ZIF-8 and its derivatives, preparation methods and applications

Abstract

This invention discloses ZIF-8 and its derivatives, preparation methods, and applications, relating to the field of biomedical nanomaterials. Using TYR inhibition rate as an indicator, this invention optimizes the preparation process of ZIF-8, obtaining ZIF-8 with stable performance. The IC50 of the TYR inhibitory activity of ZIF-8 obtained through this process is shown in the figure. 50 The concentration reached 1.67 mmol / L, showing better efficacy than nicotinamide whitening agents; at the same mass concentration, its TYR inhibition activity was significantly higher than that of UiO-66-NH2, N101, M100, and M101 nanomaterials. Further improvements can be made by doping with Ca... 2+ or Ni 2+ Preparation of ZIF-8 derivatives to enhance its TYR inhibitory activity, via Ca 2+ or Ni 2+ After modification, its IC for TYR 50 The values ​​were 0.99 mmol / L and 0.65 mmol / L, respectively, indicating an increase in inhibitory effect of over 40%. The batch-to-batch variation of the method of this invention is <5%, and the safe concentration of the obtained ZIF-8 and its derivatives for cells is <120 μg / mL. It can be used as an active ingredient to formulate cosmetics or pharmaceuticals such as emulsions, serums, masks, or creams for the prevention or treatment of pigmentation disorders such as melasma and freckles.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials, specifically to ZIF-8 and its derivatives, preparation methods, and applications. Background Technology

[0002] Tyrosinase (TYR), also known as monophenol oxidase, is the only known rate-limiting enzyme in organisms involved in melanin synthesis. It catalyzes the oxidation of catechols to benzoquinones, which participate in melanin synthesis in the human body. TYR overexpression can easily lead to skin conditions such as acanthosis nigricans, cervical dyschromia, melasma, periorbital pigmentation, and freckle-like nevi. It is also a marker of melanoma formation. Inhibiting TYR activity with TYR inhibitors can significantly reduce the efficiency of tyrosine conversion to melanin. Currently, both naturally and synthetically derived TYR inhibitors are widely used in skin whitening and the treatment of pigmentary disorders. However, most TYR inhibitors on the market suffer from poor stability and high toxicity. For example, arbutin, due to its instability, is suspected of being a precursor to hydroquinone, which has toxic side effects on the skin, and has been banned from use in cosmetics in some countries; kojic acid is easily oxidized, and long-term use may be carcinogenic; vitamin C and its derivatives have poor skin absorption and are only effective at high concentrations. Therefore, the development of safe, effective, and highly stable novel TYR inhibitors is of great significance in areas such as skin whitening cosmetics and the prevention and treatment of pigmentation disorders.

[0003] Zeolite imidazolium ester framework material-8 (ZIF-8) is a nanomaterial formed by metal-organic coordination bonds, composed of zinc ions and 2-methylimidazolium ligands. Due to its high specific surface area, tunable pore structure, good chemical stability and biocompatibility, and low toxicity, ZIF-8 or its derivatives have attracted widespread attention in the fields of antioxidant, antibacterial and anticancer drug delivery. For example, patent CN119500234A discloses FeCo-NC bimetallic single-atom nanozymes, their preparation methods, and applications, indicating that the nanomaterials prepared by this invention possess dual-enzyme activity, simultaneously mimicking catalase and NADH oxidase, and can effectively scavenge reactive oxygen species. Some studies have found that ZIF-8 has significant anti-inflammatory and antifungal effects, providing a new solution for the treatment of fungal keratitis. Patent CN119792571A discloses ZIF-8 nanoparticles loaded with mitoxantrone, their preparation, and their application in tumor chemo-immunotherapy, indicating that MIT@ZIF-8, as a novel chemo-immunotherapy regimen, can enhance the responsiveness of tumors to immune checkpoint inhibitor therapy. However, there are no reports on ZIF-8 or its derivatives as TYR inhibitors in areas such as skin whitening and prevention and treatment of pigmentary disorders. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide ZIF-8 and its derivatives, preparation methods and applications.

[0005] The technical solution of the present invention is as follows:

[0006] Application of ZIF-8 and its derivatives in the preparation of products that inhibit tyrosinase activity, wherein the active substance in the product is ZIF-8 and / or a derivative of ZIF-8, and the active substance is capable of inhibiting tyrosinase activity.

[0007] Furthermore, the product also includes pharmaceutically acceptable excipients.

[0008] Furthermore, the preparation method of ZIF-8 includes the following steps:

[0009] S1: Dissolve the zinc source and 2-methylimidazole separately in a solvent to form solution A and solution B;

[0010] S2: Add solution A dropwise to solution B while stirring, and seal the reaction to obtain the reaction product;

[0011] S3: The reaction product was centrifuged, washed and dried to obtain ZIF-8 nanoparticles.

[0012] Furthermore,

[0013] In step S1, the molar ratio of zinc source to 2-methylimidazole is 1:4-16; the zinc source is at least one of zinc nitrate hexahydrate, zinc nitrate, zinc acetate dihydrate, zinc acetate, and zinc sulfate; methanol is used as the solvent, and the total amount of methanol used is 10-40 mL.

[0014] In step S2, the reaction is carried out in a closed system for 30-210 minutes;

[0015] In step S3, methanol is used for washing, and the amount of methanol used is 10-40 mL.

[0016] Furthermore, the preparation method of the ZIF-8 derivative includes the following steps:

[0017] a: The zinc source and metal ions are mixed at a certain molar ratio and dissolved in methanol to form solution C;

[0018] b: Dissolve 2-methylimidazole in methanol to form solution D;

[0019] c: Add solution C dropwise to solution D, react for a period of time, and obtain the reaction product;

[0020] d: The reaction product was centrifuged, washed, and vacuum dried to obtain a derivative of ZIF-8.

[0021] Further, in step a, the molar ratio of zinc source to metal ions is 1:0.2-1:5; the metal ions are Ca... 2+ or Ni 2 + At least one of them; wherein Ca 2+ The source is at least one of calcium chloride and calcium carbonate; Ni 2+ The source is at least one of nickel nitrate hexahydrate, nickel nitrate, nickel chloride, nickel chloride hexahydrate, nickel bromide, and nickel sulfate;

[0022] In step c, the total amount of methanol used is 10-40 mL; the reaction time is 30-300 min.

[0023] Furthermore, the product includes tyrosinase inhibitors or skin whitening products. Specifically, skin whitening products include one of the following: lotions, serums, masks, and creams for skin whitening, or pharmaceutical products for skin whitening. The skin whitening product can also be used to prevent, alleviate, and / or treat sunspots, freckles, age spots, melasma, and post-inflammatory hyperpigmentation.

[0024] The beneficial effects of this invention are as follows: This invention creatively discovers that ZIF-8 and its derivatives can act as TYR inhibitors, and their inhibitory effect is significantly superior to UiO-66-NH2, N101, M100, and M101 nanomaterials, as well as commonly used nicotinamide whitening agents, thus broadening the function and application fields of ZIF-8. The process of this invention has strong controllability and good stability, with batch-to-batch differences of <5%, enabling large-scale production. Furthermore, this invention creatively discovers that metal ion doping can further enhance the inhibitory activity against TYR, through Ca... 2+ Ni 2+ After modification, the IC values ​​of ZIF-8 / Ca and ZIF-8 / Ni are... 50 The values ​​are respectively compared to the IC of ZIF-8 50 The values ​​decreased by 40.72% and 61.08%, and cell experiments showed that ZIF-8 and its derivatives have good biocompatibility. Attached Figure Description

[0025] Figure 1 The figures show the optimization results of the ZIF-8 synthesis process. In the figure, A represents the optimized molar ratio of 2-methylimidazole to Zn(NO3)2·6H2O, B represents the optimized reaction time, C represents the optimized methanol dosage, and D represents the stability study results. Different letters (a, b, c, d, e) indicate significant differences between different treatment groups. P <0.05, multiple letters (bc, cd, bcd, be) indicate that there is no significant difference between this group and other groups containing the same letters, but there is a significant difference between this group and groups without these letters.P <0.05).

[0026] Figure 2 Here is the FI-IR spectrum of ZIF-8;

[0027] Figure 3 This is a particle size distribution diagram of ZIF-8;

[0028] Figure 4 The zeta potential diagram for ZIF-8;

[0029] Figure 5 The inhibition rate of TYR by different MOF species;

[0030] Figure 6 IC50 of ZIF-8 for inhibiting TYR activity 50 value;

[0031] Figure 7 IC50 of nicotinamide's inhibitory activity against TYR 50 value;

[0032] Figure 8 The figure shows the inhibition rate of ZIF-8 and its derivatives against TYR; different letters (a, b, c, d, e, f) indicate significant differences between different ZIF-8 and its derivatives. P <0.05).

[0033] Figure 9 The FI-IR spectra of ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni are shown.

[0034] Figure 10 Particle size distribution diagrams for ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni;

[0035] Figure 11 Zeta potential diagrams for ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni;

[0036] Figure 12 IC50 values ​​for the inhibitory activity of ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni against TYR 50 value;

[0037] Figure 13 To determine the effect of kojic acid on the activity of mouse B16F10 melanoma cells using the CCK-8 assay;

[0038] Figure 14 To determine the effect of resveratrol on the viability of mouse B16F10 melanoma cells using the CCK-8 assay;

[0039] Figure 15To determine the effect of ZIF-8 and its derivatives on the viability of mouse B16F10 melanoma cells using the CCK-8 assay;

[0040] Figure 16 The effect of ZIF-8 on the relative activity of TYR in melanoma cells of B16F10 mice;

[0041] Figure 17 The effect of ZIF-8 on the relative melanin content in melanoma cells of B16F10 mice. Detailed Implementation

[0042] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0043] Example 1: Preparation and Characterization of ZIF-8

[0044] (1) ZIF-8 synthesis process

[0045] ZIF-8 was prepared in methanol by a room-temperature stirring method. First, 0.2975 g (1 mmol) of Zn(NO3)2·6H2O was accurately weighed and dissolved in 3 mL of methanol by sonication to form a clear solution A. Then, 0.9854 g (12 mmol) of 2-methylimidazole was accurately weighed and dissolved in 12 mL of methanol by sonication to form a clear solution B. Next, solution A was added dropwise to solution B at 1000 rpm under stirring at room temperature. The mixture was sealed and stirred at 1000 rpm for 180 min, allowing ZIF-8 nanoparticles to spontaneously form, resulting in a white suspension. Finally, the suspension was centrifuged at 12000 rpm for 5 min to obtain a white precipitate, which was washed three times with 10 mL of methanol and then vacuum-dried overnight at 60 °C to obtain ZIF-8 nanoparticles for later use.

[0046] (2) Optimization of the molar ratio of 2-methylimidazole and Zn(NO3)2·6H2O

[0047] The molar ratios of 2-methylimidazole and Zn(NO3)2·6H2O were adjusted to 4:1, 6:1, 8:1, 10:1, 12:1, 14:1, and 16:1, respectively. Other conditions were carried out as in (1). The optimal molar ratio was obtained by taking the inhibition rate of ZIF-8 on tyrosinase under different conditions as the indicator.

[0048] (3) Optimization of reaction time

[0049] Based on the optimal molar ratio of 12:1, the reaction times were adjusted to 30 min, 60 min, 90 min, 120 min, 150 min, 180 min and 210 min respectively, and other conditions were carried out as in (1). The optimal reaction time was obtained by taking the inhibition rate of ZIF-8 on tyrosinase under different conditions as the indicator.

[0050] (4) Optimization of methanol usage

[0051] Based on the optimal molar ratio of 12:1 and the optimal reaction time of 30 min, the methanol dosage was adjusted to 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL and 40 mL respectively, and other conditions were carried out as in (1). The optimal methanol dosage was obtained by taking the inhibition rate of ZIF-8 on tyrosinase under different conditions as the indicator.

[0052] (5) ZIF-8 stability test

[0053] Based on the optimal molar ratio of 12:1, the optimal reaction time of 30 min, and the methanol dosage of 15 mL, six batches of ZIF-8 were synthesized under the same conditions (1). The inhibition rate of the six batches of ZIF-8 against tyrosinase at the same mass concentration (0.714 mg / mL) was determined to evaluate the stability of ZIF-8.

[0054] (6) Characterization of ZIF-8

[0055] The functional groups and chemical bonds of ZIF-8 were determined using Fourier transform infrared spectroscopy (Thermo Fisher Scientific, USA). The particle size distribution and zeta potential of the ZIF-8 nanoparticles were determined using zetasizer (Malvin, UK).

[0056] Using the inhibition rate of ZIF-8 against TYR as an indicator, the synthesis process was optimized, and the results are as follows: Figure 1 As shown, the optimal synthesis process for ZIF-8 is a molar ratio of 2-methylimidazole to Zn(NO3)2·6H2O of 12:1, a reaction time of 30 min, and a methanol dosage of 15 mL. Under the optimal process, when the mass concentration of ZIF-8 is 0.714 mg / mL, the inhibition rate against tyrosinase is 62.45 ± 1.21%. Simultaneously, six batches of ZIF-8 were synthesized under the optimal conditions, and their inhibition rates against tyrosinase at a mass concentration of 0.714 mg / mL were determined. The results indicate that the ZIF-8 process is stable, and the inhibition rates against tyrosinase are basically consistent at the same mass concentration.

[0057] The functional groups and chemical bonds of ZIF-8 were determined using Fourier transform infrared spectroscopy. ZIF-8 showed the following values ​​at 1580 cm⁻¹. -11140 cm -1 Absorption peaks appear, corresponding to the symmetric stretching vibrations of the C=N bond in the imidazole ring and the stretching vibrations of the CN bond in the imidazole ring, respectively; at 420 cm⁻¹ -1 The appearance of an absorption peak corresponds to Zn 2+ The coordination bond vibration with the nitrogen atom of imidazole indicates that the imidazole ligand successfully binds with Zn. 2+ Coordination forms the skeletal structure; while CH stretching vibrations occur at 3135 cm⁻¹. -1 The out-of-plane bending vibration of the imidazole ring is approximately 755 cm⁻¹. -1 and 690 cm -1 nearby( Figure 2 The pore size distribution and zeta potential of ZIF-8 nanoparticles were measured using a Zetasizer nanoparticle size analyzer. The results showed that the average particle size of ZIF-8 was 176.3 nm. Figure 3 The Zeta potential is -40.94 mV. Figure 4 This indicates that ZIF-8 has a relatively uniform distribution and good dispersion.

[0058] Example 2: Evaluation of the inhibitory activity of different MOF materials and commonly used whitening agents on TYR

[0059] Commonly available MOF materials were selected, and their inhibition rates against TYR at a concentration of 0.714 mg / mL were measured to compare the inhibitory activities of different MOF materials against TYR. The results showed that at the same concentration, ZIF-8 exhibited the strongest inhibition rate against TYR, significantly stronger than common MOF materials such as UiO-66-NH2, Fe3O4-COOH@UiO-66-NH2, N101, M100, and M101 (see [link to study]). Figure 5 ).

[0060] A commercially available niacinamide whitening agent (99% purity, Shanghai Aladdin Biochemical Technology Co., Ltd.) was selected. The inhibition rates of ZIF-8 and niacinamide on TYR at different molar concentrations were measured to compare their inhibitory activities on TYR. Curve fitting was used to determine the half-maximal inhibitory rate (IC50) of ZIF-8. 50 The value was 1.67 mmol / L (see Figure 6 ), IC of nicotinamide 50 The value was 60.42 mmol / L (see...) Figure 7 Therefore, it can be concluded that ZIF-8 has a significantly stronger inhibitory activity against TYR than nicotinamide.

[0061] Example 3: Preparation and Characterization of ZIF-8 Derivatives

[0062] (1) Preparation of ZIF-8 derivatives

[0063] First, accurately weigh 1 mmol Zn(NO3)2·6H2O, and separately weigh 2 mmol Na2SeO3·5H2O, CaCl2, AlCl3·6H2O, H3BO3, Ni(NO3)2·6H2O, and FeSO4·7H2O. Dissolve CaCl2, AlCl3·6H2O, H3BO3, and Ni(NO3)2·6H2O in 3 mL of methanol using ultrasonication, and dissolve Na2SeO3·5H2O and FeSO4·7H2O in 3 mL of water, forming clear solution A. Then, accurately weigh 10 mmol 2-methylimidazole and dissolve it in 12 mL of methanol using ultrasonication, forming clear solution B. Next, at room temperature and with stirring at 1000 rpm, add solution A dropwise to solution B, seal the container, and stir at 1000 rpm for 210 min. ZIF-8 derivative nanoparticles spontaneously form, resulting in a suspension. Finally, the precipitate was obtained by centrifugation at 12000 r / min for 5 min, washed three times with 10 mL methanol, and dried under vacuum at 60℃ overnight. The ZIF-8 derivative nanoparticles were ready for use.

[0064] (2) Characterization of ZIF-8 derivatives

[0065] The functional groups and chemical bonds of the ZIF-8 derivatives were determined using Fourier transform infrared spectroscopy (Thermo Fisher Scientific, USA). The particle size distribution and zeta potential of the ZIF-8 derivative nanoparticles were determined using zetasizer (Malvin, UK).

[0066] ZIF-8 derivatives were prepared by doping with different macro- or micro-elements found in the human body. The TYR inhibition rate at a molar concentration of 2.86 mmol / L was measured, and the results are as follows: Figure 8 As shown, compared to ZIF-8, the Ca doping... 2+ and Ni 2+ Afterwards, at the same molar concentration, the inhibition rate of TYR was significantly increased by more than 6%.

[0067] The functional groups and chemical bonds of ZIF-8 / Ca and ZIF-8 / Ni were determined using Fourier transform infrared spectroscopy, such as... Figure 9 As shown, when Ca 2+ Partially replaces Zn 2+ When it coordinates with imidazole ligands, it may reduce the electron density of the imidazole ring, resulting in a 1580 cm⁻¹. -1 The intensity of the C=N peak at Ca decreases; 2+ The interaction with the imidazole ring may alter the electronic environment of the methyl group, leading to a 3135 cm⁻¹ -1 The intensity of the CH peak at that location decreases; simultaneously, the Ca... 2+Doping may cause local distortions or defects in the ZIF-8 crystal structure, 755 cm⁻¹ -1 and 690 cm -1 Out-of-plane bending vibrations may cause splitting due to reduced crystal symmetry. When Ni... 2+ Coordination with the nitrogen atom of the imidazole ligand may reduce the electron density of the imidazole ring, weaken the C=N bond strength, and lead to a decrease in vibrational frequency, with the C=N peak shifting to a lower wavenumber; simultaneously, Ni 2+ The strong coordination ability of the C-N bond may alter the local symmetry of the CN bond, causing a change in peak shape and resulting in a peak at 1140 cm⁻¹. -1 The CN peak at that location broadens or splits.

[0068] The pore size distribution and zeta potential of ZIF-8 and its derivative nanoparticles were measured using a Zetasizer nanoparticle size analyzer. The results showed that the average particle size of ZIF-8 was 176.3 nm, and the Ca-doped particles were... 2+ and Ni 2+ Subsequently, the average particle size of its bimetallic derivatives increased to 380.4 nm and 372.3 nm, respectively. Figure 10 The Zeta potential of ZIF-8 is -40.94 mV, and it is doped with Ca. 2+ and Ni 2+ Subsequently, the average Zeta potentials of its bimetallic derivatives increased, reaching -36.33 mV and -36.87 mV respectively. Figure 11 The results indicate that ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni are relatively uniformly distributed and well dispersed.

[0069] Example 4: Inhibitory effect of ZIF-8 and its derivatives on TYR in vitro

[0070] In this experiment, as shown in Table 1, 40 μL of the test solution, 40 μL of tyrosinase solution (200 U / mL), and an appropriate amount of PBS buffer (50 mM / L, pH=6.5) were added to 2 mL centrifuge tubes, mixed well, and incubated at 37℃ for 30 min. After incubation, 50 μL of L-DOPA solution (2 mM / L) was added as substrate, mixed well, and incubated at 37℃ for 20 min. After incubation, the centrifuge tubes were centrifuged at 13000 r / min for 5 min, and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance at 475 nm was measured, and the inhibition rate (%) relative to the control was calculated using the following formula.

[0071] ;

[0072] In the formula, T 样品 T represents the absorbance of the sample being tested, T0 represents the background absorbance of the sample, and T...对照 T represents the absorbance of the control sample. 溶剂 This indicates the absorbance of the PBS buffer solution.

[0073] Table 1. Reaction systems for determining the inhibitory activity of TYR inhibitors against TYR.

[0074]

[0075] The half-maximal inhibitory concentration (IC50) of ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni on the inhibitory activity of TYR. 50 The value was measured, and the result was as follows: Figure 12 The values ​​shown are 1.67 mmol / L, 0.99 mmol / L, and 0.65 mmol / L, respectively, indicating that ZIF-8 can effectively inhibit TYR activity, and its derivatives can further enhance the inhibitory activity against TYR. The IC50 values ​​of ZIF-8 / Ca and ZIF-8 / Ni are also shown. 50 The values ​​are respectively compared to the IC of ZIF-8 50 The values ​​decreased by 40.72% and 61.08%.

[0076] Example 5: Effects of ZIF-8, ZIF-8 derivatives, and commonly used skin whitening agents on mouse B16F10 melanoma cells

[0077] (1) Culture of mouse B16F10 melanoma cells

[0078] B16F10 cells, derived from mouse skin melanoma, were cultured in Durbeco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamine. These cells were grown in a humidified incubator at 37 °C and 5% CO2. To maintain the continuous exponential growth of B16F10 cells, passage was performed when the culture flask reached 80% capacity, typically every 2-3 days.

[0079] (2) Effects of ZIF-8, ZIF-8 derivatives and commonly used whitening agents on the activity of mouse B16F10 melanoma cells by CCK8 assay

[0080] When the cells reach the logarithmic growth phase, they are digested to prepare a cell suspension. 100 μL is added to each well, with a density of 8 × 10⁻⁶ cells / well. 3 Cells were seeded per well in 96-well plates. After culturing for 24 h and allowing cells to adhere, the culture medium was discarded, and the cells were washed twice with PBS.

[0081] The experiment was divided into three groups: a blank group (containing only culture medium), a control group (containing culture medium and cultured normally), and experimental groups (containing culture medium with different concentrations of the sample). Eight concentrations of each sample were used: kojic acid, ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni were administered at concentrations of 15, 30, 45, 60, 75, 90, 105, and 120 μg / mL, respectively; resveratrol was administered at concentrations of 0.2, 0.3, 0.6, 1.3, 2.5, 5.0, 10.0, and 20.0 μg / mL. Each concentration was used in six replicates. After 24 h of culture, the culture medium was discarded, and 100 μL of culture medium containing 10% CCK-8 solution was added to each well in the dark. The wells were incubated at 37°C in the dark for 1 h, and the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Cell viability (%) was calculated using the formula.

[0082] ;

[0083] In the formula, OD 实验组 The absorbance of the experimental group is represented by OD. 空白组 The absorbance of the blank group is represented by OD. 对照组 This indicates the absorbance of the control group.

[0084] (3) Effect of ZIF-8 on tyrosinase activity in B16F10 mouse melanoma cells determined by dopa oxidation method

[0085] Cell culture is the same as above. When the cells grow to the logarithmic growth phase, they are digested to prepare a cell suspension, with a volume of 1 mL per well and a density of 5 × 10⁻⁶ cells / well. 4 Seeds were inoculated per well in a 24-well plate. After incubation for 24 h, the culture medium was discarded, and the plates were washed twice with PBS.

[0086] The experiment was divided into three groups: a blank group (containing no cells but culture medium with different concentrations of samples), a control group (containing culture medium without samples and normally cultured cells), and a drug-treated group (containing culture medium with different concentrations of samples, including kojic acid (240 μg / mL), resveratrol (10 μg / mL positive control sample), and ZIF-8 (60 and 120 μg / mL samples). After 24 h of culture, the culture medium was discarded, and cells were washed with PBS. 0.3 mL of trypsin cell digestion buffer was added to each well for digestion. Cells were then collected into centrifuge tubes and centrifuged at 1500 r / min for 5 min. The supernatant was discarded, and cell pellets were obtained. Cell pellets were washed with PBS, centrifuged at 6400 r / min for 5 min, and the supernatant was discarded. 200 μL of 1% Triton X-100 PBS lysis buffer was added to each tube to resuspend the cells. The cells were placed on ice and lysed thoroughly for 60 min, mixing every 20 min. Afterwards, centrifuge at 12000 r / min for 15 min at 4℃, and collect the supernatant for TYR activity determination. Add 150 μL of the supernatant and 100 μL of 2 mmol / L L-DOPA solution to each well of a 96-well plate, incubate at 37℃ for 30 min, and measure the absorbance at 475 nm. Calculate the TYR activity (%) relative to the control using the following formula:

[0087] ;

[0088] In the formula, OD 给药组 The absorbance and OD of the drug-treated group are indicated. 空白组 The absorbance of the blank group is represented by OD. 对照组 This indicates the absorbance of the control group.

[0089] (4) Determination of the effect of ZIF-8 on melanin synthesis in B16F10 mouse melanoma cells by sodium hydroxide lysis method

[0090] The cell culture and processing steps are the same as in (3). After discarding the culture medium, wash the cells twice with PBS, then digest them with trypsin to collect the cells, centrifuge at 1500 r / min for 5 min. Discard the supernatant, resuspend the cell pellet in 500 μL PBS and centrifuge again. After discarding the supernatant, add 100 μL of 1 mol / L sodium hydroxide solution containing 10% DMSO to the cell pellet, and incubate in a metal bath at 80℃ for 1 h to allow the melanin granules to dissolve completely. Determine the amount of synthesized melanin by measuring the absorbance at 405 nm using an ELISA reader. Calculate the relative intracellular melanin content (%) relative to the control using the formula:

[0091] ;

[0092] In the formula, OD 给药组 The absorbance and OD of the drug-treated group are indicated.空白组 The absorbance of the blank group is represented by OD. 对照组 This indicates the absorbance of the control group.

[0093] The effects of kojic acid, resveratrol, ZIF-8 and its derivatives (ZIF-8 / Ca and ZIF-8 / Ni) on the activity of mouse B16F10 melanoma cells were determined using the CCK-8 assay. The results are as follows: Figures 13-15 As shown, the positive controls kojic acid, ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni had virtually no effect on the activity of mouse B16F10 melanoma cells at concentrations below 120 μg / mL, while the positive control resveratrol had virtually no effect on the activity of mouse B16F10 melanoma cells at concentrations below 20 μg / mL. These results indicate that ZIF-8 and its derivatives ZIF-8 / Ca and ZIF-8 / Ni exhibit low cytotoxicity at certain concentrations, and their biocompatibility is superior to that of the positive control resveratrol.

[0094] The effect of ZIF-8 on TYR activity in B16F10 mouse melanoma cells was determined using the dopa oxidation method. The results are as follows: Figure 16 As shown, compared with the control group, the positive controls kojic acid, resveratrol, and ZIF-8 all significantly reduced the relative activity of TYR. P <0.05), and the higher the concentration of ZIF-8, the stronger the inhibitory effect on TYR activity. The effect of ZIF-8 on melanin synthesis in B16F10 mouse melanoma cells was determined by the sodium hydroxide lysis method, and the results are as follows. Figure 17 As shown, compared with the control group, both the positive control kojic acid and ZIF-8 significantly reduced the relative production of intracellular melanin. P The concentration of ZIF-8 was <0.05%, and the higher the concentration of ZIF-8, the stronger the effect of inhibiting the relative production of intracellular melanin. These results indicate that ZIF-8 can act as a TYR inhibitor, effectively reducing intracellular TYR activity and inhibiting intracellular melanin production, making it a potential raw material for skin whitening products.

[0095] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. The application of ZIF-8 and its derivatives in the preparation of products that inhibit tyrosinase activity, characterized in that, The active substance in the product is ZIF-8 and / or a derivative of ZIF-8, which can inhibit the activity of tyrosinase. The preparation method of ZIF-8 includes the following steps: S1: Dissolve the zinc source and 2-methylimidazole separately in a solvent to form solution A and solution B; S2: Add solution A dropwise to solution B while stirring, and seal the reaction to obtain the reaction product; S3: The reaction product was centrifuged, washed and dried to obtain ZIF-8 nanoparticles; The preparation method of the ZIF-8 derivative includes the following steps: a: The zinc source and metal ions are mixed at a certain molar ratio and dissolved in methanol to form solution C; b: Dissolve 2-methylimidazole in methanol to form solution D; c: Add solution C dropwise to solution D, react for a period of time, and obtain the reaction product; d: The reaction product was centrifuged, washed, and vacuum dried to obtain a derivative of ZIF-8; In step a, the molar ratio of zinc source to metal ions is 1:0.2-1:5; the metal ions are Ca... 2+ or Ni 2+ One or more of the following; wherein Ca 2+ The source is at least one of calcium chloride and calcium carbonate; the Ni 2+ The source is at least one of nickel nitrate hexahydrate, nickel nitrate, nickel chloride, nickel chloride hexahydrate, nickel bromide, and nickel sulfate.

2. The application according to claim 1, characterized in that, The product also includes pharmaceutically acceptable excipients.

3. The application according to claim 1, characterized in that, In step S1, the molar ratio of zinc source to 2-methylimidazole is 1:4-16; the zinc source is at least one of zinc nitrate hexahydrate, zinc nitrate, zinc acetate dihydrate, zinc acetate, and zinc sulfate; methanol is used as the solvent, and the total amount of methanol used is 10-40 mL. In step S2, the reaction is carried out in a closed system for 30-210 min; In step S3, methanol is used for washing, and the amount of methanol used is 10-40 mL.

4. The application according to claim 1, characterized in that, In step c, the total amount of methanol used is 10-40 mL; the reaction time is 30-300 min.

5. The application according to claim 1, characterized in that, The products include tyrosinase inhibitors or skin whitening products.

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