Grafted compounds of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof, and methods of making and uses thereof

Abstract

The present application provides a grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof and a preparation method thereof, and it is found that the graft has a synergistic effect in inhibiting IL-6 expression, and can prevent or relieve skin discomfort caused by inflammation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof, and a preparation method and use thereof. BACKGROUND

[0002] In skin care product formulations, hyaluronic acid or a salt thereof is often used in combination with other active substances, however, in the case of combination, there can be cases where the active substances cannot fully exert their efficacy. SUMMARY

[0003] The inventors of the present application have found that a grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof having a certain structure can effectively inhibit the expression of IL-6, thereby completing the present application.

[0004] The technical solution of the present application is as follows:

[0005] 1. A grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof, wherein at least a part of the carboxyl groups of the hyaluronic acid or a salt thereof are connected to the amino groups of the glutamic acid or a salt thereof in the form of an amide bond.

[0006] 2. The grafted compound according to item 1, having a general formula as shown in formula (I):

[0007]

[0008] wherein in formula (I), R is a metal ion or H, 0≤x<1, 0

[0009] 3. The grafted compound according to item 1, wherein,

[0010] the grafting rate of the grafted compound is 20% to 100%.

[0011] 4. The grafted compound according to item 1, wherein,

[0012] the molecular weight of the grafted compound is 3k-500kDa.

[0013] 5. The grafted compound according to item 1, wherein,

[0014] the grafted compound has a 1 characteristic peak at a chemical shift δ of 4.00-4.15ppm in a H NMR spectrum obtained by nuclear magnetic resonance detection;

[0015] Preferably,

[0016] the grafted compound has a 1The grafting compound has characteristic peaks at chemical shifts of 2.10-2.25 ppm and 1.71-2.10 ppm in the H NMR spectrum.

[0017] 6. A method for preparing the grafting compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof according to any one of items 1-5, wherein,

[0018] Preparation of the quaternary ammonium base salt of hyaluronic acid or a salt thereof: hyaluronic acid or a salt thereof and a quaternary ammonium base are mixed to obtain the quaternary ammonium base salt of hyaluronic acid or a salt thereof;

[0019] Preparation of the grafting compound: the quaternary ammonium base salt of hyaluronic acid or a salt thereof is dissolved, and then glutamic acid or a salt thereof, an activating agent, and a catalyst are added to react, and the product is hydrolyzed in an alkaline environment, and the pH is adjusted to obtain the grafting compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof.

[0020] 7. A cosmetic composition comprising the grafting compound according to any one of items 1-5 or prepared by the method of item 6.

[0021] 8. Use of the grafting compound according to any one of items 1-5 or prepared by the method of item 6 in cosmetics.

[0022] 9. Use of the grafting compound according to any one of items 1-5 or prepared by the method of item 6 or the cosmetic composition of item 7 in preventing and / or relieving skin discomfort caused by inflammation.

[0023] 10. Use of the grafting compound according to any one of items 1-5 or prepared by the method of item 6 or the cosmetic composition of item 7 in anti-inflammation.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] The grafting compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof provided by the application can effectively exert the effect of anti-inflammatory skin care, and expand the application range of the grafting compound.

[0026] Further, the application obtains a preferred structure of the new raw material when used for inhibiting IL-6 expression. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 shows the H NMR spectrum of Example 6. 1 H NMR spectrum. DETAILED DESCRIPTION

[0028] The application will be further described below in conjunction with examples, which should be understood as merely further illustrating and explaining the application, and not limiting the application.

[0029] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described below. However, if there is a conflict between the definitions in the specification including that of the priority document, the priority document, and the incoφoration by reference, the specification controls. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0030] The present application provides a grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof, wherein at least a portion of carboxyl groups of the hyaluronic acid or the salt thereof is connected to the amino group of the glutamic acid or the salt thereof by an amide bond.

[0031] The grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof described in the present application can make the hyaluronic acid or the salt thereof and the glutamic acid or the salt thereof act on the skin cells simultaneously, and can achieve a better effect compared to a physical mixture of the two.

[0032] The degree of substitution refers to a molar ratio of the glutamic acid or the salt thereof connected to the hyaluronic acid or the salt thereof by an amide bond to the hyaluronic acid or the salt thereof double sugar structure.

[0033] The hyaluronic acid salt encompasses unmodified hyaluronic acid salt or modified hyaluronic acid salt (e.g., acetylated hyaluronic acid salt). For example, it can be sodium salt, zinc salt, calcium salt, magnesium salt, etc. of hyaluronic acid, or any combination of these salts.

[0034] The glutamic acid salt encompasses unmodified glutamic acid salt or modified glutamic acid salt. For example, it can be hydrochloride, acetate, sulfonate, etc. of glutamic acid, or any combination of these salts.

[0035] In some embodiments, the grafted compound has a general formula as shown in formula (I):

[0036]

[0037] In formula (I), R is a metal ion or H, 0≤x<1, 0

[0038] For example, x can be 0, 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, 0.99, or any value therebetween;

[0039] y 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, or any value therebetween;

[0040] n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or any positive integer therebetween.

[0041] In some embodiments of the present application, the grafting ratio can be represented by y / x+y.

[0042] In some embodiments of the present application, the grafting ratio is determined by nuclear magnetic resonance spectroscopy 1 H NMR; wherein the grafting ratio is determined by nuclear magnetic resonance 1 H NMR spectrum of the grafting compound, or is calculated by 1 H NMR spectrum of the grafting compound, or is calculated by

[0043] In some embodiments of the present application, the grafting rate of the grafting compound is 20% to 100%, for example, it can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therebetween.

[0044] In some embodiments of the present application, the molecular weight of the grafting compound is 3k to 500kDa; for example, the molecular weight of the grafting compound can be 3kDa, 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, 60kDa, 65kDa, 70kDa, 75kDa, 80kDa, 85kDa, 90kDa, 95kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa, 360kDa, 370kDa, 380kDa, 390kDa, 400kDa, 410kDa, 420kDa, 430kDa, 440kDa, 450kDa, 460kDa, 470kDa, 480kDa, 490kDa, 500kDa or any range therebetween.

[0045] In some embodiments of the present application, the grafting compound has characteristic peaks at chemical shifts δ of 4.00-4.15ppm in the HNMR spectrum; preferably, the grafting compound has characteristic peaks at chemical shifts δ of 4.00-4.15ppm in the HNMR spectrum. 1 HNMR spectrum, the grafting compound has characteristic peaks at chemical shifts δ of 4.00-4.15ppm; preferably, the grafting compound has characteristic peaks at chemical shifts δ of 4.00-4.15ppm in the HNMR spectrum. 1 HNMR spectrum, the grafting compound has characteristic peaks at chemical shifts δ of 2.10-2.25ppm and 1.71-2.10ppm.

[0046] In one specific embodiment of the present application, in the grafting compound, at least a part of the carboxyl groups of the hyaluronic acid or its salt are connected to the amino groups of glutamic acid or its salt in the form of amide bond, the grafting rate of the grafting compound is 20% to 95%, and the general formula of the grafting compound is shown in formula (I):

[0047]

[0048] wherein R in formula (I) is a metal ion or H, 0≤x<1, 0

[0049] In one embodiment of the present application, at least a part of the carboxyl groups of the hyaluronic acid or salt thereof in the grafted compound are connected to the amino group of glutamic acid or salt thereof in the form of an amide bond, the grafting rate of the grafted compound is 20% to 100%, the molecular weight of the grafted compound is 3k to 500kDa, and the grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15ppm in the H NMR spectrum. 1 In one embodiment of the present application, at least a part of the carboxyl groups of the hyaluronic acid or salt thereof in the grafted compound are connected to the amino group of glutamic acid or salt thereof in the form of an amide bond, the grafting rate of the grafted compound is 20% to 100%, the molecular weight of the grafted compound is 3k to 500kDa, and the grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15ppm in the H NMR spectrum.

[0050] In one embodiment of the present application, at least a part of the carboxyl groups of the hyaluronic acid or salt thereof in the grafted compound are connected to the amino group of glutamic acid or salt thereof in the form of an amide bond, the grafting rate of the grafted compound is 20% to 100%, the molecular weight of the grafted compound is 3k to 500kDa, and the grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15ppm in the H NMR spectrum. 1 In one embodiment of the present application, at least a part of the carboxyl groups of the hyaluronic acid or salt thereof in the grafted compound are connected to the amino group of glutamic acid or salt thereof in the form of an amide bond, the grafting rate of the grafted compound is 20% to 100%, the molecular weight of the grafted compound is 3k to 500kDa, and the grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15ppm, 2.10-2.25ppm and 1.71-2.10ppm in the H NMR spectrum.

[0051] The method for preparing the grafted compound of hyaluronic acid or salt thereof and glutamic acid or salt thereof described in the present application is not particularly limited, and it can be prepared according to the methods commonly used in the art, for example: mixing hyaluronic acid or salt thereof and quaternary ammonium base to obtain quaternary ammonium base salt of hyaluronic acid or salt thereof by reaction; dissolving the quaternary ammonium base salt of hyaluronic acid or salt thereof, then adding glutamic acid or salt thereof, an activating agent, a catalyst to react, hydrolyzing in an alkaline environment, adjusting pH to obtain the grafted compound of hyaluronic acid or salt thereof and glutamic acid or salt thereof (HA-GLU).

[0052] In one embodiment of the present application, the quaternary ammonium base includes tetrabutylammonium hydroxide.

[0053] In one embodiment of the present application, the glutamic acid or salt thereof includes L-glutamic acid diethyl ester hydrochloride.

[0054] In one embodiment of the present application, the activating agent includes 2-chloro-1-methylpyridine iodine salt.

[0055] In one embodiment of the present application, the catalyst includes triethylamine.

[0056] In one embodiment of the present application, the quaternary ammonium base salt of hyaluronic acid or salt thereof is dissolved with an organic solvent, and the organic solvent includes N,N-dimethylformamide (DMF).

[0057] In some embodiments of the present application, the mass ratio of the quaternary ammonium base salt of hyaluronic acid or a salt thereof to glutamic acid or a salt thereof is 1:(0.01-1); for example, it can be 1:0.01, 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, or any range therebetween.

[0058] In some embodiments of the present application, the mass ratio of glutamic acid or its salt, activator, catalyst is 1 : (0.1-10) : (0.1-5); for example, it can be 1:0.1:0.1, 1:0.1:0.2, 1:0.1:0.3, 1:0.1:0.4, 1:0.1:0.5, 1:0.1:0.6, 1:0.1:0.7, 1:0.1:0.8, 1:0.1:0.9, 1:0.1:1.0, 1:0.1:2.0, 1:0.1:3.0, 1:0.1:4.0, 1:0.1:5.0, 1:0.2:0.1, 1:0.2:0.2, 1:0.2:0.3, 1:0.2:0.4, 1:0.2:0.5, 1:0.2:0.6, 1:0.2:0.7, 1:0.2:0.8, 1:0.2:0.9, 1:0.2:1.0, 1:0.2:2.0, 1:0.2:3.0, 1:0.2:4.0, 1:0.2:5.0, 1:0.5:0.1, 1:0.5:0.2, 1:0.5:0.3, 1:0.5:0.4, 1:0.5:0.5, 1:0.5:0.6, 1:0.5:0.7, 1:0.5:0.8, 1:0.5:0.9, 1:0.5:1.0, 1:0.5:2.0, 1:0.5:3.0, 1:0.5:4.0, 1:0.5:5.0, 1:1.0:0.1, 1:1.0:0.2, 1:1.0:0.3, 1:1.0:0.4, 1:1.0:0.5, 1:1.0:0.6, 1:1.0:0.7, 1:1.0:0.8, 1:1.0:0.9, 1:1.0:1.0, 1:1.0:2.0, 1:1.0:3.0, 1:1.0:4.0, 1:1.0:5.0, 1:2.0:0.1, 1:2.0:0.2, 1:2.0:0.3, 1:2.0:0.4, 1:2.0:0.5, 1:2.0:0.6, 1:2.0:0.7, 1:2.0:0.8, 1:2.0:0.9, 1:2.0:1.0, 1:2.0:2.0, 1:2.0:3.0, 1:2.0:4.0, 1:2.0:5.0, 1:5.0:0.1, 1:5.0:0.2, 1:5.0:0.3, 1:5.0:0.4, 1:5.0:0.5, 1:5.0:0.6, 1:5.0:0.7, 1:5.0:0.8, 1:5.0:0.9, 1:5.0:1.0, 1:5.0:2.0, 1:5.0:3.0, 1:5.0:4.0, 1:5.0:5.0, 1:10:0.1, 1:10:0.2, 1:10:0.3, 1:10:0.4, 1:10:0.5, 1:10:0.6, 1:10:0.7, 1:10:0.8, 1:10:0.9, 1:10:1.0, 1:10:2.0, 1:10:3.0, 1:10:4.0, 1:10:5.0 or any range between them.

[0059] In one embodiment of the present application, hyaluronic acid or a salt thereof and tetrabutylammonium hydroxide are mixed to obtain a TBA salt of hyaluronic acid or a salt thereof (HA-TBA); the TBA salt of hyaluronic acid or a salt thereof is dissolved in N,N-dimethylformamide (DMF), and then 2-chloro-1-methylpyridine iodide, L-glutamic acid diethyl ester hydrochloride and triethylamine are added to react, hydrolyzed in an alkaline environment, and the pH is adjusted to obtain a graft copolymer of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof (HA-GLU).

[0060] In one embodiment of the present application, hyaluronic acid or a salt thereof and tetrabutylammonium hydroxide are mixed to obtain a TBA salt of hyaluronic acid or a salt thereof (HA-TBA); the TBA salt of hyaluronic acid or a salt thereof is dissolved in N,N-dimethylformamide (DMF), and then 2-chloro-1-methylpyridine iodide, L-glutamic acid diethyl ester hydrochloride and triethylamine are added to react, hydrolyzed in an alkaline environment, and the pH is adjusted to obtain a graft copolymer of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof (HA-GLU). The mass ratio of the quaternary ammonium base salt of hyaluronic acid or a salt thereof to glutamic acid or a salt thereof is 1:(0.01-1), and the molar ratio of L-glutamic acid diethyl ester hydrochloride, 2-chloro-1-methylpyridine iodide and triethylamine is 1:(0.1-10):(0.1-5).

[0061] The present application also provides a cosmetic composition, which can be one of, for example, a composition for preventing and / or relieving skin discomfort, a composition for preventing and / or relieving skin discomfort caused by inflammation, an anti-inflammatory composition, etc. The composition comprises the grafting compound described in the present application.

[0062] The present application provides the use of the grafting compound described above in cosmetics.

[0063] The present application provides the use of the grafting compound described above or the composition described above in preventing and / or relieving skin discomfort caused by inflammation.

[0064] The present application provides the use of the grafting compound described above or the composition described above in anti-inflammation.

[0065] The present application provides the use of the grafting compound described above or the composition described above in the preparation of an article for preventing and / or relieving skin discomfort caused by inflammation.

[0066] The present application provides the use of the grafting compound described above or the composition described above in the preparation of an anti-inflammatory article.

[0067] The application provides use of the above grafting compound or the above composition for preventing and / or relieving skin discomfort caused by inflammation for non-therapeutic purposes.

[0068] The application provides use of the above grafting compound or the above composition for anti-inflammation for non-therapeutic purposes.

[0069] The application provides use of the above grafting compound or the above composition for inhibiting IL-6 expression.

[0070] The application provides use of the above grafting compound or the above composition for preparing an article for inhibiting IL-6 expression.

[0071] The application provides use of the above grafting compound or the above composition for inhibiting IL-6 expression for non-therapeutic purposes.

[0072] In a specific embodiment, the skin discomfort caused by inflammation (e.g. IL-6) includes dermatitis, eczema, acne, vitiligo, sebum secretion, scarring, skin aging, etc.

[0073] Example Experimental Raw Material Sources

[0074] Table 1

[0075]

[0076]

[0077] Example 1 Preparation of Graft-1 Sample

[0078] Low molecular weight hyaluronic acid (denoted as LMW-HA) is swelled in ethanol, acidified, washed with anhydrous ethanol, and then filtered. The filter cake is dissolved in water, and the pH is adjusted to neutral using tetrabutylammonium hydroxide. The solution is filtered and dried to obtain a solid TBA salt of hyaluronic acid (denoted as HA-TBA). The low molecular weight hyaluronic acid has a molecular weight of 4.7 wDa.

[0079] 2 g of HA-TBA is dissolved in N,N-dimethylformamide (DMF). After complete dissolution, 0.79 g of 2-chloro-1-methylpyridine iodide (CMPI), 0.15 g of L-glutamic acid diethyl ester hydrochloride, and 032 g of triethylamine are added to the system. The system is stirred at room temperature overnight. After the reaction is completed, purified water and a base are added to the reaction system for hydrolysis. Subsequently, the pH is adjusted to weak acidity using an acid. The product is precipitated and purified using an organic solvent. The filter cake is obtained by filtration, and dried to obtain a sample, denoted as Graft-1.

[0080] Example 2 Preparation of Graft-2 Sample

[0081] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swelled in ethanol, acidified, washed with anhydrous ethanol, and then filtered. The filter cake was dissolved in water, and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The solution was filtered and dried to obtain a solid TBA salt of hyaluronic acid (denoted as HA-TBA). The low molecular weight hyaluronic acid had a molecular weight of 4.7 wDa;

[0082] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After complete dissolution, 0.79 g of 2-chloro-1-methylpyridine iodide (CMPI), 0.23 g of L-glutamic acid diethyl ester hydrochloride, and 0.48 g of triethylamine were added to the system. The system was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. Subsequently, the pH was adjusted to weakly acidic using an acid. The product was precipitated and purified using an organic solvent. The filter cake was obtained by filtration, and the sample was dried to obtain Graft-2.

[0083] Example 3: Preparation of Graft-3 sample

[0084] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swelled in ethanol, acidified, washed with anhydrous ethanol, and then filtered. The filter cake was dissolved in water, and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The solution was filtered and dried to obtain a solid TBA salt of hyaluronic acid (denoted as HA-TBA). The low molecular weight hyaluronic acid had a molecular weight of 4.7 wDa;

[0085] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After complete dissolution, 0.79 g of 2-chloro-1-methylpyridine iodide (CMPI), 0.23 g of L-glutamic acid diethyl ester hydrochloride, and 0.48 g of triethylamine were added to the system. The system was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. Subsequently, the pH was adjusted to weakly acidic using an acid. The product was precipitated and purified using an organic solvent. The filter cake was obtained by filtration, and the sample was dried to obtain Graft-2.

[0086] Example 4: Preparation of Graft-4 sample

[0087] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swelled in ethanol, acidified, washed with anhydrous ethanol, and then filtered. The filter cake was dissolved in water, and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The solution was filtered and dried to obtain a solid TBA salt of hyaluronic acid (denoted as HA-TBA). The low molecular weight hyaluronic acid had a molecular weight of 4.7 wDa;

[0088] Dissolve 2 g of HA-TBA in N,N-dimethylformamide (DMF), after complete dissolution, add 0.79 g of 2-chloro-1-methylpyridine iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, 0.56 g of triethylamine to the system, stir overnight at room temperature; after the reaction is completed, add purified water and base to the reaction system for hydrolysis, then adjust the pH to weak acidity with acid, precipitate and purify with organic solvent, filter the filter cake, dry to obtain the sample, and mark it as Graft-4.

[0089] Example 5: Preparation of Graft-5 sample

[0090] After swelling the low molecular weight hyaluronic acid (marked as LMW-HA) in ethanol, acidification is performed, and after washing with anhydrous ethanol, the filter cake is dissolved in water, and the pH is adjusted to neutral using tetrabutylammonium hydroxide, and the filter cake is filtered and dried to obtain the TBA salt of solid hyaluronic acid (marked as HA-TBA), and the molecular weight of the low molecular weight hyaluronic acid is 4.7 wDa;

[0091] Dissolve 2 g of HA-TBA in N,N-dimethylformamide (DMF), after complete dissolution, add 0.79 g of 2-chloro-1-methylpyridine iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, 0.56 g of triethylamine to the system, stir overnight at room temperature; after the reaction is completed, add purified water and base to the reaction system for hydrolysis, then adjust the pH to weak acidity with acid, precipitate and purify with organic solvent, filter the filter cake, dry to obtain the sample, and mark it as Graft-4.

[0092] Example 6: Preparation of Graft-6 sample

[0093] After swelling the low molecular weight hyaluronic acid (marked as LMW-HA) in ethanol, acidification is performed, and after washing with anhydrous ethanol, the filter cake is dissolved in water, and the pH is adjusted to neutral using tetrabutylammonium hydroxide, and the filter cake is filtered and dried to obtain the TBA salt of solid hyaluronic acid (marked as HA-TBA), and the molecular weight of the low molecular weight hyaluronic acid is 4.7 wDa;

[0094] Dissolve 2 g of HA-TBA in N,N-dimethylformamide (DMF), after complete dissolution, add 0.79 g of 2-chloro-1-methylpyridine iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, 0.56 g of triethylamine to the system, stir overnight at room temperature; after the reaction is completed, add purified water and base to the reaction system for hydrolysis, then adjust the pH to weak acidity with acid, precipitate and purify with organic solvent, filter the filter cake, dry to obtain the sample, and mark it as Graft-4.

[0095] Example 7: Preparation of Graft-7 sample

[0096] The small molecular weight hyaluronic acid (denoted as UMW-HA) is swelled in ethanol, acidified, washed with anhydrous ethanol, and then filtered. The filter cake is dissolved in water, and the pH is adjusted to neutral with tetrabutylammonium hydroxide. The solution is filtered and dried to obtain the TBA salt of solid hyaluronic acid (denoted as HA-TBA). The small molecular weight hyaluronic acid has a molecular weight of 3 kDa.

[0097] 2 g of HA-TBA is dissolved in N,N-dimethylformamide (DMF). After complete dissolution, 0.79 g of 2-chloro-1-methylpyridine iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, and 0.56 g of triethylamine are added to the system. The system is stirred at room temperature overnight. After the reaction is completed, purified water and a base are added to the reaction system for hydrolysis. Subsequently, the pH is adjusted to weakly acidic with an acid. The product is precipitated and purified with an organic solvent, filtered, and dried to obtain a sample, which is denoted as Graft-7.

[0098] Example 8: Preparation of Graft-8 sample

[0099] The medium molecular weight hyaluronic acid (denoted as MMW-HA) is swelled in ethanol, acidified, washed with anhydrous ethanol, and then filtered. The filter cake is dissolved in water, and the pH is adjusted to neutral with tetrabutylammonium hydroxide. The solution is filtered and dried to obtain the TBA salt of solid hyaluronic acid (denoted as HA-TBA). The medium molecular weight hyaluronic acid has a molecular weight of 36.8 wDa.

[0100] 2 g of HA-TBA is dissolved in N,N-dimethylformamide (DMF). After complete dissolution, 0.79 g of 2-chloro-1-methylpyridine iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, and 0.56 g of triethylamine are added to the system. The system is stirred at room temperature overnight. After the reaction is completed, purified water and a base are added to the reaction system for hydrolysis. Subsequently, the pH is adjusted to weakly acidic with an acid. The product is precipitated and purified with an organic solvent, filtered, and dried to obtain a sample, which is denoted as Graft-8.

[0101] Reference Example 9: Preparation of Graft-9 sample

[0102] The high molecular weight hyaluronic acid (denoted as HMW-HA) is swelled in ethanol, acidified, washed with anhydrous ethanol, and then filtered. The filter cake is dissolved in water, and the pH is adjusted to neutral with tetrabutylammonium hydroxide. The solution is filtered and dried to obtain the TBA salt of solid hyaluronic acid (denoted as HA-TBA). The high molecular weight hyaluronic acid has a molecular weight of 85 wDa.

[0103] Dissolve 2 g of HA-TBA in N, N-dimethylformamide (DMF), after dissolution, add 0.79 g of 2-chloro-1-methylpyridine iodine salt (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, 0.56 g of triethylamine to the system, stir at room temperature overnight; after the reaction is completed, add purified water and base to the reaction system for hydrolysis, then adjust the pH to weak acidity with acid, precipitate and purify with organic solvent, filter the filter cake, dry to obtain the sample, and record it as Graft-9.

[0104] Parameters of examples and reference examples in Table 2

[0105]

[0106]

[0107] Note: HA-GLU represents a grafted compound of hyaluronic acid-glutamic acid.

[0108] Experimental example

[0109] Experimental example 1

[0110] Take 10 mg of the sample of examples 1-8 and reference example 9, and 10 mg of hyaluronic acid sample with a molecular weight of 4.7 wDa and 10 mg of glutamic acid sample, dissolve the above-mentioned samples in deuterium oxide (D2O) to form a clear and transparent solution. Take an appropriate amount of transparent solution into a nuclear magnetic tube, use a nuclear magnetic resonance spectrometer to scan the hydrogen spectrum, set the scanning number to 16 times, select D2O as the solvent, and click to start the test. Obtain the nuclear magnetic resonance hydrogen spectrum of HA-GLU with different grafting rates 1 H NMR.

[0111] The characteristic peaks of examples 1-8, reference example 9 and hyaluronic acid and glutamic acid are shown in Table 3. 1 The H NMR spectrum is shown in Figure 1 .

[0112] The characteristic peaks of examples 1-8, reference example 9 and hyaluronic acid and glutamic acid are shown in Table 3.

[0113] Table 3 1 Assignment of each peak in the H NMR spectrum

[0114]

[0115]

[0116] Chemical Shift: indicates chemical shift; Coupling splitting indicates the number of coupling splitting peaks; Relative proton ratio indicates the relative number of protons

[0117] From Table 3 andFigure 1 The grafting compound is detected by nuclear magnetic resonance 1 In the H NMR spectrum, the chemical shifts of the methine and methylene groups of the grafting compound derived from glutamic acid are shifted to low field relative to glutamic acid.

[0118] The characteristic peaks of the methylene group of glutamic acid or its salt appear at δ = 1.71-2.10 ppm and δ = 2.10-2.25 ppm, respectively; preferably, the characteristic peak of the methine-CH-group of glutamic acid or its salt appears at δ = 4.00-4.15 ppm, and the characteristic peaks of the-CH-group of hyaluronic acid or its salt appear at δ = 4.30-4.40 ppm and δ = 4.40-4.48 ppm, respectively.

[0119] Calculation of the grafting rate of the HA-GLU grafting compound:

[0120] By nuclear magnetic resonance spectroscopy 1 H NMR, the grafting molar ratio in the grafting compound is determined by measuring the integral area of the hydrogen spectrum of glutamic acid in the grafting compound, so as to determine the grafting rate of the HA-GLU molecule.

[0121] Specifically, the integral area of the methine-CH-proton peak (shifted to 4.05-4.12 ppm) brought by glutamic acid in the grafting compound is measured to determine the grafting rate (100% grafting rate, the integral area at this position is 1);

[0122] Table 4

[0123] No. Grafting rate % ∫(f1＝4.05-4.12) ∫(f2.15-2.25) ∫(f1.76-2.07) Graft-1 12 0.12 0.25 3.27 Graft-2 27 0.27 0.53 3.52 Graft-3 34 0.34 0.66 3.66 Graft-4 47 0.47 0.97 3.98 Graft-5 65 0.65 1.25 4.28 Graft-6 87 0.87 1.73 4.74 Graft-7 47 0.47 0.95 3.96 Graft-8 47 0.47 0.96 3.96 Graft-9 47 0.47 0.97 3.99

[0124] Experimental Example 2

[0125] First, prepare a complete culture medium solution: mix DMEM basic culture solution, FBS fetal bovine serum, and PS double-antibody solution in a mass ratio of 89:10:1.

[0126] Weigh 0.009 g of UMW-HA, LMW-HA, MMW-HA, HMW-HA, GLU, physical mixture, and Graft-1 to Graft-9 powder, respectively, and dissolve them in the complete culture medium solution to form test mixtures, all with a content of 150 μg / mL.

[0127] No. 1 indicates that UMW-HA is added to the culture medium solution, and the content of UMW-HA is 150 μg / mL;

[0128] No. 2 indicates that LMW-HA is added to the culture medium solution, and the content of LMW-HA is 150 μg / mL;

[0129] 3 indicates that MMW-HA was added to the medium solution, wherein the content of MMW-HA was 150 μg / mL;

[0130] 4 indicates that HMW-HA was added to the medium solution, wherein the content of HMW-HA was 150 μg / mL;

[0131] 5 indicates that GLU was added to the medium solution, wherein the content of GLU was 150 μg / mL;

[0132] 6 indicates that LMW-HA and GLU were added to the medium solution, wherein the content of LMW-HA was 136 μg / mL and the content of GLU was 14 μg / mL;

[0133] 7 indicates that Graft-1 was added to the medium solution, wherein the content of Graft-1 was 150 μg / mL;

[0134] 8 indicates that Graft-2 was added to the medium solution, wherein the content of Graft-2 was 150 μg / mL;

[0135] 9 indicates that Graft-3 was added to the medium solution, wherein the content of Graft-3 was 150 μg / mL;

[0136] 10 indicates that Graft-4 was added to the medium solution, wherein the content of Graft-4 was 150 μg / mL;

[0137] 11 indicates that Graft-5 was added to the medium solution, wherein the content of Graft-5 was 150 μg / mL;

[0138] 12 indicates that Graft-6 was added to the medium solution, wherein the content of Graft-6 was 150 μg / mL;

[0139] 13 indicates that Graft-7 was added to the medium solution, wherein the content of Graft-7 was 150 μg / mL;

[0140] 14 indicates that Graft-8 was added to the medium solution, wherein the content of Graft-8 was 150 μg / mL;

[0141] 15 indicates that Graft-9 was added to the medium solution, wherein the content of Graft-9 was 150 μg / mL.

[0142] The cytological test method was as follows:

[0143] 1) Cell inoculation: 1 x 105cells were inoculated in each well of a 96-well plate. 5The fibroblasts were inoculated into the 6-well plate at a seeding density of 1x104 cells / well, and incubated in an incubator (37℃, 5% CO2) for 24 h.

[0144] 2) Modeling: According to the test grouping, the sample group and the model control group were exposed to the ultraviolet light therapy instrument (wavelength 280-320 nm, 80 mJ / cm 2 ) for UVB irradiation.

[0145] 3) Dosing: According to the test scheme in Table 5, the sample group was dosed by adding complete medium containing the working solution of the tested substance into the wells, 1 mL was added to each well, and 3 replicate wells were set for each group. After the dosing was completed, the 6-well plate was placed in an incubator (37℃, 5% CO2) for 24 h.

[0146] 4) Cell collection: After 24 h of incubation, the cell supernatant was collected, washed twice with 1 mL / well D' Hanks buffer, and the cells were lysed by blowing to collect the sample.

[0147] 5) Gene expression detection: After the cells were treated by RNA-Quick Purification Kit, the sample was collected, RNA extraction, reverse transcription and fluorescence quantitative PCR operation were carried out according to the instructions of the kit, and the results were calculated by 2 -△△Ct method to obtain the test results.

[0148] The test results are shown in Table 5, and the standard deviation of the experiment is 0.01. The lower the relative expression of IL-6, the stronger the inhibitory effect of the test sample on IL-6 expression. Compared with single hyaluronic acid or glutamic acid, if the relative expression of IL-6 of the same amount of graft is lower, it indicates that the graft has a synergistic effect. The relative expression of IL-6 of the graft of the application is lower than that of the same amount of single hyaluronic acid or glutamic acid, and the relative expression of IL-6 is also lower than that of the physical mixture of the same amount of the two, indicating that the graft has a synergistic effect.

[0149] Table 5

[0150] No Formulation for cell test IL-6 relative expression NC NA 1.000 1 150 μg / mL UMW-HA 0.518 2 150 μg / mL LMW-HA 0.538 3 150 μg / mL MMW-HA 0.717 4 150 μg / mL HMW-HA 0.562 5 150 μg / mL GLU 0.753 6 136 μg / mL LMW-HA + 14 μg / mL GLU 0.775 7 150 μg / mL Graft-1 0.582 8 150 μg / mL Graft-2 0.395 9 150 μg / mL Graft-3 0.357 10 150 μg / mL Graft-4 0.132 11 150 μg / mL Graft-5 0.165 12 150 μg / mL Graft-6 0.522 13 150 μg / mL Graft-7 0.152 14 150 μg / mL Graft-8 0.302 15 150 μg / mL Graft-9 0.880

[0151] Although the present application has been disclosed with the above embodiments, it is not intended to limit the present application, and any person skilled in the art can make some changes and modifications without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application shall be subject to the patent application scope appended hereto.

Claims

1. A grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof, wherein, at least a part of carboxyl groups of the hyaluronic acid or the salt thereof is linked to amino groups of glutamic acid or the salt thereof in the form of an amide bond; the grafting rate of the grafted compound is 20% to 100%; the molecular weight of the grafted compound is 3k to 500kDa.

2. The grafted compound according to claim 1, wherein, The grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15 ppm in a H NMR spectrum. 1 The grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15 ppm in a H NMR spectrum.

3. The grafted compound according to claim 1, wherein, The grafted compound obtained by nuclear magnetic resonance detection has characteristic peaks at chemical shifts δ of 2.10-2.25 ppm and 1.71-2.10 ppm in the H NMR spectrum. 1 The grafted compound obtained by nuclear magnetic resonance detection has characteristic peaks at chemical shifts δ of 2.10-2.25 ppm and 1.71-2.10 ppm in the H NMR spectrum.

4. A method for preparing the grafted compound of hyaluronic acid or the salt thereof and glutamic acid or the salt thereof according to any one of claims 1 to 3, wherein, Preparation of quaternary ammonium base salt of hyaluronic acid or the salt thereof: mixing hyaluronic acid or the salt thereof and quaternary ammonium base, and reacting to obtain quaternary ammonium base salt of hyaluronic acid or the salt thereof; Preparation of the grafted compound: dissolving the quaternary ammonium base salt of hyaluronic acid or the salt thereof, and then adding glutamic acid or the salt thereof, an activating agent, and a catalyst to react, hydrolyzing in an alkaline environment, adjusting pH, and obtaining the grafted compound of hyaluronic acid or the salt thereof and glutamic acid or the salt thereof.

5. A cosmetic composition comprising the grafted compound according to any one of claims 1 to 3 or prepared by the method of claim 4.

6. Use of the grafted compound according to any one of claims 1 to 3 or prepared by the method of claim 4 in cosmetics.

7. Use of the grafted compound according to any one of claims 1 to 3 or prepared by the method of claim 4 or the cosmetic composition of claim 5 in the preparation of a preparation for preventing and / or relieving skin discomfort caused by inflammation.

8. Use of the grafted compound according to any one of claims 1 to 3 or prepared by the method of claim 4 or the cosmetic composition of claim 5 in the preparation of an anti-inflammatory preparation.

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