Compound capable of reducing accumulation of advanced glycosylation end products
By using the compound Columbianetin acetate, the generation of AGEs was significantly reduced in the BSA-MGO reaction system and glucose-induced cell model, the problem of insignificant effects and side effects of existing AGEs inhibitors was solved, and safe and effective AGEs inhibitory effect was achieved, and skin health was improved.
Patent Information
- Application Number
- CN202411697767.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-26
- Publication Date
- 2025-02-28
AI Technical Summary
The existing AGEs inhibitors are not significant in anti-sugar effects and have side effects, making them difficult to use in food processing, resulting in the urgent need to find safe and effective AGEs inhibitors.
A compound Columbianetin acetate is provided, which significantly reduces the generation of AGEs through its role in the BSA-MGO reaction system and exerts the AGEs inhibitory effect in a glucose-induced cellular model.
Columbianetin acetate can effectively inhibit the production of AGEs, be better than the existing aminoguanidine, and is non-toxic to cells, significantly improves skin dullness and alleviates damage and aging caused by skin saccharification.
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Figure CN119523972A_ABST
Abstract
Description
Technical Field
[0001] The invention discloses a compound capable of reducing the accumulation of advanced glycation end products, and belongs to the field of cosmetics and functional foods. Background Art
[0002] Advanced glycation end products (AGEs) are generated through the Maillard reaction by condensation, rearrangement, cleavage, oxidation and other reactions between the carbonyl part of reducing sugar and free amino groups of proteins, amino acids, nucleic acids, etc. Excessive accumulation of AGEs in the body has attracted widespread attention because it is involved in the formation of various diseases, such as diabetes, cancer, cardiovascular disease, etc., which seriously affects human health. AGEs are widely present in food processing, and their formation mechanism is complex and diverse. Reducing the formation of AGEs in food processing is one of the key issues that the academic community focuses on.
[0003] Aminoguanidine, pyridoxamine, N-phenylacetamide bromide and its derivative ALT711 are currently effective AGEs inhibitors, but synthetic inhibitors have unavoidable side effects and cannot be used in food processing. Therefore, it is particularly important to find safe and effective AGEs inhibitors. Summary of the invention
[0004] In order to overcome the shortcoming that the existing AGEs inhibitors have a weak anti-sugar effect, the present invention discloses a compound capable of effectively inhibiting the generation of AGEs and the application of the compound in oral cosmetic products.
[0005] Specifically, the present invention is achieved through the following aspects:
[0006] In one aspect, a compound named Columbianetin acetate, whose Chinese name is dihydrocolumbianetin acetate, is provided.
[0007] On the other hand, the present invention provides the use of the compound in the field of preparing oral cosmetics;
[0008] Preferably, the compound can effectively inhibit the production of advanced glycation end products;
[0009] Preferably, the compound can effectively reduce the generation of AGEs in the BSA-MGO reaction system;
[0010] Preferably, the compound exerts an AGEs inhibitory effect in glucose-induced increase of cellular AGEs;
[0011] Preferably, the compound can reduce skin yellowing caused by AGEs accumulation;
[0012] Preferably, the compound can exert anti-aging effects by reducing AGEs.
[0013] Preferably, the above uses may appear in products including but not limited to: cosmetics, functional foods, etc.
[0014] Compared with the prior art, the compounds disclosed in the present invention have the following beneficial effects:
[0015] The compounds disclosed in the present invention can effectively reduce the accumulation of AGEs caused by a bad diet (high in oil and sugar) and an unhealthy lifestyle (staying up late and smoking), improve dull and yellow skin, and alleviate and reduce skin damage and skin aging caused by skin glycation.
[0016] The compounds disclosed in the present invention can not only reduce the generation of AGEs in the pure in vitro AGEs generation model of BSA-MGO, but also have a better AGEs inhibition effect than aminoguanidine. In addition, the compounds disclosed in the present invention can effectively reduce the increase of AGEs levels in the intracellular AGEs generation model induced by glucose, and have no toxic effect on cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:
[0018] Figure 1 The results show the inhibition rate of different substances on AGEs. DETAILED DESCRIPTION
[0019] The following examples are provided to allow those skilled in the art to more clearly understand the present invention. It should be noted that the following examples do not limit the scope of protection claimed in the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples, unless otherwise specified, can be obtained from commercial sources or by known existing methods.
[0020] Examples 1-3
[0021] Effects of different anti-glycation and anti-aging substances on the production of AGEs in the BSA-MGO model.
[0022] 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.
[0023] 1. Test materials and instruments
[0024] 1.1 Test reagents
[0025] Bovine serum albumin (BSA), methylglyoxal (MGO), and glucose: analytical grade, Sigma, USA.
[0026] 1.2 Test samples
[0027] Anti-glycation and anti-aging substances: Columbianetin acetate, CAS#: 23180-65-6; Aminoguanidine, CAS#: 1937-19-5; Purpura acetoin A, CAS#: 73069-25-7.
[0028] 1.3 Test equipment
[0029] Microplate reader (Synergy H1), Bio-Tek, USA.
[0030] 2. Test methods
[0031] 2.1 Establishment of in vitro protein AGE formation system
[0032] 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).
[0033] 2.2 Determination of inhibition of AGE formation in vitro
[0034] The mixed incubation solutions prepared in Examples 1-3 and Comparative Examples 1-3 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.
[0035] Table 1: Contents of anti-glycation and anti-aging substances in different embodiments and comparative examples
[0036]
[0037] 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).
[0038] 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).
[0039] 2.3 Data processing and statistical analysis methods (each group of experiments was repeated at least 3 times)
[0040] The AGE inhibition rate was calculated using the formula;
[0041] AGE inhibition rate = (CT) / C×100
[0042] Wherein, 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.
[0043] 3. Test results
[0044] The inhibitory effects of the test samples on AGEs are shown in Table 2.
[0045] Table 2: AGE inhibition rate in Examples 1-3, Comparative Examples 1-3 and Blank Examples
[0046] Serial number Components Example 1 12.30％ Example 2 33.31％ Example 3 58.51％ Comparative Example 1 15.51％ Comparative Example 2 53.07％ Comparative Example 3 4.17％ Blank example 1 -0.24％
[0047] Through Figure 1 The results of Table 2 were used to compare the AGEs inhibition rate. It was found that the AGEs inhibition rate of aminoguanidine at 1mM was 15.51%, the AGEs inhibition rate at 10mM was 53.11%, and the AGEs inhibition rate of praeruptorin A at 10μM was 4.17%. As the concentration of Columbianetin acetate increased at 1μM, 3μM, and 10μM, the AGEs inhibition rate gradually increased in a concentration-dependent manner, 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. The above results prove that Columbianetin acetate can significantly inhibit the generation of AGEs in the BSA-MGO system in vitro, and this inhibition is concentration-dependent.
[0048] Embodiment 4-6
[0049] Effects of different glucose concentrations on the levels of AGEs in HaCaT cells.
[0050] 1. Test materials and instruments
[0051] 1.1 Test reagents
[0052] 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.
[0053] 1.2 Test equipment
[0054] Microplate reader (Synergy H1), Bio-Tek, USA.
[0055] 2. Test methods
[0056] 2.1 Construction of cell glycosylation model
[0057] The present invention uses glucose (D-Glucose) (MCE, HY-B0389, purity: ≥98.0%) as an inducer for constructing 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 a 6 cm dish reaches 60% on the second day.
[0058] 2.2 Effects of different glucose concentrations on HaCaT cell viability
[0059] 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, so that the final concentrations of glucose were 100mM, 150mM, and 200mM, respectively. After changing the medium, continue to culture for 24h, add 10μL CellCounting Kit-8 (CCK-8, Dojindo) to each well of the 96-well plate and culture for 1-2h, and detect the OD value at 450nm with 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. In the presence of an electron coupling reagent, WST-8 can be reduced by some dehydrogenases in mitochondria to generate orange-yellow formazan. 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.
[0060] Table 3: Glucose content in different embodiments
[0061] Serial number Components Example 4 Example 5 Example 6 Blank example 1 glucose 100mM 150mM 200mM ×
[0062] Table 4: Cell viability in Examples 4-6 and blank examples
[0063] Serial number Cell viability 100 mM glucose 81.26％ 150 mM glucose 80.99％ 200 mM glucose 65.46％ Blank example 100％
[0064] 2.3 Effects of different glucose concentrations on AGE levels in HaCaT cells
[0065] 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.
[0066] The OD value of the formed AGEs was measured by an enzyme reader (Synergy H1, Bio-Tek) (detection wavelength 370nm, reference wavelength 440nm). 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 inferred based on the absorbance value.
[0067] Table 5: Effects of different examples on AGEs levels in HaCaT cells
[0068]
[0069] According to the cell viability test results in Example 2.2 and microscopic observation, 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 an induction condition for the cell glycation model.
[0070] Embodiment 7-9
[0071] Columbianetin acetate can reduce the production of intracellular AGEs in a glucose-induced cellular glycation model.
[0072] 1. Test materials and instruments
[0073] 1.1 Test reagents
[0074] 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.
[0075] 1.2 Test equipment
[0076] Microplate reader (Synergy H1), Bio-Tek, USA.
[0077] 2. Test methods
[0078] 2.1 Construction of cell glycosylation model
[0079] The present invention uses glucose (D-Glucose) (MCE, HY-B0389, purity: ≥98.0%) as an inducer for constructing 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 a 6 cm dish reaches 60% on the second day.
[0080] 2.2 Effect of Columbianetin acetate on HaCaT cell viability
[0081] 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 medium containing 0.4μM, 2μM, 10μM Columbianetin acetate and continue to culture for 24h, add 10μL Cell Counting Kit-8 (CCK-8, Dojindo) to each well of the 96-well plate and culture for 1-2h, and detect the OD value 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. In the presence of electron coupling reagents, WST-8 can be reduced by some dehydrogenases in mitochondria to generate orange-yellow formazan. 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.
[0082] Table 6: Cell viability of different concentrations of Columbianetin acetate and blank samples
[0083] Serial number Cell viability 0.4μM Columbianetin acetate 99.81％ 2μM Columbianetin acetate 106.56％ 10μM Columbianetin acetate 101.28％ Blank example 100％
[0084] 2.3 Effects of different glucose concentrations on AGE levels in HaCaT cells
[0085] 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 concentrations of Columbianetin acetate 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.
[0086] The OD value of the formed AGEs was measured by an enzyme reader (Synergy H1, Bio-Tek) (detection wavelength 370nm, reference wavelength 440nm). 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 inferred based on the absorbance value.
[0087] Table 7: Contents of glucose and Columbianetin acetate in different examples
[0088] Serial number Components Example 7 Example 8 Example 9 Comparative Example Blank example 1 glucose 150mM 150mM 150mM 150mM 0 2 Columbianetin acetate 0.4μM 2μM 10μM 0 0
[0089] Table 8: Effects of different examples on AGEs levels in HaCaT cells
[0090]
[0091] As shown in the table above, after treatment with 150 mM glucose, the level of intracellular AGEs in the comparative example increased to 1.72 times that of the blank example (control group). When glucose and Columbianetin acetate were treated simultaneously, the level of intracellular AGEs was significantly reduced compared to the group treated with glucose alone. When the concentration of Columbianetin acetate was 10 μM, the level of intracellular AGEs was only 0.87 times that of the blank example, indicating that Columbianetin acetate has a highly effective AGEs inhibition effect. When Columbianetin acetate is used in the preparation of anti-glycation foods and anti-glycation health products, especially in beauty products, it is recommended that the effective concentration of Columbianetin acetate refer to the data recorded in the above examples.
[0092] Examples 10-12
[0093] Anti-aging efficacy test of aqueous solution prepared with anti-glycation and anti-aging substances
[0094] 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 once in the morning and evening every day for 3 consecutive months, 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.
[0095] Table 9: Trial effect experimental data of aqueous solutions containing Columbianetin acetate
[0096]
[0097] From the test results, it can be seen that Example 12 has the best effect, indicating that Columbianetin acetate has an obvious effect.
[0098] Columbianetin acetate has a saturated pyrone structure, and acetylation adds an acetate group. Its dihydrogen structure means that the compound does not have a double bond on the benzene ring, which makes it different from typical coumarins. For example, umbelliferone or esculetin have different hydroxyl or methoxy substituents in their structure, which leads to differences in solubility, absorption and metabolism.
[0099] Columbianetin acetate
[0100] The above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. Use of Columbianetin acetate in the preparation of anti-glycation or anti-aging products.
2. The use according to claim 1, characterized in that: The application is an anti-glycation product for reducing the accumulation of advanced glycation end products.
3. The use according to claim 1, characterized in that: The application is an anti-glycation or anti-aging product for oral or external use.
4. The use according to claim 2, characterized in that: The anti-glycation product is an oral liquid or an external use product.
5. A method for reducing the generation of intracellular AGEs in a glucose-induced cell glycation model, characterized in that: The cell glycation model was treated with the Columbianetin acetate.