence 4 (citric acid + VCHA group) decreased by 7.87% compared with that before the samples were used, the transepidermal water loss value of Essence 5 (citric acid + HA-TLM group) decreased by 9.30% compared with that before the samples were used, and the transepidermal water loss value of Essence 6 (citric acid + VC group) decreased by 5.94% compared with that before the samples were used;

[0331] After the subjects used the test samples for 2 days, the transepidermal water loss value of Essence 3 decreased by 19.41% compared with that before the samples were used, the transepidermal water loss value of Essence 4 decreased by 22.74% compared with that before the samples were used, the transepidermal water loss value of Essence 5 decreased by 19.41% compared with that before the samples were used, and the transepidermal water loss value of Essence 6 decreased by 15.21% compared with that before the samples were used; compared with Essence 6, the difference between the transepidermal water loss value of Essence 4 and that before the use of Essence 6 after 2 days of use was lower than that of Essence 6 (P=0.001);

[0332] After the subjects used the test samples for 7 days, the transepidermal water loss value of Essence 3 decreased by 24.41% compared with before the use of the samples, the transepidermal water loss value of Essence 4 decreased by 44.29% compared with before the use of the samples, the transepidermal water loss value of Essence 5 decreased by 28.70% compared with before the use of the samples, and the transepidermal water loss value of Essence 6 decreased by 23.92% compared with before the use of the samples.

[0333] Compared with Essence 3, the difference of transepidermal water loss value of Essence 4 after 7 days of use was lower than that of Essence 3 (P<0.001); compared with Essence 5, the difference of transepidermal water loss value of Essence 4 after 7 days of use was lower than that of Essence 5 (P<0.05); compared with Essence 4 and Essence 6, the difference of transepidermal water loss value of Essence 4 after 7 days of use was lower than that of Essence 6 (P<0.001).

[0334] The above data show that Essence 2 has a repairing effect, and the repairing effect is better than Essence 1, that is, based on the citric acid brushing model, the essence containing 0.1% VCHA has a repairing effect on acid brushing damage; Essence 4 has a repairing effect, and the skin repairing effect is better than Essence 3, 5 and 6. That is, simulating the daily acid brushing skin care scene, compared with the 2% citric acid group, 0.1% VCHA and 2% citric acid can reduce transepidermal water loss when used in the same essence formula, indicating that it has a repairing effect on acid-damaged skin, and this effect is better than the 0.1% VC essence group and the 0.1% low molecular weight sodium hyaluronate (HA-TLM) group.

[0335] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A functional hyaluronic acid derivative, characterized in that: The structural formula of the functional hyaluronic acid derivative is as follows: Where n≥4, the R is selected from one of the following four structures (a), (b), (c), (d), and in the same long-chain molecule, there is no situation where all of them are a single structure of (a), (b), (c), or (d), (a): (b): metal ion or ammonium ion, (c): H, (d): alkyl group; (a) represents the junction site; When R is selected from (d) an alkyl group, the alkyl group is connected as a connection point to a carboxyl group on another molecular chain containing a disaccharide structural unit of hyaluronic acid through an ester bond.

2. The functional hyaluronic acid derivative according to claim 1, characterized in that The cross-linking degree of the functional hyaluronic acid derivative is 0.1%-25.0%.

3. The functional hyaluronic acid derivative according to claim 1, characterized in that The intrinsic viscosity of the functional hyaluronic acid derivative is less than or equal to 5.0 dL / g.

4. The functional hyaluronic acid derivative according to claim 1, characterized in that The molecular weight of the hyaluronic acid is 1 kDa-600 kDa.

5. The functional hyaluronic acid derivative according to claim 1, characterized in that In the functional hyaluronic acid derivative, the structure R (a) accounts for 1.0%-40.0% of the total substitution degree of (a), (b), (c) and (d).

6. The method for preparing a functional hyaluronic acid derivative according to any one of claims 1 to 5, characterized in that: The steps include: (1) reacting an ascorbate salt with a dihaloalkane in a molar ratio of 1-8:1 to obtain a mixed solution containing ascorbic acid ether; (2) adding quaternary ammonium salt of hyaluronic acid to the mixed solution for reaction, wherein the molar ratio of ascorbate to tetrabutylammonium salt of hyaluronic acid is 2-10:1, thereby obtaining the functional hyaluronic acid derivative.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the ascorbate to the dihaloalkane is 2-6:1; The molar ratio of the ascorbate to the tetrabutylammonium hyaluronic acid salt is 2-6:

1.

8. The preparation method according to claim 5, characterized in that: The ascorbate salt includes at least one of sodium ascorbate, potassium ascorbate, magnesium ascorbate, calcium ascorbate, zinc ascorbate, and ammonium ascorbate; The dihaloalkane includes dihalo C 2-10 alkyl.

9. A composition comprising the hyaluronate derivative according to any one of claims 1 to 4.

10. Use of the functional hyaluronic acid derivatives prepared by the method for preparing the functional hyaluronic acid derivatives according to any one of claims 1 to 4 or the functional hyaluronic acid derivatives according to any one of claims 5 to 8 in the preparation of cosmetics and medical devices; preferably, the use in the preparation of cosmetics and medical devices includes the repair of skin barrier damage caused by acid.

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