Grafted compound of hyaluronic acid or salt thereof and glutamic acid or salt thereof, and preparation method therefor and use 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 therefor. It is found that the graft has a synergistic effect in inhibiting IL-6 expression, and can prevent or alleviate skin discomfort caused by inflammation.

Description

Grafted compound of hyaluronic acid or its salt and glutamic acid or its salt, preparation method and use thereof Technical Field

[0001] The present application belongs to the field of cosmetic technology, and specifically relates to a grafted compound of hyaluronic acid or its salt and glutamic acid or its salt, and a preparation method and use thereof. Background Art

[0002] In skin care product formulations, hyaluronic acid or its salts are often mixed with other active substances. However, in such cases, the efficacy of the active substances may not be fully exerted.

[0003] Summary of the Invention

[0004] The inventors of the present application discovered 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 invention.

[0005] The technical solution of this application is as follows:

[0006] 1. A grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof, wherein at least a portion of the carboxyl groups of the hyaluronic acid or a salt thereof are linked to the amino groups of the glutamic acid or a salt thereof in the form of an amide bond.

[0007] 2. The grafted compound according to item 1, wherein the general formula thereof is as shown in formula (I):

[0008] Wherein, in formula (I), R is a metal ion or H, 0≤x<1, 0<y≤1, x+y=1, and n is a positive integer from 1 to 2000.

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

[0015] Preferably,

[0016] The grafted compound is obtained by nuclear magnetic resonance detection 1 In the H NMR spectrum, the grafted compound has characteristic peaks at chemical shifts δ of 2.10-2.25 ppm and 1.71-2.10 ppm.

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

[0018] Preparation of quaternary ammonium salt of hyaluronic acid or its salt: mixing hyaluronic acid or its salt and quaternary ammonium base, reacting to obtain quaternary ammonium salt of hyaluronic acid or its salt;

[0019] Preparation of the grafted compound: dissolve the quaternary ammonium salt of hyaluronic acid or its salt, then add glutamic acid or its salt, an activator, and a catalyst to react, hydrolyze under alkaline conditions, and adjust the pH to obtain a grafted compound of hyaluronic acid or its salt and glutamic acid or its salt.

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

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

[0022] 9. Use of the grafted compound described in any one of items 1 to 5, or the grafted compound prepared by the method described in item 6, or the cosmetic composition described in item 6 in preventing and / or alleviating skin discomfort caused by inflammation.

[0023] 10. Use of the grafted compound described in any one of items 1 to 5, or the grafted compound prepared by the method described in item 6, or the cosmetic composition described in item 7 in anti-inflammatory treatment.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The grafted compound of hyaluronic acid or its salt and glutamic acid or its salt provided in the present application can effectively exert anti-inflammatory and skin care effects, and expand the application scope of the grafted compound.

[0026] Furthermore, the present invention obtains the preferred structure of the new raw material when used to inhibit IL-6 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 shows the embodiment 6. 1 H NMR spectrum. DETAILED DESCRIPTION

[0028] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0029] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[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 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.

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

[0032] The degree of substitution refers to the molar ratio of glutamic acid or its salt connected to the disaccharide structure of hyaluronic acid or its salt through an amide bond to the disaccharide structure of hyaluronic acid or its salt.

[0033] The hyaluronate includes unmodified hyaluronate or modified hyaluronate (e.g., acetylated hyaluronate), for example, sodium salt, zinc salt, calcium salt, magnesium salt, etc. of hyaluronic acid, or any combination of these salts.

[0034] In some embodiments of the present application, the molecular weight of the hyaluronic acid or its salt is 2.5k-500kDa; for example, the molecular weight of the hyaluronic acid or its salt can be 2.5kDa, 2.6kDa, 2.7kDa, 2.8kDa, 2.9kDa, 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, 18 0kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa, or any range therebetween.

[0035] Glutamate includes unmodified glutamate or modified glutamate, such as hydrochloride, acetate, sulfonate, etc. of glutamic acid, or any combination of these salts.

[0036] In some embodiments, the general formula of the grafted compound is as shown in Formula (I):

[0037] Wherein, in formula (I), R is a metal ion or H, 0≤x<1, 0<y≤1, x+y=1, and n is a positive integer from 1 to 2000.

[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 20, 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 a positive integer therebetween.

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

[0042] In some embodiments of the present application, the general formula of the grafted compound is as shown in formula (II):

[0043] Wherein, in formula (II), R is a metal ion or H, n is a positive integer from 1 to 2000, at least a portion of OR is substituted with glutamic acid, and the substituent group of glutamic acid is shown in formula (III):

[0044] Wherein, R in formula (III) is a metal ion or H.

[0045] In some embodiments of the present application, the grafting rate is determined by nuclear magnetic resonance spectroscopy. 1 H NMR determination; wherein the grafting rate is obtained by nuclear magnetic resonance detection 1 The calculation is performed by integrating the peak area of ​​the methine (-CH- proton) of glutamic acid or its salt in the grafted compound at a shift to 4.05-4.12 ppm in the H NMR spectrum, or by 1The calculation is performed by integrating the proton peak areas of the two methylene groups (-CH2-) of glutamic acid or its salt and the methyl group (-CH3-) of hyaluronic acid or its salt in the grafted compound in the H NMR spectrum.

[0046] In some embodiments of the present application, the grafting rate of the grafted 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.

[0047] In some embodiments of the present application, the molecular weight of the grafted compound is 3k-500kDa; for example, the molecular weight of the grafted 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 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa, or any range therebetween.

[0048] In some embodiments of the present application, the grafted compound is detected by nuclear magnetic resonance. 1 In the H NMR spectrum, the grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15 ppm, for example, the grafted compound has a characteristic peak at a chemical shift δ of 4.00 ppm, 4.01 ppm, 4.02 ppm, 4.03 ppm, 4.04 ppm, 4.05 ppm, 4.06 ppm, 4.07 ppm, 4.08 ppm, 4.09 ppm, 4.10 ppm, 4.11 ppm, 4.12 ppm, 4.13 ppm, 4.14 ppm, 4.15 ppm, or any range therebetween.

[0049] In some embodiments of the present application, the grafted compound is detected by nuclear magnetic resonance. 1 In the H NMR spectrum, the grafted compound has a characteristic peak at a chemical shift δ of 2.10-2.25 ppm, for example, the grafted compound has a characteristic peak at a chemical shift δ of 2.10 ppm, 2.11 ppm, 2.12 ppm, 2.13 ppm, 2.14 ppm, 2.15 ppm, 2.16 ppm, 2.17 ppm, 2.18 ppm, 2.19 ppm, 2.20 ppm, 2.21 ppm, 2.22 ppm, 2.23 ppm, 2.24 ppm, 2.25 ppm or any range therebetween.

[0050] In some embodiments of the present application, the grafted compound is detected by nuclear magnetic resonance. 1 In the H NMR spectrum, the grafted compound has a characteristic peak at a chemical shift δ of 1.71-2.10 ppm, for example, the grafted compound has a characteristic peak at a chemical shift δ of 1.71 ppm, 1.72 ppm, 1.73 ppm, 1.74 ppm, 1.75 ppm, 1.76 ppm, 1.77 ppm, 1.78 ppm, 1.79 ppm, 1.80 ppm, 1.81 ppm, 1.82 ppm, 1.83 ppm, 1.84 ppm, 1.85 ppm, 1.86 ppm, 1.87 ppm, 1.88 There are characteristic peaks at ppm, 1.89ppm, 1.90ppm, 1.91ppm, 1.92ppm, 1.93ppm, 1.94ppm, 1.95ppm, 1.96ppm, 1.97ppm, 1.98ppm, 1.99ppm, 2.00ppm, 2.01ppm, 2.02ppm, 2.03ppm, 2.04ppm, 2.05ppm, 2.06ppm, 2.07ppm, 2.08ppm, 2.09ppm, 2.10ppm or any range therebetween.

[0051] In some embodiments of the present application, the grafted compound is detected by nuclear magnetic resonance. 1 In the H NMR spectrum, the grafted compound has a characteristic peak at a chemical shift δ of 4.30-4.40 ppm, for example, the grafted compound has a characteristic peak at a chemical shift δ of 4.30 ppm, 4.31 ppm, 4.32 ppm, 4.33 ppm, 4.34 ppm, 4.35 ppm, 4.36 ppm, 4.37 ppm, 4.38 ppm, 4.39 ppm, 4.40 ppm or any range therebetween.

[0052] In some embodiments of the present application, the grafted compound is detected by nuclear magnetic resonance. 1In the H NMR spectrum, the grafted compound has a characteristic peak at a chemical shift δ of 4.30-4.48 ppm, for example, the grafted compound has a characteristic peak at a chemical shift δ of 4.30 ppm, 4.31 ppm, 4.32 ppm, 4.33 ppm, 4.34 ppm, 4.35 ppm, 4.36 ppm, 4.37 ppm, 4.38 ppm, 4.39 ppm, 4.40 ppm, 4.41 ppm, 4.42 ppm, 4.43 ppm, 4.44 ppm, 4.45 ppm, 4.46 ppm, 4.47 ppm, 4.48 ppm, or any range therebetween.

[0053] In a specific embodiment of the present application, in the grafted compound, at least a portion of the carboxyl groups of the hyaluronic acid or its salt are connected to the amino group of the glutamic acid or its salt in the form of an amide bond, and the grafting rate of the grafted compound is 20% to 95%. The general formula of the grafted compound is shown in formula (I):

[0054] Wherein, in formula (I), R is a metal ion or H, 0≤x<1, 0<y≤1, x+y=1, and n is a positive integer from 1 to 2000.

[0055] In some embodiments of the present application, the values ​​of x, y, grafting ratio y / x+y, and n can refer to the above description.

[0056] In a specific embodiment of the present application, in the grafted compound, at least a portion of the carboxyl groups of the hyaluronic acid or its salt are connected to the amino group of the glutamic acid or its salt in the form of an amide bond, and the grafting rate of the grafted compound is 20% to 100%. The general formula of the grafted compound is shown in formula (II):

[0057] Wherein, in formula (II), R is a metal ion or H, n is a positive integer from 1 to 2000, at least a portion of OR is substituted with glutamic acid, and the substituent group of glutamic acid is shown in formula (III):

[0058] Wherein, R in formula (III) is a metal ion or H.

[0059] In a specific embodiment of the present application, in the grafted compound, at least a portion of the carboxyl groups of the hyaluronic acid or its salt are connected to the amino group of glutamic acid or its salt 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-500kDa, and in the 1H NMR spectrum obtained by nuclear magnetic resonance detection of the grafted compound, the grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15ppm.

[0060] In a specific embodiment of the present application, in the grafted compound, at least a portion of the carboxyl groups of the hyaluronic acid or its salt is connected to the amino group of the glutamic acid or its salt 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-500kDa, and the grafted compound is detected by nuclear magnetic resonance. 1 In the H NMR spectrum, the grafted compound has characteristic peaks at chemical shifts δ of 4.00-4.15 ppm, 2.10-2.25 ppm, and 1.71-2.10 ppm.

[0061] The method for preparing the grafted compound of hyaluronic acid or its salt and glutamic acid or its salt described in the present application is not particularly limited, and it can be prepared according to methods commonly used in the art, for example: mixing hyaluronic acid or its salt and a quaternary ammonium base, reacting to obtain a quaternary ammonium salt of hyaluronic acid or its salt; dissolving the quaternary ammonium salt of hyaluronic acid or its salt, then adding glutamic acid or its salt, an activator, and a catalyst to react, hydrolyzing in an alkaline environment, and adjusting the pH to obtain a grafted compound of hyaluronic acid or its salt and glutamic acid or its salt (HA-GLU).

[0062] In a specific embodiment of the present application, the quaternary ammonium base includes tetrabutylammonium hydroxide.

[0063] In a specific embodiment of the present application, glutamic acid or a salt thereof includes L-glutamic acid diethyl ester hydrochloride.

[0064] In a specific embodiment of the present application, the activating agent includes 2-chloro-1-methylpyridinium iodide.

[0065] In one embodiment of the present application, the catalyst comprises triethylamine.

[0066] In a specific embodiment of the present application, the quaternary ammonium base salt of hyaluronic acid or its salt is dissolved in an organic solvent, and the organic solvent includes N,N-dimethylformamide (DMF).

[0067] In some embodiments of the present application, the mass ratio of the quaternary ammonium base salt of hyaluronic acid or its salt to glutamic acid or its salt 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.

[0068] In some embodiments of the present application, the mass ratio of glutamic acid or its salt, activator, and 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, 1:0.1:0.5, 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 in between.

[0069] In a specific embodiment of the present application, hyaluronic acid or a salt thereof and tetrabutylammonium hydroxide are mixed and reacted 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-methylpyridinium iodide, L-glutamic acid diethyl ester hydrochloride, and triethylamine are added to react, and the mixture is hydrolyzed under 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).

[0070] In one embodiment of the present application, hyaluronic acid or a salt thereof and tetrabutylammonium hydroxide are mixed to react 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-methylpyridinium iodide, L-glutamic acid diethyl ester hydrochloride, and triethylamine are added to react, and 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-methylpyridinium iodide, and triethylamine is 1:(0.1-10):(0.1-5).

[0071] The present application also provides a cosmetic composition, which may be, for example, a composition for preventing and / or alleviating skin discomfort, a composition for preventing and / or alleviating skin discomfort caused by inflammation, an anti-inflammatory composition, etc. The composition includes the grafted compound described herein.

[0072] The present application provides use of the above-mentioned grafted compound or the above-mentioned composition in cosmetics.

[0073] The present application provides use of the grafted compound or the composition in preventing and / or alleviating skin discomfort caused by inflammation.

[0074] The present application provides the use of the above-mentioned grafted compound or the above-mentioned composition in anti-inflammatory treatment.

[0075] The present application provides use of the grafted compound or the composition in preparing a product for preventing and / or alleviating skin discomfort caused by inflammation.

[0076] The present application provides use of the above-mentioned grafted compound or the above-mentioned composition in preparing anti-inflammatory products.

[0077] The present application provides the use of the grafted compound or the composition for non-therapeutic purposes in preventing and / or alleviating skin discomfort caused by inflammation.

[0078] The present application provides the use of the grafted compound or the composition in anti-inflammatory treatment for non-therapeutic purposes.

[0079] The present application provides the use of the above-mentioned grafted compound or the above-mentioned composition in inhibiting IL-6 expression.

[0080] The present application provides use of the above-mentioned grafted compound or the above-mentioned composition in preparing a product for inhibiting IL-6 expression.

[0081] The present application provides the use of the above-mentioned grafted compound or the above-mentioned composition in inhibiting IL-6 expression for non-therapeutic purposes.

[0082] In a specific embodiment, the skin discomfort caused by inflammation (such as IL-6) includes dermatitis, eczema, acne, vitiligo, sebum secretion, scars, skin aging, etc.

[0083] The present application provides a method for preventing and / or alleviating skin inflammation, comprising administering the grafted compound or composition of the present application to a subject in need thereof.

[0084] The present application provides a method for inhibiting IL-6 expression, which comprises administering the grafted compound or composition of the present application to a subject in need thereof.

[0085] The present application provides an anti-inflammatory method, which comprises administering the grafted compound or the composition of the present application to a subject in need thereof.

[0086] In this application, the term "subject" generally refers to a cell or an animal, which can be a mammal, such as a human, a non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents and adult subjects. Subjects also include animal disease models and other animal models known to those skilled in the art.

[0087] Examples Experimental raw material sources

[0088] Table 1

[0089] Example 1 Preparation of Graft-1 Sample

[0090] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (denoted as HA-TBA). The molecular weight of the low molecular weight hyaluronic acid was 4.7 wDa.

[0091] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 0.15 g of L-glutamic acid diethyl ester hydrochloride, and 0.32 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample, which was recorded as Graft-1.

[0092] Example 2 Preparation of Graft-2 Sample

[0093] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (denoted as HA-TBA). The molecular weight of the low molecular weight hyaluronic acid was 4.7 wDa.

[0094] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 0.23 g of L-glutamic acid diethyl ester hydrochloride, and 0.48 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample designated as Graft-2.

[0095] Example 3 Preparation of Graft-3 Sample

[0096] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (denoted as HA-TBA). The molecular weight of the low molecular weight hyaluronic acid was 4.7 wDa.

[0097] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 0.38 g of L-glutamic acid diethyl ester hydrochloride, and 0.56 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample designated as Graft-3.

[0098] Example 4 Preparation of Graft-4 Sample

[0099] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (denoted as HA-TBA). The molecular weight of the low molecular weight hyaluronic acid was 4.7 wDa.

[0100] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, and 0.56 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample designated as Graft-4.

[0101] Example 5 Preparation of Graft-5 Sample

[0102] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (denoted as HA-TBA). The molecular weight of the low molecular weight hyaluronic acid was 4.7 wDa.

[0103] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 0.54 g of L-glutamic acid diethyl ester hydrochloride, and 0.62 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample designated as Graft-5.

[0104] Example 6 Preparation of Graft-6 Sample

[0105] Low molecular weight hyaluronic acid (denoted as LMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (denoted as HA-TBA). The molecular weight of the low molecular weight hyaluronic acid was 4.7 wDa.

[0106] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 1 g of L-glutamic acid diethyl ester hydrochloride, and 0.81 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample designated as Graft-6.

[0107] Example 7 Preparation of Graft-7 Sample

[0108] Low molecular weight hyaluronic acid (denoted as UMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (denoted as HA-TBA). The low molecular weight hyaluronic acid had a molecular weight of 3 kDa.

[0109] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, and 0.56 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample designated as Graft-7.

[0110] Example 8 Preparation of Graft-8 Sample

[0111] Medium molecular weight hyaluronic acid (denoted as MMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (denoted as HA-TBA). The molecular weight of the medium molecular weight hyaluronic acid was 36.8 wDa.

[0112] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, and 0.56 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample designated as Graft-8.

[0113] Reference Example 9 Preparation of Graft-9 Sample

[0114] High molecular weight hyaluronic acid (HMW-HA) was swollen in ethanol and then acidified. After washing with anhydrous ethanol, the mixture was filtered. The filter cake was dissolved in water and the pH was adjusted to neutral using tetrabutylammonium hydroxide. The mixture was filtered and dried to obtain solid hyaluronic acid TBA salt (HA-TBA). The high molecular weight hyaluronic acid had a molecular weight of 85 wDa.

[0115] 2 g of HA-TBA was dissolved in N,N-dimethylformamide (DMF). After dissolution, 0.79 g of 2-chloro-1-methylpyridinium iodide (CMPI), 0.46 g of L-glutamic acid diethyl ester hydrochloride, and 0.56 g of triethylamine were added to the system, and the mixture was stirred at room temperature overnight. After the reaction was completed, purified water and a base were added to the reaction system for hydrolysis. The pH was then adjusted to a weakly acidic state with an acid. The mixture was precipitated and purified with an organic solvent. The filter cake was filtered and dried to obtain a sample, which was recorded as Graft-9.

[0116] Table 2 Parameters of Examples and Reference Examples

[0117] Note: HA-GLU represents a hyaluronic acid-glutamic acid grafted compound.

[0118] Experimental example

[0119] Experimental Example 1

[0120] Weigh 10 mg of the samples of Examples 1-8 and Reference Example 9, 10 mg of a hyaluronic acid sample with a molecular weight of 4.7 wDa, and 10 mg of a glutamic acid sample, and dissolve the above samples in deuterated water (D2O) to form a clear transparent solution. Pipette an appropriate amount of the transparent solution into a nuclear magnetic resonance tube, use a nuclear magnetic resonance spectrometer to scan the hydrogen spectrum, set the number of scans to 16 times, select D2O as the solvent, and click to start the test. Obtain HA-GLU nuclear magnetic resonance hydrogen spectra with different grafting rates 1 H NMR.

[0121] Among them, the embodiment 6 1 The H NMR spectrum is shown in Figure 1.

[0122] The specific analysis results of the characteristic peaks shown in Examples 1-8, Reference Example 9, hyaluronic acid, and glutamic acid are shown in Table 3.

[0123] Table 3

[0124] Chemical Shift: indicates chemical shift; Coupling splitting indicates the number of coupled splitting peaks; Relative proton ratio indicates the relative number of protons

[0125] The grafted compounds described in Table 3 and Figure 1 were detected by nuclear magnetic resonance. 1 In the H NMR spectrum, relative to glutamic acid, the chemical shifts of the methine and methylene groups of the grafted compound derived from glutamic acid are both shifted to the downfield.

[0126] The characteristic peaks of the methylene group of glutamic acid or its salt appear at chemical shifts δ = 1.71-2.10 ppm and δ = 2.10-2.25 ppm, respectively; preferably, the characteristic peak of the methine -CH- of glutamic acid or its salt appears at δ = 4.00-4.15 ppm, and the characteristic peaks of -CH- of hyaluronic acid or its salt appear at δ = 4.30-4.40 ppm and δ = 4.40-4.48 ppm, respectively.

[0127] Calculation of grafting rate of HA-GLU grafted compound:

[0128] By nuclear magnetic resonance spectroscopy 1 H NMR was used to determine the grafting molar ratio in the grafted material by measuring the integrated area of ​​the hydrogen spectrum of glutamic acid in the grafted material, thereby determining the grafting rate of HA-GLU molecules.

[0129] Specifically, the peak area integral ∫ of the methine -CH- protons (shifted to 4.05-4.12 ppm) brought by glutamic acid in the grafted product is measured to make the judgment (the integral area at this point is 1 if the grafting rate is 100%);

[0130] Table 4

[0131] Experimental Example 2

[0132] First prepare the complete culture medium solution: mix DMEM basal culture medium, FBS fetal bovine serum, and PS double antibody solution in a mass ratio of 89:10:1.

[0133] 0.009 g of UMW-HA, LMW-HA, MMW-HA, HMW-HA, GLU, physical mixture, and Graft-1 to Graft-9 powders were weighed and dissolved in complete culture medium solution to form a test mixture, with a content of 150 μg / mL.

[0134] Among them, No. 1 indicates that UMW-HA was added to the culture medium solution, wherein the content of UMW-HA was 150 μg / mL;

[0135] No. 2 indicates that LMW-HA was added to the culture medium solution, wherein the content of LMW-HA was 150 μg / mL;

[0136] No. 3 indicates that MMW-HA was added to the culture medium solution, wherein the content of MMW-HA was 150 μg / mL;

[0137] No. 4 indicates that HMW-HA was added to the culture medium solution, wherein the content of HMW-HA was 150 μg / mL;

[0138] No. 5 indicates that GLU was added to the culture medium solution, wherein the content of GLU was 150 μg / mL;

[0139] No. 6 indicates that LMW-HA and GLU are added to the culture medium solution, wherein the content of LMW-HA is 136 μg / mL and the content of GLU is 14 μg / mL;

[0140] No. 7 indicates that Graft-1 was added to the culture medium solution, wherein the content of Graft-1 was 150 μg / mL;

[0141] No. 8 indicates that Graft-2 is added to the culture medium solution, wherein the content of Graft-2 is 150 μg / mL;

[0142] No. 9 indicates that Graft-3 is added to the culture medium solution, wherein the content of Graft-3 is 150 μg / mL;

[0143] No. 10 indicates that Graft-4 is added to the culture medium solution, wherein the content of Graft-4 is 150 μg / mL;

[0144] No. 11 indicates that Graft-5 is added to the culture medium solution, wherein the content of Graft-5 is 150 μg / mL;

[0145] No. 12 indicates that Graft-6 is added to the culture medium solution, wherein the content of Graft-6 is 150 μg / mL;

[0146] No. 13 indicates that Graft-7 is added to the culture medium solution, wherein the content of Graft-7 is 150 μg / mL;

[0147] No. 14 indicates that Graft-8 is added to the culture medium solution, wherein the content of Graft-8 is 150 μg / mL;

[0148] No. 15 indicates that Graft-9 is added to the culture medium solution, wherein the content of Graft-9 is 150 μg / mL.

[0149] The cytology test method is as follows:

[0150] 1) Cell seeding: 1×10 5 Fibroblasts were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator (37° C., 5% CO 2 ) for 24 h.

[0151] 2) Modeling: According to the experimental groups, the sample group and the model control group were exposed to ultraviolet light therapy device (wavelength 280-320nm, 80mJ / cm 2 ) for UVB irradiation.

[0152] 3) Dosing: Dosing was performed in groups according to the test protocol in Table 5. Complete medium containing the test substance working solution was added to the sample group wells, with 1 mL of sample added to each well. Three replicates were set up for each group. After dosing, the 6-well plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 hours.

[0153] 4) Cell collection: After culturing for 24 hours, collect the cell supernatant, wash twice with 1 mL / well D'Hanks buffer, lyse the cells by pipetting, and collect the sample.

[0154] 5) Gene expression detection: After the cells were treated with RNA-Quick Purification Kit, samples were collected and RNA extraction, reverse transcription and fluorescence quantitative PCR were performed according to the kit instructions. -△△Ct Method is used to calculate the results and obtain the test results.

[0155] The test results are shown in Table 5, and the standard deviation of the experimental test is 0.01. The lower the relative expression of IL-6, the stronger the effect of the test sample in inhibiting IL-6 expression. Compared with a single hyaluronic acid or glutamic acid, if the relative expression of IL-6 of the graft of the same amount is lower, it means that the graft has a synergistic effect. Compared with the single components of hyaluronic acid or glutamic acid of the same amount, the relative expression of IL-6 of the graft of the present application is reduced, and compared with the physical mixture of the two of the same amount, the relative expression of IL-6 is also reduced, indicating that the graft has a synergistic effect.

[0156] Table 5

[0157] Although the present invention has been disclosed above with reference to the embodiments, they are not intended to limit the present invention. Anyone with ordinary knowledge in the art may make slight changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the patent application attached 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 the carboxyl groups of the hyaluronic acid or its salt is linked to the amino group of glutamic acid or its salt in the form of an amide bond.

2. The grafted compound according to claim 1, wherein the general formula is as shown in formula (I): in, In the formula (I), R is a metal ion or H, 0≤x<1, 0<y≤1, x+y=1, and n is a positive integer from 1 to 2000.

3. The grafted compound according to claim 1, wherein The grafting rate of the grafted compound is 20% to 100%.

4. The grafted compound according to claim 1, wherein The molecular weight of the grafted compound is 3k-500kDa.

5. The grafted compound according to claim 1, wherein The grafted compound is detected by nuclear magnetic resonance. 1 In the H NMR spectrum, the grafted compound has a characteristic peak at a chemical shift δ of 4.00-4.15 ppm; Preferably, The grafted compound is detected by nuclear magnetic resonance. 1 In the H NMR spectrum, the grafted compound has characteristic peaks at chemical shifts δ of 2.10-2.25 ppm and 1.71-2.10 ppm.

6. A method for preparing a grafted compound of hyaluronic acid or a salt thereof and glutamic acid or a salt thereof according to any one of claims 1 to 5, wherein: Preparation of quaternary ammonium salt of hyaluronic acid or its salt: mixing hyaluronic acid or its salt and quaternary ammonium base, and reacting to obtain quaternary ammonium salt of hyaluronic acid or its salt; Preparation of grafted compound: dissolve the quaternary ammonium salt of hyaluronic acid or its salt, then add glutamic acid or its salt, activator and catalyst to react, hydrolyze in alkaline environment, adjust pH, and obtain grafted compound of hyaluronic acid or its salt and glutamic acid or its salt.

7. A cosmetic composition, comprising the grafted compound according to any one of claims 1 to 5 or the grafted compound prepared by the method according to claim 6.

8. Use of the grafted compound according to any one of claims 1 to 5 or the grafted compound prepared by the method according to claim 6 in cosmetics.

9. Use of the grafted compound according to any one of claims 1 to 5, the grafted compound prepared by the method according to claim 6, or the cosmetic composition according to claim 7 in preventing and / or alleviating skin discomfort caused by inflammation.

10. Use of the grafted compound according to any one of claims 1 to 5, the grafted compound prepared by the method according to claim 6, or the cosmetic composition according to claim 7 in anti-inflammatory treatment.

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