A bimetallic complex with whitening and anti-aging effects, its preparation method and application

Abstract

This invention discloses a bimetallic complex with whitening and anti-aging effects, its preparation method, and its application, belonging to the field of biomaterial preparation technology. The preparation method includes S1, dissolving an amino acid in water to obtain solution A; S2, dissolving a metal salt in ethanol to obtain solution B; S3, mixing solutions A and B evenly, and then reacting them through a solvothermal reaction to obtain solution C; S4, washing and drying solution C to obtain the final product. The amino acid in step S1 is at least one of glutamic acid and aspartic acid; the metal salt in step S2 is copper acetate and zinc acetate, copper acetate and manganese chloride, or copper acetate and cobalt nitrate; the solvothermal reaction conditions in step S3 are: temperature 60-110℃, reaction time 2-48h. The prepared bimetallic complex has both whitening and anti-aging effects, providing a new direction for the application of BioMOFs materials.

Description

Technical Field

[0001] This invention belongs to the field of biomaterial preparation technology, specifically, it relates to a bimetallic complex with whitening and anti-aging effects, its preparation method and application. Background Technology

[0002] Metal-organic frameworks (MOFs) are crystalline porous materials formed by the self-assembly of metal ions or metal clusters with organic ligands through coordination bonds. They possess advantages such as high specific surface area, tunable pore structure, and abundant functional sites, showing broad application prospects in gas storage and separation, catalysis, sensing, and drug delivery. Biometal-organic frameworks (BioMOFs), as an important branch of MOFs, are typically constructed using biocompatible, low-toxicity metal ions and biodegradable organic ligands (such as amino acids, sugars, and proteins).

[0003] Among the metal ions that can be used to construct MOFs, copper (Cu) and zinc (Zn) have become research hotspots due to their unique physicochemical properties and biological activities. Copper ions are cofactors for many key enzymes (such as superoxide dismutase SOD and tyrosinase), possessing good biocompatibility, low toxicity, and certain antibacterial and anti-inflammatory capabilities; zinc ions, as an essential trace element for the human body, not only have excellent biocompatibility but also possess inherent antibacterial activity.

[0004] However, single-metal MOFs have relatively limited functionality, making it difficult to meet the high demands for comprehensive material performance in complex applications. In recent years, the design and synthesis of bimetallic MOFs has become an important research direction. By introducing two different metal ions, their respective advantages can be effectively integrated to generate synergistic effects, thereby regulating the electronic structure, pore microenvironment, and active sites of the material, achieving significant performance improvements.

[0005] Patent document CN119074956A discloses a defective metal-organic framework (MOF) antioxidant nanozyme, its preparation method, and its application, belonging to the field of nanobiotechnology. The preparation method involves dissolving a copper source, a zirconium source, and organic ligands containing pyrazole and carboxyl groups in an organic solvent. Then, a competing ligand is added, and after thorough mixing, a solvothermal reaction is carried out at 80℃-120℃ to prepare a defective MOF. The defective MOF aqueous solution and a kidney-targeting molecule solution are then thoroughly mixed, and the defective MOF antioxidant nanozyme is obtained through coordination and electrostatic adsorption. This invention adjusts the particle size and defects by using organic ligands containing pyrazole and carboxyl groups, competing ligands containing carboxylic acids, and organic solvents to prepare a small-particle-size, defective MOF, thereby improving antioxidant activity. By loading kidney-targeting molecules, a defective MOF antioxidant nanozyme is obtained, achieving kidney-targeting action. However, this invention does not involve any application in the cosmetic field.

[0006] The generation of free radicals and the accumulation of reactive oxygen species are among the main factors leading to skin aging. Therefore, cosmetics need to have antioxidant capabilities, and effectively scavenging free radicals has become a core strategy for anti-aging. At the same time, the market demand for whitening and spot-fading products continues to grow. Although traditional MOF materials have been widely used in fields such as biocatalysis, medical imaging, tissue engineering, and drug delivery, their application in cosmetics is still in its initial stage. Patent document CN118126339A discloses a complex with antioxidant and whitening effects, its preparation method, and its application. The preparation method of the complex includes the following steps: (1) mixing water with one or more of organic acids, amino acids, and amine reagents to obtain solution A; (2) mixing copper salt with alcohol solution to obtain solution B; (3) adding solution A to solution B, adjusting the pH to obtain solution C; (4) adding surfactant to solution C, reacting, washing, and drying to obtain the complex. The raw materials used in the complex prepared in this invention have low toxicity and good biocompatibility, and can be widely used in the biomedical field, but the antioxidant capacity of the complex in this invention is relatively weak. Against this backdrop, the development of bio-metal-organic frameworks (BioMOFs) that combine whitening and anti-aging effects not only broadens the application scope of MOF materials, but also plays an important role in promoting their large-scale application in cosmetics. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a bimetallic complex with whitening and anti-aging effects, its preparation method, and its application. This invention uses biocompatible amino acids as organic ligands and successfully prepares the bimetallic complex via a solvothermal synthesis method. This bimetallic complex possesses both whitening and anti-aging effects, providing a new direction for the application of BioMOFs materials.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing a bimetallic complex with whitening and anti-aging effects, comprising the following steps:

[0010] S1. Dissolve amino acids in water to obtain solution A;

[0011] S2, metal salt is dissolved in ethanol and mixed evenly to obtain solution B;

[0012] S3. After mixing solutions A and B thoroughly, solution C is obtained through a solvothermal reaction.

[0013] S4. Wash and dry solution C to obtain the bimetallic complex.

[0014] In some embodiments, the amino acid in step S1 is at least one of glutamic acid and aspartic acid.

[0015] In some embodiments, the molar concentration of solution A in step S1 is 0.05-0.6 mol / L; preferably 0.3-0.6 mol / L.

[0016] In some embodiments, the metal salt in step S2 is copper acetate and zinc acetate, copper acetate and manganese chloride, or copper acetate and cobalt nitrate. Preferably, the molar ratio of copper acetate to zinc acetate is 1-3:1; more preferably 1-2:1; even more preferably 1-1.5:1; the molar ratio of copper acetate to manganese chloride is 1-3:1; more preferably 1-2:1; even more preferably 1-1.5:1; the molar ratio of copper acetate to cobalt nitrate is 1-3:1; more preferably 1-2:1; even more preferably 1-1.5:1. Preferably, the metal salt is copper acetate and zinc acetate or copper acetate and manganese chloride, more preferably copper acetate and zinc acetate.

[0017] In some embodiments, the molar concentration of solution B in step S2 is 0.04-0.3 mol / L; preferably 0.04-0.2 mol / L; and more preferably 0.04-0.15 mol / L.

[0018] In some embodiments, the molar ratio of the metal salt to the amino acid in step S3 is 1:1-3, preferably 1:1-2.

[0019] In some embodiments, the conditions for the solvothermal reaction in step S3 are: temperature 60-110℃, reaction time 2-48h; preferably, temperature 80-100℃, reaction time 12-24h.

[0020] Secondly, the present invention provides a bimetallic complex with whitening and anti-aging effects obtained by the above preparation method.

[0021] The bimetallic complex of the present invention has excellent antioxidant activity and SOD-like activity, which can effectively inhibit the production of melanin in cells and has significant whitening and anti-aging effects. Moreover, the raw materials have low toxicity and good biocompatibility, which meets the safety requirements for application in the biomedical field.

[0022] Thirdly, the present invention provides the application of the above-mentioned bimetallic complex in the preparation of cosmetics.

[0023] The bimetallic complex of the present invention can be used as a whitening and anti-aging ingredient in the preparation of cosmetics. The bimetallic complex can be used as the sole whitening and anti-aging ingredient or in combination with other whitening and anti-aging ingredients.

[0024] Fourthly, the present invention provides a cosmetic product with whitening and anti-aging effects, comprising the above-mentioned bimetallic complex.

[0025] In some embodiments, the bimetallic complex has a mass fraction of 0.01%-5% in the cosmetic; preferably 0.01%-3%; more preferably 0.01%-2%.

[0026] In some implementations, the cosmetic is a toner, serum, emulsion, cream, gel, or mask.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. By screening amino acids and metal salts and controlling parameters such as the molar ratio of amino acids to metal salts and solvothermal reaction, this invention can effectively control the morphology and size of the product, with good repeatability and easy large-scale preparation.

[0029] 2. The bimetallic complex prepared by this invention has excellent antioxidant activity and SOD-like activity, which can effectively inhibit the production of melanin in cells and has significant whitening and anti-aging effects. Moreover, the raw materials have low toxicity and good biocompatibility, which meets the safety requirements for application in the biomedical field.

[0030] 3. Compared with monometallic complex materials, the bimetallic MOFs of the present invention have better antioxidant effects, especially in scavenging hydroxyl radicals and DPPH radicals.

[0031] 4. The bimetallic complex provided by this invention has good application potential in the cosmetics field, and provides a new direction for expanding the application scope of bimetallic complexes. Attached Figure Description

[0032] Figure 1 Scanning electron microscope image of the bimetallic complex prepared in Example 3.

[0033] Figure 2 Scanning electron microscope image of the bimetallic complex prepared in Example 4.

[0034] Figure 3 The image shows the infrared spectrum of the bimetallic complex in Example 4.

[0035] Figure 4 The results show the toxicity of the bimetallic complex (Example 4) to HaCaT cells at the experimental concentration. Compared with the control group, ****P < 0.0001, and ns indicates P > 0.05.

[0036] Figure 5The survival rate of B16 cells after treatment with the bimetallic complex (Example 4) at the experimental concentration is shown in the figure. Compared with the control group, **P < 0.01 and ****P < 0.0001.

[0037] Figure 6 The values ​​represent the melanin production rate of B16 cells after treatment with different concentrations of arbutin and the control group. Compared with the model group, ****P<0.0001, ns indicates P>0.05. Detailed Implementation

[0038] The following description of the embodiments is merely to aid in understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various modifications and improvements to the present invention without departing from its principles, and these modifications and improvements also fall within the scope of the claims. The following description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not limited to the embodiments shown herein, but can be applied to a wider scope consistent with the principles and novel features disclosed herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0039] The following examples and comparative examples illustrate the present invention in a more detailed manner, but the invention is not limited thereto. Unless otherwise specified, all percentages in the present invention are mass percentages, and all temperatures are room temperature (20-25°C). The present invention does not limit the source of the raw materials used; unless otherwise specified, all raw materials used in the present invention are commercially available products commonly used in this field.

[0040] Examples 1-6 and Comparative Examples 1-6

[0041] According to the process parameters in Tables 1 and 2, the bimetallic complexes of the corresponding examples and comparative examples were prepared using the following preparation methods.

[0042] The preparation method is as follows:

[0043] S1. Take A1 mL of water, add A2 mmol of amino acid A3 and stir to dissolve to obtain solution A;

[0044] S2. Take B1 mL of ethanol, add B2 mmol of metal salt 1 and B3 mmol of metal salt 2, and stir magnetically for 500 rpm until dissolved to obtain solution B.

[0045] S3. Slowly add solution A to solution B, and stir magnetically at 500 rpm for 20 minutes to obtain solution C;

[0046] S4. Transfer solution C to a polytetrafluoroethylene-lined reactor, set the temperature to D1℃, and the reaction time to D2 h. After the reaction is complete, remove the reaction solution and separate it to obtain a solid product. Wash it three times with a mixture of deionized water and anhydrous ethanol (volume ratio 1:1), and dry it to obtain a bimetallic complex.

[0047] Table 1. Process parameters for Examples 1-6

[0048]

[0049] Table 2. Process parameters for Comparative Examples 1-6

[0050]

[0051] The bimetallic complex prepared in Example 3 was analyzed by SEM, and the results are as follows: Figure 1 As shown in the figure. The results show that the bimetallic complexes prepared in Example 3 have a particle size of about 7 μm, are mostly spherical, and have a structure similar to nanoflower clusters on the surface.

[0052] The bimetallic complex prepared in Example 4 was analyzed by SEM, and the results are as follows: Figure 2 As shown in the figure. The results show that the particle size of the bimetallic complex prepared in Example 4 is about 5 μm, mostly spherical, and has a fine porous structure on the surface. The infrared spectrum of the bimetallic complex prepared in Example 4 is shown in the figure. Figure 3 As shown, the results indicate that at 3314 and 3206 cm... -1 The broad absorption peak at 1619 cm⁻¹ is attributed to the stretching vibrations of amino and hydroxyl groups. -1 The strong absorption peak appearing at this point is due to the carboxylate group (-COO). - The asymmetric stretching vibration of 1393cm -1 The absorption peak at 1572 cm⁻¹ corresponds to its symmetrical stretching vibration. Furthermore, the absorption peak at 1572 cm⁻¹... -1 The absorption peak at 1135 cm⁻¹ is attributed to the bending vibration of NH₃. -1 The absorption peak at that point originates from the in-plane rocking vibration of NH2.

[0053] Compared to free glutamic acid, the asymmetric and symmetric stretching vibration peaks of the carboxyl group after coordination showed significant shifts, and the difference between them (Δν) decreased, indicating that the carboxyl oxygen atom participated in coordination. Simultaneously, peaks at 479 and 529 cm⁻¹... -1 Absorption peaks at 422 and 500 cm⁻¹, attributed to stretching vibrations of Cu-N and Zn-N bonds, were observed. -1Absorption peaks attributable to stretching vibrations of Cu-O and Zn-O bonds were observed. These findings collectively indicate that glutamic acid successfully coordinated with copper and zinc ions via its amino nitrogen atom and carboxyl oxygen atom.

[0054] Example 7 Whitening and Anti-aging Face Cream

[0055] The whitening and anti-aging face cream is prepared using the bimetallic complex prepared in the examples as raw material.

[0056] The specific ingredients and formulation of the whitening and anti-aging face cream are shown in Table 3.

[0057] Table 3. Specific ingredients and proportions of whitening and anti-aging face cream

[0058]

[0059] The preparation method of the above-mentioned whitening face cream is as follows:

[0060] (1) Mix xanthan gum with a small amount of glycerol in a small beaker and stir thoroughly with a glass rod to form a uniform xanthan gum paste without particles. Let it stand for later use. Dissolve potassium sorbate in a small amount of deionized water and stir until completely dissolved. Mix the bimetallic complex powder with most of the remaining glycerol in a small beaker and stir thoroughly with a glass rod to form a uniform bimetallic complex / glycerol paste.

[0061] (2) Preparation of aqueous phase (A): Add the weighed remaining deionized water and betaine to a large main beaker, and heat the aqueous phase to 70°C in a water bath. Stir until the betaine is completely dissolved.

[0062] Preparation of oil phase / emulsifier phase (B): In another small beaker, add octadecanol, glyceryl stearate, and hydrogenated castor oil, heat the oil phase to 70°C, and stir until all solids are completely melted.

[0063] (3) Mixing and emulsification: Slowly pour the heated oil phase (B) into the heated aqueous phase (A). Emulsify using a high-speed stirrer for 5 minutes. Add the surfactant (C) and maintain the temperature at 70°C. Add the coconut oil diethanolamide to the emulsified system from the previous step. Continue stirring for 5 minutes to ensure uniform mixing, then add the pre-dispersed xanthan gum paste.

[0064] (4) Stop heating and begin cooling. Continue stirring gently until the temperature drops to 45°C. Add the pre-dissolved potassium sorbate solution and stir until homogeneous. Add the pre-dispersed bimetallic complex / glycerol paste. Stir for 5 minutes, then fill the finished product into opaque containers and store in a cool, dry place, away from direct sunlight and high temperatures.

[0065] Effect test

[0066] 1. Antioxidant performance test

[0067] (1) Hydroxyl radical scavenging effect test

[0068] The salicylic acid method was used to determine the scavenging effect of materials on hydroxyl radicals. Hydroxyl radicals are a form of reactive oxygen species with high chemical reactivity. They attack various biomolecules in cells and cause significant harm to organisms. The scavenging of hydroxyl radicals is typically determined by competitively capturing them with salicylic acid. In the experiment, hydrogen peroxide generates hydroxyl radicals under the action of ferrous ions. Salicylic acid is added to the system to capture these radicals, producing a purple product with characteristic absorption in the visible light region. Subsequently, an antioxidant is added to the reaction system to compete with salicylic acid for hydroxyl radical capture, reducing the formation of the purple product and altering the absorbance of the solution. By comparing this result with a blank control, the antioxidant activity of the sample can be determined.

[0069] The testing method is as follows:

[0070] Preparation of sample solutions: Before testing, the bimetallic complexes (MOFs) obtained in Examples 1-6 and the bimetallic complexes (MOFs) prepared in Comparative Examples 1-6 were prepared into sample solutions with a concentration of 2 mg / ml. The solvent was pure water and the concentration of vitamin C was 2 mg / ml.

[0071] Add 250 μL of sample solutions of different concentrations to a 1.5 mL centrifuge tube, including 250 μL of 6 mmol / L FeSO4 solution and 250 μL of 6 mmol / L hydrogen peroxide solution. Mix well and let stand for 10 min. Then add 250 μL of 6 mmol / L salicylic acid (95% v / v ethanol solution) and mix well. Incubate at room temperature in the dark for 30 min. After the reaction is complete, measure the absorbance at 510 nm. The hydroxyl radical scavenging rate is calculated using the following formula:

[0072] Hydroxyl radical scavenging rate P(%) = (A1-A2) / A1×100%,

[0073] In the formula: A1 is the absorbance of the control group 250μL FeSO4+250μL H2O2+250μL SA+250μL H2O; A2 is the absorbance of the sample group 250μL MOFs+250μL FeSO4+250μL H2O2+250μL SA.

[0074] The hydroxyl radical scavenging effect test is shown in Table 4.

[0075] Table 4 Results of the test on the effect of scavenging hydroxyl radicals

[0076]

[0077] As shown in Table 4, the bimetallic complexes prepared in Examples 1-6 exhibited hydroxyl radical scavenging rates of 70.25%-86.37%, with the bimetallic complex prepared in Example 4 showing the highest hydroxyl radical scavenging rate, approaching that of vitamin C. This indicates that the bimetallic complexes prepared in Examples 1-6 all possess good hydroxyl radical scavenging effects.

[0078] Comparison of Comparative Examples 1, 3 and 5 shows that the bimetallic complexes prepared using conventional amino acids have a hydroxyl radical scavenging effect comparable to that of monometallic complexes.

[0079] Comparing Comparative Examples 1, 3, and 4, it can be seen that the bimetallic complexes prepared by combining copper acetate and zinc acetate in conventional amounts exhibit slightly weaker hydroxyl radical scavenging effects compared to the monometallic complexes. However, the bimetallic complex prepared by combining copper acetate and zinc acetate in a specific amount in Example 1 of this invention demonstrates a synergistic effect in hydroxyl radical scavenging compared to the monometallic complexes, achieving unexpected technical results.

[0080] (2) SOD-like activity test

[0081] The SOD-like activity of the material was determined by the NBT photoreduction method. Under the combined action of light irradiation and photocatalyst, dissolved oxygen in the system was reduced to superoxide anion radicals (·O2). - It can reduce colorless NBT to form a blue formazan precipitate. The maximum characteristic absorption peak of formazan in aqueous solution is around 560 nm. The stronger the SOD activity of the material, the more it can remove O2. - The stronger the SOD-like activity, the less blue formazan is produced, and the lower the absorbance at 560 nm. The SOD-like activity of the sample can be determined by comparing it with the blank control group.

[0082] The testing method is as follows:

[0083] Sample solution preparation: Before testing, the bimetallic complexes (MOFs) prepared in Examples 1-6 and the bimetallic complexes (MOFs) prepared in Comparative Examples 1-6 were prepared into sample solutions with a concentration of 2 mg / ml. The solvent was pure water and the concentration of vitamin C was 2 mg / ml.

[0084] At room temperature, add 1.5 mL of PBS solution (0.05 M, pH=7.8), 0.3 mL of methionine solution (130 mM), 0.3 mL of nitrotetrazolium chloride (NBT) solution (1.25 mM), 0.3 mL of disodium ethylenediaminetetraacetate solution (100 μM), 0.3 mL of riboflavin (VB2) solution (20 μM), 0.05 mL of the material dispersion (2 mg / mL), and 0.25 mL of deionized water to a 5 mL centrifuge tube. Irradiate the centrifuge tube under an LED lamp with a light intensity of 400 lux for 20 min and record the absorbance at 560 nm.

[0085] Superoxide anion radical scavenging rate = (A1 - A2) / A1 × 100%

[0086] In the formula: A1 is the average absorbance of the blank; A2 is the average absorbance of the sample.

[0087] The test results for SOD-like substances are shown in Table 5.

[0088] Table 5 shows the test results of SOD activity.

[0089]

[0090] As shown in Table 5, the superoxide ion scavenging rates of the bimetallic complexes prepared in Examples 1-6 ranged from 23.81% to 49.94%. Among them, the bimetallic complex prepared in Example 4 had the highest superoxide ion scavenging rate, which was close to that of vitamin C. This indicates that the bimetallic complexes prepared in Examples 1-6 all have good superoxide ion scavenging effects.

[0091] Comparing Example 1 with Comparative Examples 1 and 3, it can be seen that, compared with monometallic complexes, the bimetallic complex of the present invention has a synergistic effect in superoxide ion scavenging, achieving unexpected technical results.

[0092] (3) Total antioxidant capacity test

[0093] The DPPH method is widely used for the quantitative determination of the antioxidant capacity of biological samples or food. The principle of this method is based on the characteristic that DPPH free radicals have a single electron and a strong absorption peak at 517 nm, resulting in a purple color in their alcoholic solution. When a free radical scavenger is present, it will pair with this single electron, causing the absorption to gradually disappear. The degree of fading is quantitatively related to the number of electrons accepted. This method has become a commonly used method for evaluating the antioxidant activity of natural products.

[0094] Test method:

[0095] Preparation of sample solutions: Before testing, the bimetallic complexes (MOFs) obtained in Examples 1-6 and the bimetallic complexes (MOFs) prepared in Comparative Examples 1-6 were prepared into sample solutions with a concentration of 2 mg / ml. The solvent was pure water and the concentration of vitamin C was 2 mg / ml.

[0096] A 0.4 mM DPPH solution with a volume fraction of 95% (v / v) in ethanol was prepared and stored at low temperature (4°C) protected from light. 100 μL of the DPPH solution and 100 μL of sample solutions of different concentrations were mixed thoroughly and reacted at 37°C in the dark for 30 minutes. After the reaction, the absorbance of the reaction solution was measured at 517 nm using a microplate reader. The DPPH free radical scavenging rate was calculated using the following formula:

[0097] P(%)=[(1-(A2-A1) / A0)]×100%,

[0098] In the formula: A2 is the absorbance of 100 μL DPPH ethanol solution + 100 μL sample solution; A1 is the absorbance of 100 μL sample solution + 100 μL sample solvent; A0 is the absorbance of 100 μL DPPH ethanol solution + 100 μL distilled water.

[0099] The results of the total antioxidant capacity test are shown in Table 6.

[0100] Table 6 Results of Total Antioxidant Capacity Test

[0101]

[0102] As shown in Table 6, the DPPH radical scavenging rates of the bimetallic complexes prepared in Examples 1-6 ranged from 65.42% to 79.91%, with the bimetallic complex prepared in Example 4 exhibiting the highest DPPH radical scavenging rate. Although all of these rates were slightly lower than the antioxidant performance of Vitamin C, considering that Vitamin C is completely soluble in water while the bimetallic complexes in the examples are poorly soluble in water, the antioxidant capacity of the samples was not fully demonstrated within the experimental conditions and time frame. Therefore, it can be inferred that the bimetallic complexes prepared in Examples 1-6 all have good DPPH radical scavenging effects.

[0103] Comparing Comparative Examples 1, 3 and 5, it can be seen that the bimetallic complexes prepared using conventional amino acids have DPPH radical scavenging effects comparable to those of monometallic complexes.

[0104] Comparing Example 1 with Comparative Examples 1 and 3, it can be seen that, compared with monometallic complexes, the bimetallic complex of the present invention has a synergistic effect in DPPH free radical scavenging, achieving unexpected technical effects.

[0105] 2. Tyrosinase inhibition rate test

[0106] In the process of melanin production, tyrosine is first converted to dopa, and dopa is then converted to dopaquinone, with tyrosinase playing a major catalytic role. Dopaquinone is a colored substance with characteristic absorption at a wavelength of 475 nm. When a sample solution that inhibits tyrosinase is added, the amount of dopaquinone synthesized decreases. The decrease is dose-dependent, and therefore, the change in absorbance at 475 nm can be used to compare the inhibitory effect of the sample solution on tyrosinase.

[0107] Test method:

[0108] Preparation of sample solutions: Before testing, samples 3-6 prepared in Examples 3-6 and Comparative Examples 1 and 3-6 were prepared into solutions with a concentration of 1 mg / ml. The solvent was pure water. The concentrations of vitamin C and glutamic acid in the control group were also 1 mg / ml.

[0109] Before testing, the bimetallic complexes (MOFs) prepared in Examples 3-6 and the bimetallic complexes (MOFs) prepared in Comparative Examples 1, 3-6 were prepared into sample solutions with a concentration of 1 mg / ml. The solvent was pure water. The concentrations of vitamin C and glutamic acid in the control group were also 1 mg / ml.

[0110] Prepare phosphate-buffered saline (PBS) solution at pH 6.83, a tyrosinase solution with a mass concentration of 0.07 mg / mL, and an L-tyrosine solution with a mass concentration of 1.0 mg / mL, respectively. Accurately prepare the following solutions: Solution 1: 2.0 mL PBS + 0.5 mL tyrosinase solution + 0.5 mL L-tyrosine solution; Solution 2: 2.5 mL PBS + 0.5 mL tyrosinase solution; Solution 3: 0.5 mL sample solution + 1.5 mL PBS + 0.5 mL tyrosinase solution + 0.5 mL L-tyrosine solution; Solution 4: 0.5 mL sample solution + 2.0 mL PBS + 0.5 mL tyrosinase solution. Incubate solutions 1-4 in a 37°C water bath for 30 min, then immediately measure the absorbance at 475 nm. Record the absorbance values ​​as A1, A2, A3, and A4, respectively. Calculate the tyrosinase inhibition rate using the following formula.

[0111] I = [(A1-A2)-(A3-A4)] / (A1-A2)×100%

[0112] The results of the tyrosinase inhibition rate test are shown in Table 7.

[0113] Table 7 Results of Tyrosinase Inhibition Rate Test

[0114]

[0115] As shown in Table 7, the tyrosinase inhibition rates of the bimetallic complexes prepared in the examples ranged from 65.62% to 76.71%, with the bimetallic complex prepared in Example 5 exhibiting the highest tyrosinase inhibition rate. The tyrosinase inhibition of vitamin C is mainly attributed to its antioxidant properties, and its instability and susceptibility to oxidation generally mean its actual whitening effect is not considered. Glutamic acid, a commonly used active ingredient in cosmetics, showed a relatively low tyrosinase inhibition rate in in vitro tests, even significantly lower than that of the bimetallic complex provided in this invention. Therefore, the bimetallic complex prepared in this invention possesses a good tyrosinase inhibition rate and exhibits excellent whitening potential.

[0116] Furthermore, a comparison of Example 1 with Comparative Examples 1 and 3 shows that, compared with monometallic complexes, the bimetallic complexes of the present invention exhibit a synergistic effect in tyrosinase inhibition, achieving unexpected technical results.

[0117] 3. HaCAT cytotoxicity assay

[0118] Test principle:

[0119] This experiment is based on a standardized method for in vitro cytotoxicity assessment. Its core principle is that metabolic enzymes such as succinate dehydrogenase in the mitochondria of living cells can reduce yellow MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) to water-insoluble blue-purple formazan crystals. This biochemical reduction reaction is carried out only by living cells, and the amount of formazan produced is directly proportional to the number of living cells and their metabolic activity in the cell population. By dissolving the crystals and measuring their absorbance at a specific wavelength (usually 490 nm), cell viability can be quantitatively characterized, thereby inferring the degree of inhibition of cellular metabolic activity by the test material, i.e., its cytotoxicity.

[0120] Test method:

[0121] HaCat cells were cultured at 37°C and 5% CO2 in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10000 U / ml), respectively. For cell viability assays, 1 × 10⁶ cells were cultured per well. 5 Cells were seeded at a density of 100% in 96-well plates. Once the cell density reached 80%, the cells were washed three times with phosphate-buffered saline (PBS), and the sample solution was diluted with serum-free high-glucose DMEM. Different concentrations of the sample were added, and after incubation for 24 hours, cell viability was measured using the MTT assay. This was used to evaluate the effect of the bimetallic complex (Example 4) on HaCat cell viability.

[0122] Test results are as follows Figure 4 As shown.

[0123] like Figure 4 As shown, at the experimental concentrations, the bimetallic complex did not exhibit significant cytotoxicity to HaCaT cells. Furthermore, concentrations between 2 mg / L and 128 mg / L showed a certain growth-promoting effect on cells, with the highest growth-promoting effect observed at a concentration of 8 mg / L, resulting in a cell survival rate of 129%. This suggests that low concentrations of the bimetallic complex may have a certain growth-promoting effect on cells, which may be related to the role of the glutamate ligand.

[0124] 4. Intracellular melanin production inhibition experiment

[0125] Test principle:

[0126] The B16 cytotoxicity assay uses the MTT assay to assess the potential toxicity of samples to mouse melanoma cells (B16 cells), a prerequisite for evaluating their biological activity (such as melanin inhibition). The principle is that succinate dehydrogenase in the mitochondria of living cells reduces the MTT reagent to water-insoluble blue-purple formazan crystals. The amount of these crystals is directly proportional to cell viability and number. By detecting the absorbance of the formazan, cell viability can be quantitatively calculated, thus eliminating false positives such as decreased melanin content due to sample cytotoxicity, ensuring that the subsequent melanin inhibition effect stems from genuine biological activity regulation rather than cell death.

[0127] The intracellular melanin production inhibition assay aims to quantitatively evaluate the inhibitory effect of a sample on intracellular melanin synthesis. The principle is as follows: First, B16-F10 cells are stimulated with melanocyte-stimulating hormone (α-MSH) to activate the intracellular melanin synthesis pathway, establishing a cell model with high melanin content (model group). After co-incubation of the sample and model cells, quantification is performed using a cytochemical lysis method: cells are thoroughly lysed and the intracellular melanin is dissolved using an alkaline DMSO solution (1mM NaOH containing 10% DMSO) at high temperature, ultimately forming a homogeneous and stable solution. Melanin exhibits broad-spectrum absorption characteristics around 490 nm, and the absorbance value of the solution is directly proportional to the melanin content. By detecting this absorbance value using a microplate reader and comparing it with the untreated model group, the inhibition rate of the sample on α-MSH-induced melanin production at different concentrations can be accurately calculated.

[0128] Test method:

[0129] (1) B16 cytotoxicity test

[0130] B16 cells were cultured at 37°C and 5% CO2 in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10000 U / ml), respectively. For cell viability assays, 1 × 10⁶ cells were cultured per well. 5Cells were seeded at a density of 100% in 96-well plates. Once the cell density reached 80%, the cells were washed three times with phosphate-buffered saline (PBS), and the sample solution was diluted with serum-free high-glucose DMEM. Different concentrations of the sample were added, and after incubation for 24 hours, cell viability was measured using the MTT assay. This was used to evaluate the effect of the bimetallic complex (Example 4) on B16 cell viability.

[0131] In an experiment on intracellular melanin production using B16-F10 cells, the toxicity of different concentrations of samples to the cells was first investigated, such as... Figure 5 As shown, the bimetallic complex did not exhibit significant toxicity to B16 cells at the experimental concentration.

[0132] (2) Test for inhibiting intracellular melanin production

[0133] B16-F10 cells were seeded at a density of 70% in 96-well plates and then treated with different concentrations (25-500 mg / L) of sample drugs. Except for the control group, the model group and experimental group were treated with 200 nM melanocyte-stimulating hormone (α-MSH) for 72 hours, followed by washing twice with ice-cold PBS buffer. Then, 1 mM sodium hydroxide solution containing 10% DMSO was added to each well, and the solution was heated in a 90°C water bath for 1 hour. Finally, the absorbance of the solution was measured at 490 nm using a microplate reader, with arbutin as a reference.

[0134] Test results:

[0135] Experimental results are as follows Figure 6 As shown in the figure. Except for the control and model groups, all other groups were treated with α-MSH to induce melanin production. The inhibitory effect of the bimetallic complex (25–200 mg / L) on melanin production showed a concentration-dependent variation. The increased melanin production observed at lower concentrations (25 mg / L) may be related to the stress response of cells to mild stimulation, a phenomenon also reported in some bioactive substances. However, at effective concentrations (≥100 mg / L), this product showed a clear concentration-dependent inhibitory effect. At 50 mg / L, the bimetallic complex had no significant effect on melanin production, and there was no significant difference between the sample and model groups; however, when the concentration was increased to 100 mg / L and 200 mg / L, it showed a significant melanin-inhibiting effect. At a concentration of 200 mg / L, its inhibitory effect on melanin production was particularly significant, approaching that of the positive control arbutin.

[0136] In conclusion, higher concentrations (≥100 mg / L) of the bimetallic complex significantly inhibit α-MSH-induced melanin production, indicating that this bimetallic complex has potential application value in the field of cosmetic whitening.

[0137] The above description, in conjunction with specific embodiments, further illustrates the present invention. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention can be made without departing from the spirit and scope of the invention, and all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. A method for preparing a double metal complex having whitening and anti-aging efficacy, characterized in that, The preparation method comprises the following steps: S1, dissolving amino acid in water to obtain solution A; S2, dissolving metal salt in ethanol to obtain solution B; S3, mixing solution A and solution B, stirring uniformly, and then obtaining solution C through solvothermal reaction; S4, washing and drying solution C to obtain the double metal complex; The amino acid in step S1 is at least one of glutamic acid and aspartic acid; The metal salt in step S2 is copper acetate and manganese chloride; the molar ratio of copper acetate to manganese chloride is 1-3:1; The molar ratio of the metal salt to the amino acid in step S3 is 1:1-3; The solvothermal reaction in step S3 is carried out at a temperature of 60-110℃ for 12-48h.

2. The production method according to claim 1, characterized by, The molar concentration of solution A in step S1 is 0.05-0.6mol / L.

3. The preparation method according to claim 1, characterized in that, The molar ratio of copper acetate to manganese chloride in step S1 is 1-2:

1.

4. The method of claim 1, wherein, The molar concentration of solution B in step S2 is 0.04-0.3mol / L.

5. The preparation method according to claim 1, characterized in that, The solvothermal reaction in step S3 is carried out at a temperature of 80-100℃ for 12-24h.

6. The double metal complex with whitening and anti-aging effects obtained by the preparation method in any one of claims 1-5.

7. The use of the double metal complex in claim 6 in the preparation of cosmetics.

8. A cosmetic having whitening and anti-aging effects, characterized by, The cosmetic comprises the double metal complex in claim 6.

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