Polycyclic substituted methylene acetone compound and anti-saccharification application thereof

Abstract

The invention discloses application of a compound shown as a formula I or salt or deuterated substance thereof in preparation of an anti-saccharification agent, in which ring A is a benzene ring or a five-membered ring containing O or S; the ring A may be substituted by m Ra, m being 0, 1, 2, 3 or 4; ra is hydroxyl, methyl, methoxyl, nitro or dimethylamino, and the deuterated substance is a partial deuterated substance and / or a perdeuterated substance. In the experiment of inhibiting generation of AGEs, compared with an Amino guanidine AG (Amino guanidine, AG), the anti-saccharification effect is obvious: under the low concentration, the anti-saccharification inhibition rate (the final administration concentration is 33 mu M) of the aromatic methylene acetone active small molecule compound protected by the invention is between 10% and 58%, and is far higher than the anti-saccharification inhibition rate (8.4%) of the Amino guanidine AG under the same final administration concentration (33 mu M), and the anti-saccharification inhibition rate (8.4%) of the Amino guanidine AG is far higher than the anti-saccharification inhibition rate (8.4%) of the Amino guanidine AG under the same final administration concentration (33 mu M) of the Amino guanidine AG. And the anti-saccharification inhibition rate of the radix codonopsis AG approaches to the final administration concentration (1mM) and is 57%. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a polycyclic substituted methyleneacetone compound and an anti-glycation application thereof. Background Art

[0002] Protein glycation occurs slowly and continuously throughout the life cycle of an organism, leading to the continuous accumulation of advanced glycation end products (AGEs) and their involvement in the pathogenesis of age-related diseases.

[0003] The process of protein glycation is complex. The process is initiated by the reaction of the following substances: the carbonyl group of reducing sugars (such as glucose, fructose or ribose, etc.) forms Schiff bases with proteins with free amino groups, and then undergoes Amadori rearrangement. The resulting Amadori products are further degraded to form active dicarbonyl compounds such as methylglyoxal, glyoxal and 3-deoxyglucose ketone. The dicarbonyl compounds then react with a single amino group on the side chain of lysine or arginine, or the sulfhydryl group of cysteine ​​in the protein, or cross-link two of the residues. These reactions lead to the formation of various cross-linked adducts, collectively referred to as advanced glycation end products (AGEs). Amadori products can also directly rearrange to form AGEs.

[0004] AGEs tend to bind to proteins with high lysine and arginine unit content and long half-life, such as collagen, serum albumin, elastin, myelin, low-density lipoprotein, plasminogen activator, fibrinogen, etc. These glycated proteins lose their function and are resistant to proteolytic clearance by proteases. The accumulation of AGEs in the extracellular matrix leads to its abnormal cross-linking and reduced vascular elasticity, leading to atherosclerosis. In addition, AGEs receptors (RAGE) are expressed on many cell types. AGE-RAGE interactions lead to the production of reactive oxygen species, inflammation, angiogenesis, and proliferation through various signal transduction pathways. The activation of RAGE stimulates the phosphorylation of extracellular signal-regulated kinases, ultimately leading to the stimulation of the transcription factor NF-κB and the production of proinflammatory cytokines. In addition, the stimulation of RAGE leads to the activation of the transforming growth factor (TGF-β) pathway and induces the overexpression of vascular endothelial growth factor. The formation of intracellular AGEs can also lead to nitric oxide quenching and impaired growth factor function.

[0005] Protein glycation that occurs in heat-treated foods is called the Maillard reaction, and the AGEs produced are introduced into the human body through the exogenous route of diet. AGEs are naturally present in uncooked animal-derived foods, and cooking forms new AGEs in these foods. Nε-(carboxymethyl)lysine (CML) levels have been found to be a useful marker of AGE content in the diet and have been selected to characterize the AGE load of foods. A survey of the CML content of 549 foods showed that grilling, baking, scorching and frying accelerate the formation of new AGEs. These modified proteins are not digested but can be fermented by colon bacteria. After fermentation, AGEs form toxic compounds that have negative effects on human health.

[0006] Skin aging is a complex process, which is manifested by the decline of body function and regeneration capacity. Skin aging includes exogenous and intrinsic aging. Exogenous aging is mainly related to ultraviolet radiation, while intrinsic aging is an inevitable physiological change that occurs over time. In recent years, studies have shown that intrinsic aging is closely related to skin glycation. Glycation is a non-enzymatic reaction between nucleic acids, proteins and lipids and sugars, which ultimately produces stable and irreversible products, namely advanced glycation end products (AGEs). Skin glycation acts on the body through the following pathways, leading to the occurrence of aging. First, the extracellular matrix (ECM) is one of the main targets of glycation. Collagen reacts with sugar to produce AGEs. The AGEs deposited in the matrix cross-link with the ECM, which damages the function of collagen and fibrin, resulting in a decrease in the elasticity and contractility of connective tissue. At the same time, glycated collagen inhibits the degradation of matrix metalloproteinases (MMPs), accelerates the degradation of collagen and reduces the synthesis of functional collagen. In addition, AGEs have the characteristics of yellow-dark color, which plays a key role in the process of skin color changing to dark yellow.

[0007] In short, the accumulation of AGEs in the body, especially on the skin, will cause cross-linking damage to proteins and binding to specific receptors, causing many skin problems, leading to dull skin, wrinkles, decreased collagen fiber elasticity, dark brown spots, and even promoting the production of matrix metalloproteinases that can decompose collagen and elastin. These factors will eventually lead to problems such as accelerated skin aging.

[0008] Although some existing oral medicines, health products, functional foods, and skin care products have anti-glycation effects, they cannot solve the changes in the aging level in the body, and lack dietary protein anti-glycation active ingredients. Therefore, there is an urgent need for safe and effective active ingredients for human anti-glycation and dietary protein anti-glycation as active additives for cosmetics, health products, functional foods, medicines and other products that maintain human health.

[0009] The prior art does not find the use of the compound or its salt or deuterated product required by the present invention in the preparation of an anti-glycation agent. Summary of the invention

[0010] Based on this, the present invention provides a use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of an anti-glycation agent:

[0011]

[0012] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0013] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0014] Wherein, Ra is hydroxy, methyl, halogen, methoxy, nitro or dimethylamino;

[0015] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0016] Further, the compound of formula I is

[0017]

[0018] Further, the compound of formula II is

[0019]

[0020] wherein R1, R2, R3, R4 and R5 are each independently selected from the group consisting of hydrogen, hydroxy, halogen, methyl, methoxy, nitro and dimethylamino.

[0021] Further, R1 is hydrogen, methoxy, halogen or methyl, R2 is hydrogen, hydroxyl, methyl or methoxy, R3 is hydrogen, hydroxyl, halogen, methoxy, nitro or dimethylamino, R4 is hydrogen, hydroxyl or methoxy, and R5 is hydrogen, hydroxyl, halogen or methyl.

[0022] Further, the compound of formula I is any one of the following compounds:

[0023]

[0024]

[0025] According to another aspect of the present invention, there is provided an anti-glycation composition, the anti-glycation composition comprising a compound of formula I or a salt or deuterated substance thereof;

[0026] Wherein, the compound of formula I is a compound having anti-glycation activity that inhibits the generation of the final product of non-enzymatic glycation reaction.

[0027] Furthermore, the compound of formula I is synthesized or derived from an extract, a crude extract and / or an enriched fraction of a natural raw material.

[0028] Furthermore, the natural raw materials include plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products thereof.

[0029] According to another aspect of the present invention, there is provided a use of the anti-glycation composition in the preparation of an anti-glycation agent, wherein the anti-glycation composition is a skin composition, a pharmaceutical composition, a health product, a functional food and / or a food additive.

[0030] Furthermore, the skin composition further comprises one or more skin care substances for preventing and / or improving skin aging and / or accelerated skin aging, such as carnosine, acetylated hyaluronic acid, adenosine and / or kava extract.

[0031] Furthermore, the skin composition is in the form of an emulsion, an essence, a toner, a face cream or a face mask.

[0032] Furthermore, the food additive is used to resist glycation during the cooking and storage of food and its raw materials, or to slow down the deterioration of food and its raw materials during the storage process.

[0033] Furthermore, the skin aging and / or accelerated skin aging includes one or more of the following: dull skin, skin wrinkles, decreased elasticity of skin collagen fibers, and skin pigmentation patches.

[0034] Furthermore, the skin composition further comprises other auxiliary materials.

[0035] Furthermore, the auxiliary material is selected from one or more of the following: a thickener, a pH adjuster, a preservative, an antibacterial agent and a stabilizer.

[0036] Furthermore, the thickener is selected from one or more of the following: carbomer, sodium polyacrylate, xanthan gum and hydroxyethyl cellulose.

[0037] Further, the pH adjuster is selected from one or more of the following: triethanolamine, aminomethyl propanol, citric acid and lactic acid.

[0038] Further, the preservative is selected from one or more of the following: methylparaben, propylparaben, butylparaben, p-hydroxyacetophenone and phenoxyethanol.

[0039] Furthermore, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0040] Furthermore, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, retention agent, lubricant, dispersant, plasticizer, opacifier and antioxidant.

[0041] Furthermore, the dosage form of the pharmaceutical composition is a patch, emulsion, tablet, pill, capsule, powder, film, lozenge, granule or oral solution.

[0042] Furthermore, the health care product and / or functional food is packaged food and food raw material.

[0043] Beneficial effects of the present invention:

[0044] In the experiment of inhibiting AGEs generation, compared with the Yangshen drug aminoguanidine (AG), the anti-glycation effect is significant: at low concentrations, the anti-glycation inhibition rate of the aromatic methyleneacetone active small molecule compound protected by the present invention (final administration concentration of 33 μM) is between 10% and 58%, which is much higher than the anti-glycation inhibition rate (8.4%) of Yangshen AG at the same final administration concentration (33 μM), and is close to the anti-glycation inhibition rate (57%) of Yangshen AG at the final administration concentration (1 mM). BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0046] Figure 1 The figure is a schematic diagram of the screening results of the anti-glycation activity (AGEs inhibition rate %) of the polycyclic substituted methylene acetone compounds of Example 1. Among them, ***P<0.001 vs. control (only adding the same amount of solvent DMSO as that of the sample to be tested in the glycation reaction system). DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0048] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0049] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0050] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0051] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0052] As described in the background technology section, the prior art does not find the use of the compound or its salt or deuterated product required by the present invention in the preparation of an anti-glycation agent. In order to solve the above problem, the present invention provides a compound of the following formula I or its salt or deuterated product for use in the preparation of an anti-glycation agent,

[0053]

[0054] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0055] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0056] Wherein, Ra is hydroxy, methyl, halogen, methoxy, nitro or dimethylamino;

[0057] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0058] Such anti-glycation active molecules are aromatic methylene acetone active small molecule compounds, which are derived from synthetic or natural plants and have good safety. In addition, the structure of aromatic methylene acetone active small molecule compounds is relatively simple, and chemical synthesis or extraction and separation are relatively easy. In particular, chemical synthesis can obtain target products with high yields through reactions with cheap raw materials, and the production cost is relatively low, which is suitable for large-scale production. Furthermore, aromatic methylene acetone active small molecule compounds have relatively good stability and are suitable for large-scale applications in various fields such as medical drugs, food, food additives, health products, skin care products, and cosmetics.

[0059] In a preferred embodiment, the compound of formula I is

[0060]

[0061] In a preferred embodiment, the compound of formula II is

[0062]

[0063] wherein R1, R2, R3, R4 and R5 are each independently selected from the group consisting of hydrogen, hydroxy, halogen, methyl, methoxy, nitro and dimethylamino.

[0064] In a preferred embodiment, R1 is hydrogen, methoxy, halogen or methyl, R2 is hydrogen, hydroxyl, methyl or methoxy, R3 is hydrogen, hydroxyl, halogen, methoxy, nitro or dimethylamino, R4 is hydrogen, hydroxyl or methoxy, and R5 is hydrogen, hydroxyl, halogen or methyl.

[0065] In a preferred embodiment, the compound of formula I is any one of the following compounds:

[0066]

[0067]

[0068] According to another aspect of the present invention, there is provided an anti-glycation composition, the anti-glycation composition comprising a compound of formula I or a salt or deuterated substance thereof;

[0069] Wherein, the compound of formula I is a compound having anti-glycation activity that inhibits the generation of the final product of non-enzymatic glycation reaction.

[0070] In a preferred embodiment, the compound of formula I is synthesized or is derived from an extract, a crude extract and / or an enriched fraction of a natural raw material.

[0071] In a preferred embodiment, the natural raw material includes plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products thereof.

[0072] According to another aspect of the present invention, there is provided a use of the anti-glycation composition in the preparation of an anti-glycation agent, wherein the anti-glycation composition is a skin composition, a pharmaceutical composition, a health product, a functional food and / or a food additive.

[0073] In a preferred embodiment, the skin composition further comprises one or more skin care substances for preventing and / or improving skin aging and / or accelerated skin aging, such as carnosine, acetylated hyaluronic acid, adenosine and / or kava extract.

[0074] In a preferred embodiment, the skin composition is in the form of an emulsion, an essence, a toner, a cream or a mask.

[0075] In a preferred embodiment, the food additive is used to resist glycation during the cooking and storage of food and its raw materials, or to slow down the deterioration of food and its raw materials during the storage process.

[0076] In a preferred embodiment, the skin aging and / or accelerated skin aging includes one or more of the following: dull skin, skin wrinkles, decreased elasticity of skin collagen fibers, and skin pigmentation patches.

[0077] In a preferred embodiment, the skin composition further comprises other auxiliary materials.

[0078] In a preferred embodiment, the auxiliary material is selected from one or more of the following: a thickener, a pH adjuster, a preservative, an antibacterial agent and a stabilizer.

[0079] In a preferred embodiment, the thickener is selected from one or more of the following: carbomer, sodium polyacrylate, xanthan gum and hydroxyethyl cellulose.

[0080] In a preferred embodiment, the pH adjuster is selected from one or more of the following: triethanolamine, aminomethyl propanol, citric acid and lactic acid.

[0081] In a preferred embodiment, the preservative is selected from one or more of the following: methylparaben, propylparaben, butylparaben, p-hydroxyacetophenone and phenoxyethanol.

[0082] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0083] In a preferred embodiment, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, retention agent, lubricant, dispersant, plasticizer, opacifier and antioxidant.

[0084] In a preferred embodiment, the pharmaceutical composition is in the form of a patch, emulsion, tablet, pill, capsule, powder, film, lozenge, granule or oral solution.

[0085] In a preferred embodiment, the health care product and / or functional food is packaged food and food raw material.

[0086] According to another aspect of the present invention, there is provided a use of a compound of the following formula I or a salt or deuterated product thereof, or a composition comprising a compound of the following formula I or a salt or deuterated product thereof, in the preparation of an in vitro anti-glycation agent or an in vitro elastase inhibitor,

[0087]

[0088] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0089] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0090] Wherein, Ra is hydroxy, methyl, halogen, methoxy, nitro or dimethylamino.

[0091] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0092] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0093] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in the present specification can be used in any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0094] Example 1

[0095] Anti-glycation test (inhibition of the formation of non-enzymatic glycation end products AGEs) experimental method

[0096] 1. Purpose of the experiment

[0097] The ability of the compounds to inhibit the synthesis of AGEs was evaluated by fluorescence detection of non-enzymatic glycation end products (AGEs).

[0098] 2. Experimental Principle

[0099] Advanced glycation end products (AGEs) refer to a group of stable end products produced by the condensation, rearrangement, cleavage, and oxidation modification of free amino groups of macromolecules such as proteins, amino acids, lipids, or nucleic acids with aldehyde groups of reducing sugars under non-enzymatic conditions. The production of AGEs can be inhibited by preventing the formation of early glycation products of reducing sugars.

[0100] AGEs have the property of spontaneous fluorescence, so the content of AGEs can be reflected by fluorescence detection.

[0101] 3. Reagents and consumables

[0102] Main reagents: bovine serum albumin (BSA; Absin, 9048-46-8), PBS buffer (Absin, abs962-500ml), aminoguanidine hydrochloride (AG; MCE, HY-B1041), methylglyoxal (MGO; Aladdin, M109035-250g), 96 ELISA plate (JET BIOFIT, FEP100096), the aromatic methyleneacetone compound ZK-1 to be tested was homemade (identified as osmundacetone by hydrogen spectrum and mass spectrometry, purity>95%), ZK-2, ZK-4, ZK-17, ZK-19, ZK-20, ZK-21, ZK-22, ZK-23, and ZK-25 were purchased from Anhui Zesheng Technology Co., Ltd. (Annaiji brand) with a purity of>95%. ZK-3, ZK-5, ZK-6, ZK-7, ZK-8, ZK-9, ZK-10, ZK-11, ZK-12, ZK-13, ZK-14, ZK-15, ZK-16, ZK-24, and ZK-26 were purchased from Nanchang Chennuo Biotechnology Co., Ltd. with a purity of >95%.

[0103]

[0104]

[0105] 4. Experimental equipment

[0106] The experimental equipment is shown in Table 1.

[0107] Table 1 Experimental equipment

[0108] Device Name brand Part Number Constant temperature oscillator Hangzhou Aosheng MB100-4A ELISA reader Thermo VL0L00D0

[0109] 5. Reagent preparation

[0110] a) BSA: Prepare 20 mg / ml BSA in PBS and filter with 0.22 μm filter membrane after complete dissolution;

[0111] b) MGO: The original solution was 40% concentration, which was diluted with PBS to 1% MGO solution;

[0112] c) The positive control compound was aminoguanidine hydrochloride (AG), with final concentrations of 1 mM and 33 μM;

[0113] d) The sample to be tested is dissolved in DMSO to a 20-40 mM stock solution, and the final reaction concentration is 33 μM.

[0114] 6. Experimental process

[0115] a) Add 20 mg / ml BSA solution and 1% MGO solution into a 96-well ELISA plate at a ratio of 1:1;

[0116] b) Add the Yangshen compound / test sample / DMSO to a 96-well ELISA plate, shake and mix, and use a fluorescence ELISA reader to detect the sample fluorescence value RFU under the conditions of an excitation wavelength of 370 nm and an emission wavelength of 440 nm;

[0117] c) Place the 96-well plate in a constant temperature oscillator at 45°C in the dark and incubate for 16 hours;

[0118] d) After 16 hours, the 96-well plate was centrifuged at 1000 rpm for 5 minutes, and the fluorescence value RFU of the sample was detected using a fluorescence microplate reader under the conditions of an excitation wavelength of 370 nm and an emission wavelength of 440 nm.

[0119] 7. Data Analysis

[0120] The AGEs inhibition rate was calculated using the formula for the inhibition rate of the compound on AGEs generation, and the experimental results were expressed as Mean±SD:

[0121]

[0122] Note: The negative control is the fluorescence value of BSA and MGO detected after 16 hours of incubation, the test group is the fluorescence value of the test object detected after 16 hours of incubation, and the blank control is the fluorescence value of BSA and MGO detected at 0 hours.

[0123] T-test is calculated using the T.TEST function of Microsoft Office365 Excel. The specific parameter selection is T.TEST(data-1, data-2, tails=2, type=2), i.e. T.TEST(array-1, array-2, tails=2, type=2).

[0124] 8. Experimental results

[0125] The experimental results are as follows Figure 1As shown in Table 2, Table 3 and Table 4, in the experiment of inhibiting AGEs generation, compared with the Yangshen drug aminoguanidine (AG), the anti-glycation effect is significant: at low concentrations, the anti-glycation inhibition rate of the aromatic methylene acetone active small molecule compound protected by the present invention (final administration concentration of 33 μM) is between 10% and 58%, which is much higher than the anti-glycation inhibition rate (8.4%) of Yangshen AG at the same final administration concentration (33 μM), and is close to the anti-glycation inhibition rate (57%) of Yangshen AG at the final administration concentration (1 mM). The same type of aromatic methylene acetone compounds ZK-25 and ZK-26 have no anti-glycation activity. Compared with the negative control, the significant T test P values ​​of Yangshen AG (test concentration 1 mM) and compounds ZK-1 to ZK-24 are all less than 0.001, while the significant T test P values ​​of compounds ZK-25 and ZK-26 are all greater than 0.05.

[0126] Table 2 Anti-glycation activity of compounds (AGEs inhibition rate %)

[0127]

[0128]

[0129] Table 3 T test of fluorescence values ​​of compounds ZK-25, ZK-26 and negative control (DMSO)

[0130]

[0131] Table 4 Significance T test of anti-glycation inhibition rate of compounds ZK-25, ZK-26 and negative control (DMSO)

[0132]

[0133] Example 2

[0134] Elastase inhibitory activity

[0135] 1. Principle of Elastase Inhibition Assay

[0136] The main characteristics of aging skin are thinning of the epidermis, flattening of the dermal-epidermal junction, and overall degradation of the extracellular matrix (ECM) of the dermis. Elastic fibers play an important role in skin aging. Their degradation and destruction can cause skin elasticity loss, sagging, various wrinkles, and even patches and nodules with lost skin color. Elastase is one of the most important enzymes in the matrix metalloproteinase family. It is mainly synthesized and secreted by fibroblasts. It is a highly selective and specific proteolytic enzyme that can degrade elastin in the skin, leading to a series of aging symptoms such as reduced skin elasticity, sagging, and wrinkles.

[0137] Elastase reacts with the enzyme substrate SucAla3-pNA (N-succinyl-tri-L-alanine 4-nitroanilide) to generate p-nitroaniline (pNA), which is yellow and absorbs at 410nm. The absorbance changes after adding the active substance, and the magnitude of the absorbance change can reflect the inhibition rate of the elastase inhibitor.

[0138] 2. Main materials, reagents and instruments

[0139] Elastase (Porcine pancreas), MedChemExpress; SucAla3-pNA, Tris, Sigma-Aldrich, Co.; Quercetin (purity>98%), Anhui Zesheng Technology Co., Ltd.; Oleanolic acid (purity>98%), Nanjing Yuanzhi Biotechnology Co., Ltd.; ELISA plate, sample tank, Guangzhou Jiete Biofiltration Co., Ltd.; Microplate constant temperature oscillator, Hangzhou Aosheng Instrument Co., Ltd.; XSR205DU / A analytical balance, Mettler-Toledo Instrument Co., Ltd.; Varioskan TM LUX Multi-Mode Microplate Reader, Thermo Fisher Scientific Inc.

[0140] 3. Experimental methods

[0141] 3.1 Solution preparation

[0142] Buffer: 100 mM Tris-HCl, pH 8.0

[0143] SucAla3-pNA: weigh an appropriate amount and dissolve it in Tris-HCl buffer to 1.6 mM

[0144] Elastase: Weigh an appropriate amount and dissolve it in ddH2O to 1 mg / mL, then dilute it with an equal volume of Tris-HCl buffer solution.

[0145] Quercetin: weigh an appropriate amount and dissolve it in DMSO to 3.02 mg / mL

[0146] Oleanolic acid: weigh an appropriate amount and dissolve in DMSO to 5.00 mg / mL

[0147] 3.2 Reaction system and sample testing

[0148] As shown in Table 5, 140 μL Tris-HCl buffer solution, 5 μL sample solutions of different concentrations (test sample solution, or positive control solution, or DMSO as negative control solution) and 5 μL 0.5 mg / mL elastase were added to a 96-well plate, shaken and mixed, and incubated at room temperature for 15 min, then 50 μL 1.6 mM elastase substrate SucAla3-pNA was added, and immediately shaken and mixed, and the absorbance at 0 min was measured at 410 nm using an enzyme reader, followed by incubation at room temperature for 15 min. After 15 min, the absorbance at 15 min was measured at 410 nm using an enzyme reader, and the inhibition rate of the test sample on elastase was calculated by analyzing and processing the data.

[0149] Table 5 Reaction system and sample detection

[0150]

[0151] Note: “-” means not included

[0152] Inhibition rate calculation formula:

[0153]

[0154] Where:

[0155] A 样品0 The absorption wavelength OD value of the product pNA generated by the sample under test at 410nm at 0min;

[0156] A 样品15 The characteristic absorption wavelength OD value of the product pNA generated when the sample to be tested is measured at 410nm at the 15th minute;

[0157] A 空白0 The characteristic absorption wavelength OD value of the generated product pNA was measured at 410 nm for the negative control group (DMSO) at 0 min;

[0158] A 空白15 At 15 minutes, the negative control group (DMSO) was measured at 410 nm for the characteristic absorption wavelength OD value of the generated product pNA.

[0159] 4. Experimental results

[0160] The experimental results are shown in Table 6.

[0161] Table 6 Elastase inhibitory activity measurement results (inhibition rate %)

[0162]

[0163]

[0164] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of an anti-glycation agent, characterized in that: Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S; wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4; Wherein, Ra is hydroxy, methyl, halogen, methoxy, nitro or dimethylamino; Wherein, the deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

2. The use according to claim 1, characterized in that The compound of formula I is Preferably, the compound of formula II is wherein R1, R2, R3, R4 and R5 are each independently selected from the group consisting of hydrogen, hydroxyl, halogen, methyl, methoxy, nitro and dimethylamino; Preferably, R1 is hydrogen, methoxy, halogen or methyl, R2 is hydrogen, hydroxy, methyl or methoxy, R3 is hydrogen, hydroxy, halogen, methoxy, nitro or dimethylamino, R4 is hydrogen, hydroxy or methoxy, and R5 is hydrogen, hydroxy, halogen or methyl; Preferably, the compound of formula I is any one of the following compounds:

3. An anti-glycation composition, characterized in that: The anti-glycation composition comprises a compound of formula I or a salt or deuterated substance thereof; Wherein, the compound of formula I is a compound having anti-glycation activity that inhibits the formation of end products of non-enzymatic glycation reactions.

4. The anti-glycation composition according to claim 3, characterized in that The compound of formula I is synthesized or derived from an extract, crude extract and / or enriched fraction of a natural raw material; Preferably, the natural raw materials include plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products thereof.

5. Use of the anti-glycation composition according to claim 3 or 4 in the preparation of an anti-glycation agent, characterized in that: The anti-glycation composition is a skin composition, a pharmaceutical composition, a health product, a functional food and / or a food additive.

6. The use according to claim 5, characterized in that The skin composition further comprises one or more skin care substances for preventing and / or improving skin aging and / or accelerated skin aging, such as carnosine, acetylated hyaluronic acid, adenosine and / or kava extract; Preferably, the skin composition is in the form of a lotion, essence, toner, cream or mask; More preferably, the food additive is used to resist glycation during the cooking and storage of food and its raw materials, or to slow down the deterioration of food and its raw materials during the storage process.

7. The use according to claim 6, characterized in that The skin aging and / or accelerated skin aging include one or more of the following: dull skin, skin wrinkles, decreased elasticity of skin collagen fibers, and skin pigmentation patches; Preferably, the skin composition further comprises other auxiliary materials; More preferably, the auxiliary material is selected from one or more of the following: including thickeners, pH regulators, preservatives, antibacterial agents and stabilizers; Preferably, the thickener is selected from one or more of the following: carbomer, sodium polyacrylate, xanthan gum and hydroxyethyl cellulose; Also preferably, the pH adjuster is selected from one or more of the following: triethanolamine, aminomethyl propanol, citric acid and lactic acid; Also preferably, the preservative is selected from one or more of the following: methylparaben, propylparaben, butylparaben, p-hydroxyacetophenone and phenoxyethanol.

8. The use according to claim 5, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, retention agent, lubricant, dispersant, plasticizer, opacifier and antioxidant.

9. The use according to claim 5, characterized in that The dosage form of the pharmaceutical composition is a patch, emulsion, tablet, pill, capsule, powder, film, lozenge, granule or oral solution.

10. The use according to claim 5, characterized in that The health care products and / or functional foods are packaged foods and food raw materials.

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