Application of coumarin compound in preparation of product for reducing accumulation of advanced glycosylation end product
By using coumarin-like compound Apigravin to inhibit the generation of AGEs, the problem of insufficient effect of existing anti-saccharifying agents was solved, and efficient AGEs inhibition effect was achieved, which was suitable for a variety of application scenarios.
Patent Information
- Application Number
- CN202411649123.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-02-21
AI Technical Summary
The existing anti-saccharifying agents have no significant effect in inhibiting the generation of advanced glycosylated terminal products (AGEs) and cannot meet the application requirements.
The coumarin compound Apigravin is used to inhibit the generation of AGEs through its structure and is used in the preparation of products that reduce AGEs accumulation.
Apigravin can significantly inhibit the generation of AGEs and has an efficient inhibitory effect of AGEs. It is suitable for the treatment of diseases related to AGEs accumulation, and is suitable for the application of anti-saccharified foods or health products.
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Abstract
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 coumarin compounds in the preparation of products for reducing the accumulation of advanced glycation end products. Background Art
[0002] Advanced Glycation End Products (AGEs) are compounds generated by a series of complex reactions between proteins, lipids and sugars in non-enzymatic reactions. The accumulation of AGEs is associated with a variety of pathological processes related to aging and chronic diseases. Studies have shown that food-borne AGEs can induce pathological biological reactions, including diabetes and its complications, cardiovascular disease, neurodegenerative diseases, orthopedic diseases, and female ovarian dysfunction. AGEs not only damage the structure and function of proteins, but also trigger oxidative stress and inflammatory responses by binding to their receptors, further exacerbating tissue damage and disease progression.
[0003] Since the formation of AGEs is irreversible and difficult to eliminate through conventional metabolic pathways, researchers have been committed to developing anti-glycemic compounds in recent years to delay the formation of AGEs, promote their degradation or reduce their damage to tissues. Such anti-glycemic compounds can not only be used to prevent and treat diseases related to AGEs, but also have the potential to delay aging. Patents in related fields are mostly focused on the synthesis of new anti-glycation agents, the application of plant extracts, and the inhibition and removal methods for AGEs, but the existing AGEs inhibitors have insignificant anti-glycemic effects and cannot meet application requirements. Summary of the invention
[0004] Based on this, the main purpose of this application is to provide the use of coumarin compounds in the preparation of products that reduce the accumulation of advanced glycation end products. The coumarin compound Apigravin can significantly inhibit the formation of AGEs, has a high AGEs inhibitory effect, can be used to treat diseases related to the accumulation of AGEs, and can meet the application requirements of anti-glycation foods or health products.
[0005] In a first aspect of the present application, a use of a coumarin compound in preparing a product for reducing accumulation of advanced glycation end products is provided, wherein the structure of the coumarin compound is as follows:
[0006] .
[0007] 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.
[0008] In some embodiments, the diseases associated with the accumulation of AGEs include aging and / or chronic diseases.
[0009] In some embodiments, the chronic disease comprises diabetes, cardiovascular disease, neurodegenerative disease, orthopedic disease, or female ovarian dysfunction.
[0010] In some embodiments, the neurodegenerative disease is Alzheimer's disease.
[0011] In some embodiments, the product that reduces accumulation of advanced glycation end products comprises a cosmetic, a food, or a pharmaceutical.
[0012] In some embodiments, the administration of the product for reducing accumulation of advanced glycation end products includes oral administration and / or topical administration.
[0013] In some embodiments, the dosage form of the product includes a solid dosage form, a semisolid dosage form, or a liquid dosage form.
[0014] In some embodiments, the liquid dosage form comprises a solution, a syrup, an emulsion, or a tincture.
[0015] In some embodiments, the product for reducing accumulation of advanced glycation end products has an effective concentration of 0.1-100 μM.
[0016] In a second aspect, the present application provides a product for reducing the accumulation of advanced glycation end products, including a coumarin compound, wherein the structure of the coumarin compound is as follows:
[0017] ;
[0018] Optionally, the product for reducing accumulation of advanced glycation end products further comprises auxiliary materials.
[0019] In a third aspect, the present application provides a product for preventing and / or treating diseases associated with the accumulation of AGEs, including a coumarin compound, wherein the structure of the coumarin compound is as follows:
[0020] ;
[0021] Optionally, the product for preventing and / or treating diseases associated with the accumulation of AGEs further comprises excipients.
[0022] Compared with the prior art, this application has the following beneficial effects:
[0023] 1. Apigravin, a coumarin compound, can significantly inhibit the generation of AGEs and has a highly effective AGE inhibitory effect. It can be used to treat or improve diseases related to the accumulation of AGEs, such as aging and chronic diseases such as diabetes, cardiovascular disease, neurodegenerative diseases, orthopedic diseases, or female ovarian dysfunction.
[0024] 2. The coumarin compound Apigravin not only reduces AGEs production in the BSA-MGO pure in vitro AGEs production model, but also effectively reduces AGEs levels in the glucose-induced intracellular AGEs production model, and has no toxic effect on cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] 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:
[0026] Figure 1 The results show the inhibition rate of different substances on AGEs. DETAILED DESCRIPTION
[0027] 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.
[0028] 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.
[0029] 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.
[0030] the term
[0031] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:
[0032] 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.
[0033] 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.
[0034] 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.
[0035] In a first aspect of the present application, a use of a coumarin compound in preparing a product for reducing accumulation of advanced glycation end products is provided, wherein the structure of the coumarin compound is as follows:
[0036] .
[0037] The English name of the coumarin compound in this application is Apigravin, and the molecular formula is C 15 H 16 O4. Apigravin is an effective antibacterial agent with antibacterial activity against Bacillus subtilis, with a MIC value of 61 μM. This application has been verified through experiments and found that Apigravin can significantly inhibit the generation of AGEs and has a highly effective AGEs inhibitory effect. It can be used to treat diseases related to the accumulation of AGEs and can meet the application requirements of anti-glycation foods or health products.
[0038] Specifically, Apigravin can effectively reduce the generation of AGEs in the BSA-MGO reaction system, inhibit AGEs in the increase of cellular AGEs induced by glucose, reduce skin yellowing caused by AGEs accumulation, and exert anti-aging effects by reducing AGEs. Therefore, Apigravin has a value that cannot be ignored in the field of oral beauty.
[0039] 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.
[0040] In some embodiments, the diseases associated with the accumulation of AGEs include aging and / or chronic diseases.
[0041] In some embodiments, the chronic disease comprises diabetes, cardiovascular disease, neurodegenerative disease, orthopedic disease, or female ovarian dysfunction.
[0042] In some embodiments, the neurodegenerative disease is Alzheimer's disease.
[0043] Understandably, Apigravin has a significant inhibitory effect on the generation of AGEs and can avoid the accumulation of AGEs. Therefore, it is highly feasible for preventing, treating or improving diseases caused by the accumulation of AGEs, such as aging, as well as chronic diseases such as diabetes, cardiovascular disease, neurodegenerative diseases, orthopedic diseases or female ovarian dysfunction.
[0044] In some embodiments, the product that reduces accumulation of advanced glycation end products comprises a cosmetic, a food, or a pharmaceutical.
[0045] In some embodiments, the administration of the product for reducing accumulation of advanced glycation end products includes oral administration and / or topical administration.
[0046] In some embodiments, the dosage form of the product includes a solid dosage form, a semisolid dosage form, or a liquid dosage form.
[0047] In some embodiments, the solid dosage form includes tablets, capsules, pills and granules; the semisolid dosage form includes ointments, gel pastes and patches; the liquid dosage form includes solutions, syrups, emulsions or tinctures.
[0048] In some embodiments, the effective concentration of the product for reducing the accumulation of advanced glycation end products is 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.
[0049] It is understandable that the product for reducing the accumulation of advanced glycation end products is not limited to food, it can also be a medicine or cosmetic, and there is no particular limitation on the dosage form and the mode of administration. When used as a cosmetic, it is more of an external use product, preferably a liquid dosage form or a semi-solid dosage form, which is applied by spraying, smearing, etc.; when used as a food or medicine, it can be a solid dosage form, a semi-solid dosage form or a liquid dosage form, and is mostly administered orally. When prepared into different dosage forms, in addition to Apigravin, excipients and carriers commonly used in the art can also be added.
[0050] In a second aspect, the present application provides a product for reducing the accumulation of advanced glycation end products, including a coumarin compound, wherein the structure of the coumarin compound is as follows:
[0051] ;
[0052] Optionally, the product for reducing accumulation of advanced glycation end products further comprises auxiliary materials.
[0053] In a third aspect, the present application provides a product for preventing and / or treating diseases associated with the accumulation of AGEs, including a coumarin compound, wherein the structure of the coumarin compound is as follows:
[0054] ;
[0055] Optionally, the product for preventing and / or treating diseases associated with the accumulation of AGEs further comprises excipients.
[0056] It is understandable that the coumarin compounds can be added with auxiliary materials to prepare different dosage forms, and the auxiliary materials can specifically include carriers, fillers, diluents, excipients, solvents, functional additives, coating materials or encapsulation materials, etc.
[0057] Unless otherwise specified, the raw materials used in the following experiments can be purchased from the market.
[0058] The following are specific embodiments.
[0059] Examples 1-3
[0060] Effects of different anti-glycation and anti-aging substances on the production of AGEs in the BSA-MGO model.
[0061] 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.
[0062] 1. Test materials and instruments
[0063] 1.1 Test reagents
[0064] Bovine serum albumin (BSA), methylglyoxal (MGO), glucose: analytical grade, Sigma, USA.
[0065] 1.2 Test samples
[0066] Anti-glycation and anti-aging substances: Apigravin, CAS#:72963-64-5; Aminoguanidine, CAS#:1937-19-5.
[0067] 1.3 Test equipment
[0068] Microplate reader (Synergy H1), Bio-Tek, USA.
[0069] 2. Test methods
[0070] 2.1 Establishment of in vitro protein AGE formation system
[0071] 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).
[0072] 2.2 Determination of inhibition of AGE formation in vitro
[0073] The mixed incubation solutions prepared in Examples 1-3 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.
[0074] Table 1: Contents of anti-glycation and anti-aging substances in different embodiments and comparative examples
[0075]
[0076] 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).
[0077] 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).
[0078] 2.3 Data processing and statistical analysis methods (each group of experiments was repeated at least 3 times)
[0079] The AGE inhibition rate was calculated using the formula;
[0080] AGE inhibition rate = (CT) / C × 100%;
[0081] 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.
[0082] 3. Test results
[0083] The inhibitory effect of the test samples on AGEs, such as Figure 1 And as shown in Table 2.
[0084] Table 2: AGE inhibition rates in Examples 1-3, Comparative Examples 1-2 and Blank Examples
[0085]
[0086] Through Figure 1 The results in Table 2 were used to compare the AGEs inhibition rate. It was found that the AGEs inhibition rate of aminoguanidine at 1mM was 9.24%, and the AGEs inhibition rate at 10mM was 51.84%. The AGEs inhibition rate of Apigravin increased gradually with the increase of concentration at 1μM, 3μM, and 10μM, which were 26.36%, 53.33%, and 76.34%, respectively. The AGEs inhibition effect was higher than that of 1mM aminoguanidine. The above results prove that in the in vitro BSA-MGO system, Apigravin can significantly inhibit the generation of AGEs, and this inhibition is concentration-dependent.
[0087] Embodiment 4-6
[0088] Effects of different glucose concentrations on the levels of AGEs in HaCaT cells.
[0089] 1. Test materials and instruments
[0090] 1.1 Test reagents
[0091] 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.
[0092] 1.2 Test equipment
[0093] Microplate reader (Synergy H1), Bio-Tek, USA.
[0094] 2. Test methods
[0095] 2.1 Construction of cell glycosylation model
[0096] 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.
[0097] 2.2 Effects of different glucose concentrations on HaCaT cell viability
[0098] HaCaT was inoculated in a 96-well plate, and 100 μl of a suspension at a concentration of 1.5×105 / ml was added to each well, and cultured until the next day. The drug was diluted with culture medium to obtain Examples 4-6 and blanks as shown in Table 3, so that the final concentrations of glucose were 100 mM, 150 mM, and 200 mM, respectively. After the liquid was replaced, the culture was continued for 24 hours. 10 μl CellCounting Kit-8 (CCK-8, Dojindo) was added to each well of the 96-well plate and cultured for 1~ 2 hours, and the OD value was detected at 450 nm 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.
[0099] Table 3: Glucose content in different embodiments
[0100]
[0101] Table 4: Cell viability in Examples 4-6 and blank examples
[0102]
[0103] 2.3 Effects of different glucose concentrations on AGE levels in HaCaT cells
[0104] 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.
[0105] 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, and the absorbance and protein concentration have a good linear relationship in a wide range. Therefore, the protein concentration can be inferred based on the absorbance value.
[0106] Table 5: Effects of different examples on AGEs levels in HaCaT cells
[0107]
[0108] 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.
[0109] Embodiment 7-9
[0110] Apigravin can reduce the production of intracellular AGEs in a glucose-induced cellular glycation model.
[0111] 1. Test materials and instruments
[0112] 1.1 Test reagents
[0113] 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.
[0114] 1.2 Test equipment
[0115] Microplate reader (Synergy H1), Bio-Tek, USA.
[0116] 2. Test methods
[0117] 2.1 Construction of cell glycosylation model
[0118] 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.
[0119] 2.2 Effect of Apigravin on HaCaT cell viability
[0120] HaCaT was inoculated in a 96-well plate, and 100 μl of a suspension at a concentration of 1.5×105 / ml was added to each well and cultured until the next day. After treatment with complete culture medium containing 0.4 μM, 2 μM, and 10 μM Apigravin, the culture was continued for 24 h. 10 μl of Cell Counting Kit-8 (CCK-8, Dojindo) was added to each well of the 96-well plate and cultured for 1 to 2 h. The OD value was detected at 450 nm using an enzyme microplate reader.
[0121] Table 6: Cell viability in different concentrations of Apigravin and blank samples
[0122]
[0123] 2.3 Effects of different glucose concentrations on AGE levels in HaCaT cells
[0124] The drugs were diluted with complete medium (10% FBS (Gibco) + DMEM (Gibco)) to obtain Examples 7-9 and blanks as shown in Table 7, so that the final concentration of glucose was 150 mM, and the final concentration of Apigravin was 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.
[0125] 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).
[0126] Table 7: Contents of glucose and Apigravin in different embodiments
[0127]
[0128] Table 8: Effects of different examples on AGEs levels in HaCaT cells
[0129]
[0130] As can be seen from the above table, after treatment with 150mM glucose, the level of intracellular AGEs in the comparative example increased to 1.34 times that of the blank example (control group). When glucose and Apigravin were treated simultaneously, the level of intracellular AGEs was significantly reduced compared to the group treated with glucose only. When the concentration of Apigravin was 10μM, the level of intracellular AGEs was only 0.78 times that of the blank example, indicating that Apigravin has a highly effective AGEs inhibition effect. When Apigravin is used in the preparation of anti-glycation foods and anti-glycation health products, especially in the specific application of beauty products, the effective concentration of Apigravin is recommended to refer to the data recorded in the above examples.
[0131] 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.
[0132] 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 coumarin compounds in the preparation of products for reducing accumulation of advanced glycation end products, characterized in that: The coumarin compound structure is as follows: 。 2. The use according to claim 1, 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.
3. The use according to claim 2, characterized in that The diseases associated with the accumulation of AGEs include aging and / or chronic diseases.
4. The use according to claim 3, characterized in that The chronic diseases include diabetes, cardiovascular diseases, neurodegenerative diseases, orthopedic diseases or female ovarian dysfunction.
5. The use according to claim 4, characterized in that The neurodegenerative disease is Alzheimer's disease.
6. The use according to any one of claims 1 to 5, characterized in that: The product for reducing the accumulation of advanced glycation end products includes cosmetics, food or medicine.
7. The use according to any one of claims 1 to 5, characterized in that: The administration of the product for reducing the accumulation of advanced glycation end products includes oral administration and / or topical administration.
8. The use according to any one of claims 1 to 5, characterized in that: The dosage form of the product includes solid dosage form, semisolid dosage form or liquid dosage form; and / or the liquid dosage form comprises a solution, a syrup, an emulsion or a tincture; And / or the effective concentration of the product for reducing the accumulation of advanced glycation end products is 0.1-100 μM.
9. A product for reducing accumulation of advanced glycation end products, characterized in that Including coumarin compounds, the coumarin compound structure is as follows: ； 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 Including coumarin compounds, the coumarin compound structure is as follows: ； Optionally, the product for preventing and / or treating diseases associated with the accumulation of AGEs further comprises excipients.