Application of furo-benzopyrone derivative in preparation of product for reducing accumulation of advanced glycosylation end product

Abstract

The invention discloses application of a furo-benzopyrone derivative in preparation of a product for reducing accumulation of advanced glycosylation end products, and belongs to the technical field of preparation of anti-saccharification products. The furo-benzopyrone derivative not only has a remarkable AGEs inhibition effect, can avoid accumulation of AGEs, but also can reduce skin yellowing caused by accumulation of AGEs, plays an anti-aging role by reducing AGEs, has no toxic effect on cells, has good safety, can meet the general requirements of anti-saccharification, and has good application prospects. The compound is used for preventing and / or treating diseases related to AGEs accumulation, such as senescence and diabetes, cardiovascular diseases, metabolic syndrome, obesity, hypertension, arteriosclerosis, neurodegenerative diseases and other degenerative diseases.

Description

Technical Field

[0001] The present application relates to the technical field of preparation of anti-glycation products, and in particular to the application of furanobenzopyrone derivatives in the preparation of products that reduce the accumulation of advanced glycation end products. Background Art

[0002] Glycation, also known as non-enzymatic glycation or Maillard reaction, refers to a non-enzymatic reaction that occurs in a biological environment. It can insert sugar chains into macromolecules such as proteins, DNA, and lipids to form very stable amino-sugar, nucleic acid-sugar, and lipid-sugar covalent structures; while glycosylation refers to the process or reaction of covalent binding of non-sugar biological molecules to sugars under the action of glycosyltransferases. Glycation can occur both in vitro and in vivo during physiological processes. Under non-enzymatic conditions in vivo, the free amino groups of macromolecules such as proteins, lipids, or nucleic acids and the aldehyde groups of reducing sugars undergo condensation, rearrangement, cleavage, and oxidation to produce a group of stable, non-degradable end products, which are called "Advanced Glycation End Products (AGEs)".

[0003] The accumulation of AGEs is closely related to a variety of chronic diseases and the aging process. The accumulation of AGEs in the body can destroy cell structure and function, leading to hardening of the extracellular matrix, especially in the skin, blood vessels, eyes and nervous system. Its cross-linking effect on collagen can lead to decreased skin elasticity, wrinkle formation, vascular hardening and other degenerative changes. In addition, AGEs are highly related to the occurrence and development of a variety of metabolic diseases such as diabetes, cardiovascular disease, and Alzheimer's disease.

[0004] Anti-glycation and anti-aging is a long-term process. Long-term use of chemically synthesized anti-glycation agents will bring challenges to the health of the body. Therefore, the use of natural products to fight glycation and aging has gradually attracted widespread attention. Studies have found that phenolic substances can prevent the formation of early fat oxidation products-Schiff bases, and play an anti-glycation role; studies have found that brown algae extracts are a natural source of anti-glycation agents, and gallic acid in brown algae extracts can be used as a carbohydrate inhibitor in the cosmetics field; studies have found that when the concentration of ziziphus elata polysaccharide ranges from 0.25-4.00 mg / mL, the ziziphus elata polysaccharide extracted by microwave-assisted low eutectic solvent method shows the strongest anti-glycation activity.

[0005] However, existing anti-glycation agents have limited effects in reducing the accumulation of AGEs and cannot meet the general requirements of anti-glycation. Summary of the invention

[0006] Based on this, the main purpose of the present application is to provide the use of furanobenzopyrone derivatives in the preparation of products for reducing the accumulation of advanced glycation end products. The furanobenzopyrone derivatives have significant AGEs inhibitory effects, can meet the general demand for anti-glycation, and are used to prevent, treat or improve diseases related to the accumulation of AGEs.

[0007] In a first aspect of the present application, a furanobenzopyrone derivative is provided for use in preparing a product for reducing accumulation of advanced glycation end products, wherein the furanobenzopyrone derivative is a compound represented by formula I:

[0008] (Formula I);

[0009] Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group;

[0010] Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H.

[0011] In some embodiments, the compound of Formula I is selected from at least one of the following compounds:

[0012] or .

[0013] In some embodiments, in some embodiments, R1 is -C(CH3)2-R6; R6 is -OH or -O-CO-CH3; R2 is H or methoxy.

[0014] In some embodiments, the furanobenzopyrone derivative is selected from at least one of the following compounds:

[0015] , .

[0016] In some embodiments, the product for reducing the accumulation of advanced glycation end products is a product for preventing and / or treating diseases associated with the accumulation of AGEs.

[0017] In some embodiments, the diseases associated with the accumulation of AGEs include aging and / or degenerative diseases; optionally, the degenerative diseases include at least one of diabetes, cardiovascular disease, metabolic syndrome, obesity, hypertension, arteriosclerosis, neurodegenerative diseases, orthopedic diseases and female ovarian dysfunction; optionally, the neurodegenerative diseases include Alzheimer's disease and / or peripheral neuropathy.

[0018] In some embodiments, the products that reduce accumulation of advanced glycation end products include cosmetics, foods, and pharmaceuticals.

[0019] In some embodiments, the cosmetic comprises at least one of lotion, cream, emulsion, essence, facial mask, and eye cream.

[0020] In some embodiments, the administration of the product for reducing accumulation of advanced glycation end products includes oral administration and / or topical administration.

[0021] In some embodiments, the external application includes topical administration and / or systemic transdermal administration.

[0022] In a second aspect, the present application provides a product for reducing the accumulation of advanced glycation end products, including a furanobenzopyrone derivative, wherein the furanobenzopyrone derivative has a structure of a compound shown in Formula I:

[0023] (Formula I);

[0024] Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group;

[0025] Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H;

[0026] Optionally, the product for reducing accumulation of advanced glycation end products further comprises auxiliary materials.

[0027] In a third aspect, the present application provides a product for preventing and / or treating diseases associated with the accumulation of AGEs, including a furanobenzopyrone derivative, wherein the furanobenzopyrone derivative has a structure of a compound shown in Formula I:

[0028] (Formula I);

[0029] Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group;

[0030] Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H;

[0031] Optionally, the product for preventing and / or treating diseases associated with the accumulation of AGEs further comprises excipients.

[0032] Compared with the prior art, this application has the following beneficial effects:

[0033] 1. The furanobenzopyrone derivative can not only reduce the generation of AGEs in the BSA-MGO pure in vitro AGEs generation model, but also effectively reduce the level of AGEs in the glucose-induced intracellular AGEs generation model, and has a significant AGEs inhibitory effect, and can be used to prevent, treat or improve diseases related to the accumulation of AGEs.

[0034] 2. The furanobenzopyrone derivatives can reduce skin yellowing caused by AGEs accumulation and exert anti-aging effects by reducing AGEs.

[0035] 3. The furanobenzopyrone derivatives have no toxic effect on cells and have good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art description. The drawings are only used to illustrate the preferred implementation methods and are not considered to be limitations of the present application. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0037] Figure 1 The results are the inhibition rate of Columbianetin acetate on AGEs;

[0038] Figure 2 This is the inhibition rate of 8-Methoxymarmesin on AGEs. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of this application clearer and to provide a more thorough and comprehensive understanding of the disclosed content of this application, the technical solution of this application will be clearly and completely described below in conjunction with the specific embodiments of this application and the corresponding drawings. The described embodiments are only part of the embodiments of this application, not all of them.

[0040] The implementation of the present application is described in detail below in conjunction with the accompanying drawings. This embodiment is implemented based on the technical solution of the present application, and a detailed implementation method and specific operation process are given, but the protection scope of the present application is not limited to the following embodiments.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0042] the term

[0043] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0044] In the present application, "multiple", "multiple", "at least one" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" or "at least one" means one or greater than or equal to two.

[0045] In the present application, the terms "preferable", "particularly", etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0046] The term "alkyl" refers to a saturated hydrocarbon containing primary (normal) carbon atoms, or secondary carbon atoms, or tertiary carbon atoms, or quaternary carbon atoms, or a combination thereof. Phrases containing this term, for example, "C1-C5 alkyl" refers to an alkyl containing 1 to 5 carbon atoms, and each occurrence can be independently C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl or C5 alkyl; "C2-C6 alkyl" refers to an alkyl containing 2 to 6 carbon atoms, and each occurrence can be independently C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl. Suitable examples include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -C H(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl -1-butyl (-CH2CH2CH(CH3)2) and 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl ( -CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3.

[0047] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0048] In a first aspect of the present application, a furanobenzopyrone derivative is provided for use in preparing a product for reducing accumulation of advanced glycation end products, wherein the furanobenzopyrone derivative is a compound represented by formula I:

[0049] (Formula I);

[0050] Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group;

[0051] Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H.

[0052] The furanobenzopyrone derivatives of the present application include Columbianetin acetate and 8-Methoxymarmesin, wherein Columbianetin acetate is called dihydrocarbamazepine acetate in Chinese, and its CAS number is 23180-65-6; 8-Methoxymarmesin has a molecular formula of C 15 H 16 O5, CAS number is 13895-93-7.

[0053] In the present application, Columbianetin acetate and 8-Methoxymarmesin can not only reduce AGEs generation in the BSA-MGO pure in vitro AGEs generation model, but also effectively reduce AGEs levels in the glucose-induced intracellular AGEs generation model, have a significant AGEs inhibitory effect, and can also reduce skin yellowing caused by AGEs accumulation, play an anti-aging effect by reducing AGEs, and have no toxic effect on cells, have good safety, can meet the general demand for anti-glycation, and are used to prevent and / or treat diseases related to the accumulation of AGEs, such as aging and diabetes, cardiovascular disease, metabolic syndrome, obesity, hypertension, arteriosclerosis, neurodegenerative diseases and other degenerative diseases.

[0054] It is understandable that C1-C5 alkoxy refers to -O-R7, wherein R7 is a C1-C5 alkyl group, and the C1-C5 alkoxy group may be a straight-chain alkoxy group, such as methoxy, ethoxy, propoxy, butoxy and pentoxy, or a branched alkoxy group, without specific limitation; among the C2-C6 alkyl groups substituted by hydroxyl or acetoxy groups, the C2-C6 alkyl groups may be straight-chain alkyl groups or branched alkyl groups, without specific limitation.

[0055] In some embodiments, the compound of Formula I is selected from at least one of the following compounds:

[0056] or .

[0057] In some embodiments, in some embodiments, R1 is -C(CH3)2-R6; R6 is -OH or -O-CO-CH3; R2 is H or methoxy.

[0058] In some embodiments, the furanobenzopyrone derivative is selected from at least one of the following compounds:

[0059] , .

[0060] In some embodiments, the product for reducing the accumulation of advanced glycation end products is a product for preventing and / or treating diseases associated with the accumulation of AGEs.

[0061] In some embodiments, the diseases associated with the accumulation of AGEs include aging and / or degenerative diseases; optionally, the degenerative diseases include at least one of diabetes, cardiovascular disease, metabolic syndrome, obesity, hypertension, arteriosclerosis, neurodegenerative diseases, orthopedic diseases and female ovarian dysfunction; optionally, the neurodegenerative diseases include Alzheimer's disease and / or peripheral neuropathy.

[0062] It can be understood that the furanobenzopyrone derivatives can not only reduce the generation of AGEs in the BSA-MGO pure in vitro AGEs generation model, but also effectively reduce the level of AGEs in the glucose-induced intracellular AGEs generation model, and have a significant inhibitory effect on the generation of AGEs, which can avoid the accumulation of AGEs and reduce the yellowing of the skin caused by the accumulation of AGEs. It plays an anti-aging effect by reducing AGEs, and is therefore highly feasible for preventing, treating or improving diseases caused by the accumulation of AGEs, such as aging, as well as degenerative diseases such as diabetes, cardiovascular disease, metabolic syndrome, obesity, hypertension, arteriosclerosis, neurodegenerative diseases, orthopedic diseases and female ovarian dysfunction.

[0063] In some embodiments, the products that reduce accumulation of advanced glycation end products include cosmetics, foods, and pharmaceuticals.

[0064] In some embodiments, the cosmetic comprises at least one of lotion, cream, emulsion, essence, facial mask, and eye cream.

[0065] In some embodiments, the administration of the product for reducing accumulation of advanced glycation end products includes oral administration and / or topical administration.

[0066] In some embodiments, the external application includes local topical administration and / or systemic transdermal administration.

[0067] It is understandable that the dosage form and administration mode of the product of the present invention are not particularly limited, for example, the dosage form can be selected from solid dosage forms, semi-solid dosage forms and liquid dosage forms, wherein solid dosage forms include but are not limited to tablets, capsules, pills and granules; semi-solid dosage forms include but are not limited to ointments, gel pastes and plasters; liquid dosage forms include but are not limited to solutions, syrups, emulsions and tinctures. Preferably, a liquid dosage form is used.

[0068] When used as an external product, local surface administration or systemic transdermal administration can be achieved by spraying, smearing, etc. When taken orally, it is most convenient to prepare an oral solution, and the medication compliance is good. When prepared into various dosage forms, necessary excipients, carriers or solvents can be added to adjust the uniformity, stability, storage performance, taste, etc. of the product. When the product for reducing the accumulation of advanced glycation end products is administered in a liquid dosage form, the effective concentration can be 0.1-100 μM, preferably 0.4-100 μM, specifically 0.4 μM, 1 μM, 3 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, etc.

[0069] In a second aspect, the present application provides a product for reducing the accumulation of advanced glycation end products, including a furanobenzopyrone derivative, wherein the furanobenzopyrone derivative has a structure of a compound shown in Formula I:

[0070] (Formula I);

[0071] Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group;

[0072] Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H;

[0073] Optionally, the product for reducing accumulation of advanced glycation end products further comprises auxiliary materials.

[0074] In a third aspect, the present application provides a product for preventing and / or treating diseases associated with the accumulation of AGEs, including a furanobenzopyrone derivative, wherein the furanobenzopyrone derivative has a structure of a compound shown in Formula I:

[0075] (Formula I);

[0076] Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group;

[0077] Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H;

[0078] Optionally, the product for preventing and / or treating diseases associated with the accumulation of AGEs further comprises excipients.

[0079] It is understandable that the furanobenzopyrone derivatives can be added with excipients to prepare different dosage forms. The excipients may specifically include carriers, fillers, diluents, excipients, solvents, functional additives, coating materials or encapsulation materials, etc.

[0080] Unless otherwise specified, the raw materials used in the following experiments can be purchased from the market.

[0081] The following are specific embodiments.

[0082] 1. The effects of different anti-glycation and anti-aging substances on the amount of AGEs produced in the BSA-MGO model.

[0083] Conventional AGEs have fluorescent properties. This experimental example uses the principle that methyl glycol and bovine serum albumin undergo glycation reaction in vitro to form AGEs for modeling. After adding anti-glycation and anti-aging substances, the AGE inhibition rate is calculated by the fluorescent quantitative method to evaluate its anti-glycation effect.

[0084] 1. Test materials and instruments

[0085] 1.1 Test reagents

[0086] Bovine serum albumin (BSA), methylglyoxal (MGO), glucose: analytical grade, Sigma, USA.

[0087] 1.2 Test samples

[0088] Anti-glycation and anti-aging substances: Columbianetin acetate, CAS#: 23180-65-6; 8-Methoxymarmesin, CAS#: 13895-93-7; Aminoguanidine, CAS#: 1937-19-5.

[0089] 1.3 Test equipment

[0090] Microplate reader (Synergy H1), Bio-Tek, USA.

[0091] 2. Test methods

[0092] 2.1 Establishment of in vitro protein AGE formation system

[0093] BSA (bovine serum albumin) and MGO (methylglyoxal) were prepared into 10 mg / ml and 25 mM reaction solutions, respectively, and the solvent was phosphate buffer (10 mM, pH 7.4) to form a BSA-MGO reaction system as a model group for AGEs generation. The OD value of the formed AGEs was measured by a microplate reader (Synergy H1, Bio-Tek) (detection wavelength 370 nm, reference wavelength 440 nm).

[0094] 2.2 Determination of inhibition of AGE formation in vitro

[0095] The mixed incubation solutions prepared in Examples 1A-3A, Examples 1B-3B, and Comparative Examples 1-2 were prepared with liquids containing 10 mg / ml BSA and 25 mM MGO. The control group (blank example) was a BSA-MGO reaction system without the test substance. The above liquids were sterile filtered with a 0.2 μm filter membrane and dispensed into 1.5 mL conical bottom centrifuge tubes.

[0096] Table 1: Contents of anti-glycation and anti-aging substances in different embodiments and comparative examples

[0097]

[0098] Then 100 μl of the reaction solution was placed in a black 96-well ELISA plate to detect the fluorescence value on day 0. The OD value of the formed AGEs was measured by an ELISA reader (Synergy H1, Bio-Tek) (detection wavelength 370 nm, reference wavelength 440 nm).

[0099] The remaining reaction solution was placed in a 37°C incubator for 4 days, and 100 μl of the reaction solution was placed in a black 96-well ELISA plate on the 4th day to detect the fluorescence value on the 4th day. The OD value of the formed AGEs was measured by an ELISA reader (Synergy H1, Bio-Tek) (detection wavelength 370 nm, reference wavelength 440 nm).

[0100] 2.3 Data processing and statistical analysis methods (each group of experiments was repeated at least 3 times)

[0101] The AGE inhibition rate was calculated using the formula;

[0102] AGE inhibition rate = (CT) / C × 100%;

[0103] Among them, C is the fluorescence intensity of the control group (blank case), that is, the fluorescence intensity of the BSA-MGO reaction system in the absence of anti-glycation and anti-aging substances; T is the fluorescence intensity of the BSA-MGO reaction system in the presence of anti-glycation and anti-aging substances.

[0104] 3. Test results

[0105] The inhibitory effect of the test samples on AGEs, such as Figure 1-2 And as shown in Table 2.

[0106] Table 2: AGE inhibition rate in examples, comparative examples and blank examples

[0107]

[0108] Through Figure 1-2 The AGEs inhibition rate was compared and analyzed with the results in Table 2. It was found that the AGEs inhibition rate of aminoguanidine at 1 mM was 9.24%, and the AGEs inhibition rate at 10 mM was 51.84%.

[0109] Columbianetin acetate showed a concentration-dependent increase in AGEs inhibition rate at 1μM, 3μM, and 10μM concentrations, which were 12.3%, 33.31%, and 58.51%, respectively. Moreover, when the drug concentration of Columbianetin acetate was higher than 3μM, the AGEs inhibition effect was higher than that of 1mM aminoguanidine.

[0110] As the concentration of 8-Methoxymarmesin increased at 1μM, 3μM, and 10μM, the AGEs inhibition rate gradually increased in a concentration-dependent manner, reaching 12.97%, 32.44%, and 51.37%, respectively. The AGEs inhibition effect was higher than that of 1mM aminoguanidine.

[0111] The above results prove that in the in vitro BSA-MGO system, Columbianetin acetate and 8-Methoxymarmesin can significantly inhibit the formation of AGEs, and this inhibitory effect is concentration-dependent.

[0112] 2. Effects of different glucose concentrations on AGEs levels in HaCaT cells.

[0113] 1. Test materials and instruments

[0114] 1.1 Test reagents

[0115] Human keratinocytes (HaCaT) cells, glucose: analytical grade, Sigma, USA. BCA (bicinchoninic acid) protein assay kit: Bio-Tech Biotechnology Co., Ltd. Human advanced glycation end products (AGEs) enzyme-linked immunosorbent assay kit: Wuhan Huamei Biotechnology.

[0116] 1.2 Test equipment

[0117] Microplate reader (Synergy H1), Bio-Tek, USA.

[0118] 2. Test methods

[0119] 2.1 Construction of cell glycosylation model

[0120] This application uses glucose (D-Glucose) (MCE, HY-B0389, purity: ≥98.0%) as an inducer for the construction of a human keratinocyte (HaCaT) cell saccharification model. The cells are grown to the logarithmic growth phase and plated so that the confluence of the cells in the 6 cm dish reaches 60% on the second day.

[0121] 2.2 Effects of different glucose concentrations on HaCaT cell viability

[0122] HaCaT was inoculated into a 96-well plate at a concentration of 1.5 × 10 5 / ml suspension 100μl, culture until the next day. The drug was diluted with culture medium to obtain Examples 4-6 and blank examples as shown in Table 3, respectively, so that the final concentrations of glucose were 100mM, 150mM, and 200mM, respectively. After the liquid was replaced, the culture was continued for 24h, and 10μl CellCounting Kit-8 (CCK-8, Dojindo) was added to each well of the 96-well plate and cultured for 1~ 2h, and the OD value was detected at 450nm using an enzyme microplate reader. CCK8 is a kit based on WST-8 and widely used for rapid and highly sensitive detection of cell proliferation and cytotoxicity. WST-8 can be reduced by some dehydrogenases in mitochondria to generate orange-yellow formazan in the presence of an electron coupling reagent. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color is linearly related to the number of cells.

[0123] Table 3: Glucose content in different embodiments

[0124]

[0125] Table 4: Cell viability in Examples 4-6 and blank examples

[0126]

[0127] 2.3 Effects of different glucose concentrations on AGE levels in HaCaT cells

[0128] The drug was diluted with complete medium (10% FBS (Gibco) + DMEM (Gibco)) to obtain Examples 4-6 and blank examples as shown in Table 3, so that the final concentrations of glucose were 100 mM, 150 mM, and 200 mM, respectively. After incubating the cells for 48 hours, the cell samples were collected for BCA and intracellular AGEs content determination, and the AGEs content per unit protein was calculated.

[0129] The OD value of the formed AGEs was measured by a microplate reader (Synergy H1, Bio-Tek) (detection wavelength 370 nm, reference wavelength 440 nm). BCA (bicinchoninic acid) was measured using the known technology in the art. Under alkaline conditions, divalent copper ions can be reduced to monovalent copper ions by proteins. The monovalent copper ions interact with the unique BCA Solution A to produce a sensitive color reaction. The water-soluble complex shows strong absorbance at 562nm. The absorbance and protein concentration have a good linear relationship in a wide range, so the protein concentration can be calculated based on the absorbance value. The results are shown in Table 5.

[0130] Table 5: Effects of different examples on AGEs levels in HaCaT cells

[0131]

[0132] According to the cell viability test results in Section 2.2 and microscopic observations, when the glucose concentration reached 200 mM, the cells showed obvious cell death. Combined with Table 5, it can be seen that when the final glucose concentration reached 150 mM, the AGEs level increased to 1.45 times the normal level. Therefore, 150 mM glucose can be used as the induction condition for the cell glycation model.

[0133] 3. Columbianetin acetate and 8-Methoxymarmesin can reduce the production of intracellular AGEs in a glucose-induced cellular glycation model.

[0134] 1. Test materials and instruments

[0135] 1.1 Test reagents

[0136] Human keratinocytes (HaCaT) cells, glucose: analytical grade, Sigma, USA. BCA (bicinchoninic acid) protein assay kit: Bio-Tech Biotechnology Co., Ltd. Human advanced glycation end products (AGEs) enzyme-linked immunosorbent assay kit: Wuhan Huamei Biotechnology.

[0137] 1.2 Test equipment

[0138] Microplate reader (Synergy H1), Bio-Tek, USA.

[0139] 2. Test methods

[0140] 2.1 Construction of cell glycosylation model

[0141] This application uses glucose (D-Glucose) (MCE, HY-B0389, purity: ≥98.0%) as an inducer for the construction of a human keratinocyte (HaCaT) cell saccharification model. The cells are grown to the logarithmic growth phase and plated so that the confluence of the cells in the 6 cm dish reaches 60% on the second day.

[0142] 2.2 Effects of Columbianetin acetate and 8-Methoxymarmesin on HaCaT cell viability

[0143] HaCaT was inoculated into a 96-well plate at a concentration of 1.5 × 10 5 / ml suspension 100μl, culture until the next day, add complete culture medium containing 0.4μM, 2μM, 10μM Columbianetin acetate or 8-Methoxymarmesin and continue to culture for 24h, add 10μl Cell Counting Kit-8 (CCK-8, Dojindo) to each well of 96-well plate and culture for 1~2h, and detect OD value at 450nm with enzyme microplate reader.

[0144] Table 6: Cell viability in different concentrations of Columbianetin acetate, 8-Methoxymarmesin and blank samples

[0145]

[0146] 2.3 Effects of different glucose concentrations on AGE levels in HaCaT cells

[0147] The drugs were diluted with complete medium (10% FBS (Gibco) + DMEM (Gibco)) to obtain Examples 7A-9A, Examples 7B-9B and blanks as shown in Table 7, so that the final concentration of glucose was 150 mM, and the final concentrations of Columbianetinacetate and 8-Methoxymarmesin were 0.4 μM, 2 μM, and 10 μM, respectively. After incubating the cells for 48 hours, the cell samples were collected for BCA and intracellular AGEs content determination, and the AGEs content per unit protein was calculated.

[0148] The OD value of the formed AGEs was measured by a microplate reader (Synergy H1, Bio-Tek) (detection wavelength 370 nm, reference wavelength 440 nm).

[0149] Table 7: Contents of glucose, Columbianetin acetate and 8-Methoxymarmesin in different examples

[0150]

[0151] Table 8: Effects of different examples on AGEs levels in HaCaT cells

[0152]

[0153] As can be seen from the above table, after treatment with 150 mM glucose, the intracellular AGEs level in the comparative example increased to 1.34 times that of the blank example (control group).

[0154] When glucose and a certain concentration of Columbianetin acetate were treated simultaneously, the level of intracellular AGEs was significantly reduced compared to the group treated with glucose only. When the concentration of Columbianetin acetate was 10 μM, the intracellular AGEs was only 0.99 times that of the blank case, indicating that Columbianetin acetate has a highly efficient AGEs inhibitory effect.

[0155] When glucose and 8-Methoxymarmesin were treated simultaneously, the level of intracellular AGEs was significantly reduced compared with the group treated with glucose only. When the concentration of 8-Methoxymarmesin was 10 μM, the intracellular AGEs was only 1.15 times that of the blank case, indicating that 8-Methoxymarmesin has a high efficiency in inhibiting AGEs.

[0156] When Columbianetin acetate or 8-Methoxymarmesin is used in the preparation of anti-glycation foods and anti-glycation health products, especially in beauty products, the effective concentrations of Columbianetin acetate and 8-Methoxymarmesin are recommended to refer to the data recorded in the above examples.

[0157] IV. Anti-aging efficacy test of aqueous solution prepared using Columbianetin acetate

[0158] Subjects: 40 subjects were divided into 4 groups, 10 in each group, aged 30-50 years old. All subjects had dark yellow skin, wrinkles, decreased collagen fiber elasticity, dark brown spots and other phenomena. The subjects were in good health, had no history of skin diseases and allergies, had non-sensitive skin, and were able to use skin care products according to specifications. Each group of subjects sprayed their faces with 0μM, 0.4μM, 2μM, and 10μM Columbianetin acetate every morning and evening (about 20 cm away from the face, sprayed on the face in a Z shape 3 times), once a day in the morning and evening, for 3 consecutive months, and compared the skin conditions before and after 3 months, and calculated the wrinkle reduction rate of each group of subjects. The test results are shown in Table 9. The VISIA-CR facial skin image analyzer was used to test the improvement of wrinkles, observe the wrinkle depth analysis of the skin, the wrinkle reduction rate, and measure the change rate before and after 3 months.

[0159] Table 9: Trial effect experimental data of aqueous solutions containing Columbianetin acetate

[0160]

[0161] It can be seen from the test results that Columbianetin acetate can effectively reduce wrinkles, and the wrinkle reduction rate is positively correlated with its dosage. Among them, Example 12 has the best effect. It can be seen that Columbianetin acetate can significantly reduce wrinkles and has a good anti-aging effect.

[0162] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0163] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. Use of a furanobenzopyrone derivative in the preparation of a product that reduces accumulation of advanced glycation end products, characterized in that: The furanobenzopyrone derivative is a compound represented by formula I: (Formula I); Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group; Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H.

2. The use according to claim 1, characterized in that The compound of formula I is selected from at least one of the following compounds: or .

3. The use according to claim 1, characterized in that R1 is -C(CH3)2-R6; R6 is -OH or -O-CO-CH3; R2 is H or methoxy.

4. The use according to claim 1, characterized in that The furanobenzopyrone derivative is selected from at least one of the following compounds: 、 。 5. The use according to any one of claims 1 to 4, characterized in that: The product for reducing the accumulation of advanced glycation end products is a product for preventing and / or treating diseases associated with the accumulation of AGEs.

6. The use according to claim 5, characterized in that The diseases associated with the accumulation of AGEs include aging and / or degenerative diseases; Optionally, the degenerative disease includes at least one of diabetes, cardiovascular disease, metabolic syndrome, obesity, hypertension, arteriosclerosis, neurodegenerative disease, orthopedic disease and female ovarian dysfunction; Optionally, the neurodegenerative disease comprises Alzheimer's disease and / or peripheral neuropathy.

7. The use according to any one of claims 1 to 4, characterized in that: The products that reduce the accumulation of advanced glycation end products include cosmetics, foods and medicines; Optionally, the cosmetics include at least one of lotion, facial cream, emulsion, essence, facial mask, and eye cream.

8. The use according to any one of claims 1 to 4, characterized in that: The administration of the product for reducing accumulation of advanced glycation end products includes oral administration and / or topical administration; Optionally, the external application includes local surface administration and / or systemic transdermal administration.

9. A product for reducing accumulation of advanced glycation end products, characterized in that It includes furanobenzopyrone derivatives, and the structure of the furanobenzopyrone derivative is a compound shown in formula I: (Formula I); Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group; Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H; Optionally, the product for reducing accumulation of advanced glycation end products further comprises auxiliary materials.

10. A product for preventing and / or treating diseases associated with the accumulation of AGEs, characterized in that: It includes furanobenzopyrone derivatives, and the structure of the furanobenzopyrone derivative is a compound shown in formula I: (Formula I); Among them, R4 is fused with R3 or R5 ; R1 is a C2-C6 alkyl group substituted by a hydroxyl group or an acetoxy group; R2 is H or a C1-C5 alkoxy group; Provided that, when R4 is fused with R3 When R4 and R5 are fused together, When R3 is H; Optionally, the product for preventing and / or treating diseases associated with the accumulation of AGEs further comprises excipients.

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