ZIF-8 as well as derivative, preparation method and application thereof

Abstract

The invention discloses ZIF-8 as well as a derivative, a preparation method and application thereof, relates to the field of biomedical nanomaterials, and aims to optimize a preparation process of ZIF-8 by taking TYR inhibition rate as an index and obtain ZIF-8 with stable performance. The IC50 value of the ZIF-8 obtained by the process to the TYR inhibitory activity reaches 1.67 mmol / L, and the effect is superior to that of a nicotinamide whitening agent; under the same mass concentration, the TYR inhibition activity of the nano material is obviously higher than that of UiO-66-NH2, N101, M100 and M101 nano materials. Furthermore, a ZIF-8 derivative can be prepared by doping Ca < 2 + > or Ni < 2 + >, so that the TYR inhibition activity of the ZIF-8 derivative is improved, the IC50 values of the ZIF-8 derivative to TYR modified by Ca < 2 + > or Ni < 2 + > are 0.99 mmol / L and 0.65 mmol / L respectively, and the inhibition effect is improved by 40% or above. According to the method disclosed by the invention, the batch difference is less than 5%, the safe concentration of the obtained ZIF-8 and the derivative thereof to cells is less than 120mu g / mL, and the ZIF-8 and the derivative thereof can be used as active ingredients to be prepared into cosmetics or medicines such as emulsion, essence, facial masks or cream and the like, and are used for preventing or treating hyperpigmentation diseases such as chloasma and freckles and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical nanomaterials, and particularly to ZIF-8 and its derivatives, a preparation method and applications thereof. Background Art

[0002] Tyrosinase (TYR), also known as monophenol oxidase, is the only known key rate-limiting enzyme involved in melanin synthesis in organisms. It can catalyze the oxidation of catechol substances to o-benzoquinone compounds and participate in the synthesis of melanin in the human body. When TYR is overexpressed, skin diseases such as acanthosis nigricans, poikiloderma of Civatte, melasma, periorbital hyperpigmentation, and nevus spilus are likely to form, and it is one of the markers for the formation of melanoma. By inhibiting the activity of TYR with TYR inhibitors, the efficiency of the conversion of tyrosine to melanin can be significantly reduced. Currently, TYR inhibitors from natural and synthetic sources have been widely used in skin whitening and the treatment of pigmentation diseases. However, most of the commercially available TYR inhibitors have problems such as poor stability and high toxicity. For example, arbutin is suspected to be a precursor of hydroquinone, which is toxic to the skin, due to its instability and has been prohibited from being added to cosmetics in some countries; kojic acid is easily oxidized and may also cause cancer after long-term use; vitamin C and its derivatives have poor skin absorbability and are only effective at high concentrations. Therefore, the development of new TYR inhibitors with high safety, effectiveness, and stability is of great significance in whitening cosmetics, the prevention and treatment of pigmentation diseases, etc.

[0003] Zeolitic imidazolate framework material-8 (ZIF-8) is a nanomaterial formed by metal-organic coordination bonds and consists of zinc ions and 2-methylimidazole ligands. Due to its high specific surface area, adjustable pore structure, good chemical stability, good biocompatibility, and low toxicity, ZIF-8 or its derivatives have received extensive attention in the fields of antioxidant, antibacterial, and anticancer drug delivery. For example, the FeCo-NC bimetallic single-atom nanozyme and its preparation method and applications disclosed in the patent with the publication number CN119500234A show that the nanomaterial prepared by this invention has dual enzyme activities, can simultaneously simulate 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; the ZIF-8 nanoparticles loaded with mitoxantrone and its preparation and applications in tumor chemo-immunotherapy disclosed in the patent with the publication number CN119792571A show that MIT@ZIF-8, as a new chemo-immunotherapy regimen, can enhance the response of tumors to immune checkpoint inhibitor therapy. However, there has been no report on ZIF-8 or its derivatives as TYR inhibitors in the fields of skin whitening and the prevention and treatment of pigmentation diseases. Summary of the Invention

[0004] The object of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide ZIF-8 and its derivatives, a preparation method and an application thereof.

[0005] The technical solution of the present invention is as follows: Application of ZIF-8 and its derivatives in the preparation of a product for inhibiting tyrosinase activity, wherein the active substance in the product is ZIF-8 and / or a derivative of ZIF-8, and the active substance can inhibit the activity of tyrosinase.

[0006] Furthermore, the product further comprises a pharmaceutically acceptable excipient.

[0007] Furthermore, the preparation method of the ZIF-8 comprises the following steps: S1: Dissolve a zinc source and 2-methylimidazole in solvents respectively to form solution A and solution B; S2: Dropwise add solution A into solution B while stirring, and carry out a closed reaction to obtain a reaction product; S3: Centrifuge, wash and dry the reaction product to obtain ZIF-8 nanoparticles.

[0008] Furthermore, In step S1, the molar ratio of the 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 closed reaction is carried out for 30-210 min; In step S3, methanol is used for washing, and the amount of methanol used is 10-40 mL.

[0009] Furthermore, the preparation method of the ZIF-8 derivative comprises the following steps: a: Mix a zinc source and a metal ion in a certain molar ratio and dissolve them in methanol to form solution C; b: Dissolve 2-methylimidazole in methanol to form solution D; c: Dropwise add solution C into solution D, react for a period of time to obtain a reaction product; d: Centrifuge, wash and vacuum dry the reaction product to obtain the ZIF-8 derivative.

[0010] Furthermore, in step a, the molar ratio of the zinc source to the metal ion is 1:0.2-1:5; the metal ion is Ca 2+ or Ni 2 + at least one of them; wherein the Ca 2+ source is at least one of calcium chloride and calcium carbonate; Ni2+ The source is at least one of nickel nitrate hexahydrate, nickel nitrate, nickel chloride, nickel chloride hexahydrate, nickel bromide, and nickel sulfate; In step c, the total amount of methanol used is 10 - 40 mL; the reaction time is 30 - 300 min.

[0011] Furthermore, the product includes a tyrosinase inhibitor or a whitening product. Specifically, the whitening product includes one of a lotion, essence, facial mask, and cream for skin whitening, or a medicine for skin whitening. This whitening product can also be used for products that prevent, relieve, and / or treat solar lentigines, freckles, senile lentigines, melasma, and post - inflammatory hyperpigmentation.

[0012] The beneficial effects of the present invention are as follows: The present invention creatively discovers that ZIF - 8 and its derivatives can be used as TYR inhibitors, and the inhibitory effect is significantly better than that of UiO - 66 - NH2, N101, M100, and M101 nanomaterials, as well as the commonly used niacinamide whitening agent on the market, broadening the functions and application fields of ZIF - 8. The process of the present invention has strong controllability and good stability, and the difference between batches is <5%, enabling large - scale production. In addition, the present invention creatively discovers that metal ion doping can further enhance the inhibitory activity against TYR. After being modified by Ca 2+ , Ni 2+ , the IC 50 values of ZIF - 8 / Ca and ZIF - 8 / Ni are respectively reduced by 40.72% and 61.08% compared with the IC 50 value of ZIF - 8, and cell experiments show that ZIF - 8 and its derivatives have good biocompatibility. Description of the Drawings

[0013] Figure 1 is the optimization result of the ZIF - 8 synthesis process. Among them, A is the optimization result of the molar ratio of 2 - methylimidazole and Zn(NO3)2·6H2O, B is the optimization result of the reaction time, C is the optimization result of the methanol dosage, and D is the stability investigation result; in the figure, different letters (a, b, c, d, e) indicate significant differences between different treatment groups ( P <0.05), and multiple letters (bc, cd, bcd, be) indicate that there is no significant difference between this group and other groups with the same letters, but there is a significant difference from the groups without these letters ( P <0.05).

[0014] Figure 2 is the FI - IR spectrogram of ZIF - 8; Figure 3 is the particle size distribution diagram of ZIF - 8; Figure 4 is the Zeta potential diagram of ZIF - 8; Figure 5 is the inhibition rate of different types of MOFs on TYR; Figure 6 is the IC 50 value of the inhibitory activity of ZIF-8 on TYR; Figure 7 is the IC 50 value of the inhibitory activity of nicotinamide on TYR; Figure 8 is the inhibition rate of ZIF-8 and its derivatives on TYR; In the figure, different letters (a, b, c, d, e, f) indicate significant differences between different ZIF-8 and its derivatives ( P <0.05).

[0015] Figure 9 are the FI-IR spectrograms of ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni; Figure 10 are the particle size distribution diagrams of ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni; Figure 11 are the Zeta potential diagrams of ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni; Figure 12 are the IC 50 values of the inhibitory activity of ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni on TYR; Figure 13 is the effect of kojic acid on the activity of mouse B16F10 melanoma cells determined by the CCK-8 method; Figure 14 is the effect of resveratrol on the viability of mouse B16F10 melanoma cells determined by the CCK-8 method; Figure 15 is the effect of ZIF-8 and its derivatives on the viability of mouse B16F10 melanoma cells determined by the CCK-8 method; Figure 16 is the effect of ZIF-8 on the relative activity of TYR in mouse B16F10 melanoma cells; Figure 17 is the effect of ZIF-8 on the relative content of melanin in mouse B16F10 melanoma cells. Specific implementation methods

[0016] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0017] Example 1: Preparation and Characterization of ZIF-8 (1)ZIF-8 synthesis process ZIF-8 was prepared in methanol by the room temperature stirring method. First, 0.2975 g (1 mmol) of Zn(NO3)2·6H2O was accurately weighed and ultrasonically dissolved in 3 mL of methanol to form a clear solution A; 0.9854 g (12 mmol) of 2-methylimidazole was accurately weighed and ultrasonically dissolved in 12 mL of methanol to form a clear solution B. Then, at room temperature and under stirring at 1000 rpm, solution A was added dropwise to solution B, sealed, and stirred at 1000 rpm for 180 min. ZIF-8 nanoparticles were spontaneously formed to obtain a white suspension. Finally, centrifugation was carried out at 12000 r / min for 5 min to obtain a white precipitate, which was washed 3 times with 10 mL of methanol each time and vacuum dried at 60 °C overnight to obtain ZIF-8 nanoparticles for use.

[0018] (2)Optimization of the molar ratio of 2-methylimidazole and Zn(NO3)2·6H2O 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, and other conditions were carried out according to (1). Taking the inhibition rate of ZIF-8 obtained under different conditions on tyrosinase as an index, the optimal molar ratio was obtained.

[0019] (3)Optimization of reaction time On the basis of obtaining 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 according to (1). Taking the inhibition rate of ZIF-8 obtained under different conditions on tyrosinase as an index, the optimal reaction time was obtained.

[0020] (4)Optimization of methanol dosage On the basis of obtaining the optimal molar ratio of 12:1 and the optimal reaction time of 30 min, the methanol dosages were adjusted to 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, and 40 mL respectively, and other conditions were carried out according to (1). Taking the inhibition rate of ZIF-8 on tyrosinase obtained under different conditions as an index, the optimal methanol dosage was obtained.

[0021] (5)ZIF-8 Stability Experiment On the basis of obtaining 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 according to other conditions in (1). The inhibition rates of six batches of ZIF-8 on tyrosinase at the same mass concentration (0.714 mg / mL) were measured to evaluate the stability of ZIF-8.

[0022] (6)Characterization of ZIF-8 The functional groups and chemical bonds of ZIF-8 were measured using a Fourier transform infrared spectrometer (Thermo Fisher, USA). The particle size distribution and zeta potential of ZIF-8 nanoparticles were measured using a zetasizer (Malvern, UK).

[0023] Taking the inhibition rate of ZIF-8 on TYR as an index, its synthesis process was optimized, and the results were as Figure 1 shown. The optimal synthesis process of ZIF-8 was that the molar ratio of 2-methylimidazole to Zn(NO3)2·6H2O was 12:1, the reaction time was 30 min, and the methanol dosage was 15 mL. Under the optimal process, when the mass concentration of ZIF-8 was 0.714 mg / mL, the inhibition rate on tyrosinase was 62.45 ± 1.21%. At the same time, six batches of ZIF-8 were synthesized under the optimal conditions, and the inhibition rates on tyrosinase at a mass concentration of 0.714 mg / mL were measured. The results showed that the ZIF-8 process was stable, and the inhibition rates on tyrosinase were basically the same at the same mass concentration.

[0024] The functional groups and chemical bonds of ZIF-8 were measured using a Fourier transform infrared spectrometer. ZIF-8 showed absorption peaks at 1580 cm -1 and 1140 cm -1 , corresponding to the symmetric stretching vibration of the C=N bond in the imidazole ring and the stretching vibration of the C-N bond in the imidazole ring respectively; an absorption peak appeared at 420 cm -1 , corresponding to the coordination bond vibration between Zn 2+ and the imidazole nitrogen atom, indicating that the imidazole ligand was successfully coordinated with Zn 2+ to form a framework structure; while the C-H stretching vibration appeared at around 3135 cm -1 , and the out-of-plane bending vibration of the imidazole ring was at 755 cm -1and 690 cm -1 near Figure 2 ). The pore size distribution and zeta potential of ZIF-8 nanoparticles were measured using a nano particle size analyzer Zetasizer. The results showed that the average particle size of ZIF-8 was 176.3 nm ( Figure 3 ), and the zeta potential was -40.94 mV ( Figure 4 ), indicating that ZIF-8 was relatively uniformly distributed and had good dispersibility.

[0025] Example 2: Evaluation of the inhibitory activities of different MOF materials and common skin whiteners against TYR Common MOF materials on the market were selected, and the inhibition rates against TYR were measured at a mass concentration of 0.714 mg / mL respectively to compare the inhibitory activities of different MOF materials against TYR. The results showed that at the same mass concentration, the inhibition rate of ZIF-8 against TYR activity was the strongest, significantly stronger than common MOF materials such as UiO-66-NH2, Fe3O4-COOH@UiO-66-NH2, N101, M100, and M101 (see Figure 5 ).

[0026] Common niacinamide (purity 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.) skin whiteners on the market were selected, and the inhibition rates of ZIF-8 and niacinamide against TYR were measured at different molar concentrations respectively to compare the inhibitory activities of ZIF-8 and niacinamide against TYR. Through curve fitting, the half inhibitory concentration (IC 50 value) of ZIF-8 was 1.67 mmol / L (see Figure 6 ), and the IC 50 value of niacinamide was 60.42 mmol / L (see Figure 7 ). It can be seen from this that the inhibitory activity of ZIF-8 against TYR was significantly stronger than that of niacinamide.

[0027] Example 3: Preparation and characterization of ZIF-8 derivatives (1) Preparation of ZIF-8 derivatives First, accurately weigh 1 mmol of Zn(NO3)2·6H2O, 2 mmol of Na2SeO3·5H2O, CaCl2, AlCl3·6H2O, H3BO3, Ni(NO3)2·6H2O, and FeSO4·7H2O respectively. Among them, CaCl2, AlCl3·6H2O, H3BO3, and Ni(NO3)2·6H2O are ultrasonically dissolved in 3 mL of methanol, and Na2SeO3·5H2O and FeSO4·7H2O are dissolved in 3 mL of water to form clear solution A respectively; accurately weigh 10 mmol of 2-methylimidazole and ultrasonically dissolve it in 12 mL of methanol to form clear solution B. Then, at room temperature and under stirring at 1000 rpm, solution A is gradually added dropwise to solution B, sealed, and stirred at 1000 rpm for 210 min. ZIF-8 derivative nanoparticles are spontaneously formed to obtain a suspension. Finally, centrifuge at 12000 r / min for 5 min to obtain a precipitate, wash it 3 times with 10 mL of methanol respectively, and vacuum dry it overnight at 60 °C. The ZIF-8 derivative nanoparticles are ready for use.

[0028] (2)Characterization of ZIF-8 derivatives The functional groups and chemical bonds of ZIF-8 derivatives are determined using a Fourier transform infrared spectrometer (Thermo Fisher, USA). The particle size distribution and zeta potential of ZIF-8 derivative nanoparticles are determined using a zetasizer (Malvern, UK).

[0029] ZIF-8 derivatives are prepared by doping with different macro or trace elements contained in the human body, and the TYR inhibition rate at a molar concentration of 2.86 mmol / L is measured. The results are as Figure 8 shown. Compared with ZIF-8, after doping with Ca 2+ and Ni 2+ , at the same molar concentration, the inhibition rate of TYR is significantly increased by more than 6%.

[0030] The functional groups and chemical bonds of ZIF-8 / Ca and ZIF-8 / Ni are determined using a Fourier transform infrared spectrometer. As Figure 9 shown, when Ca 2+ partially replaces Zn 2+ or coordinates with the imidazole ligand, it may reduce the electron density of the imidazole ring, resulting in a decrease in the intensity of the C=N peak at 1580 cm -1 ; the interaction between Ca 2+ and the imidazole ring may change the electron environment of the methyl group, resulting in a decrease in the intensity of the C-H peak at 3135 cm -1 ; at the same time, the doping of Ca 2+ may cause local distortion or defects in the crystal structure of ZIF-8, and 755 cm -1 and 690 cm-1 The out-of-plane bending vibration may split due to the reduction of crystal symmetry. When Ni 2+ coordinates with the nitrogen atoms of imidazole ligands, it may reduce the electron density of the imidazole ring, weaken the C=N bond strength, resulting in a decrease in the vibration frequency and the C=N peak shifting towards lower wavenumbers; meanwhile, Ni 2+ 's strong coordination ability may change the local symmetry of the C-N bond, causing changes in the peak shape, resulting in the broadening or splitting of the C-N peak at 1140 cm -1 .

[0031] The pore size distribution and zeta potential of ZIF-8 and its derivative nanoparticles were measured using a nanoparticle size analyzer Zetasizer. The results showed that the average particle size of ZIF-8 was 176.3 nm. After doping with Ca 2+ and Ni 2+ , the average particle sizes of their bimetallic derivatives increased, reaching 380.4 nm and 372.3 nm respectively ( Figure 10 ); the Zeta potential of ZIF-8 was -40.94 mV. After doping with Ca 2+ and Ni 2+ , the average Zeta potentials of their bimetallic derivatives increased, reaching -36.33 mV and -36.87 mV respectively ( Figure 11 ), indicating that ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni were relatively evenly distributed and had good dispersibility.

[0032] Example 4: Inhibitory effect of ZIF-8 and its derivatives on TYR in vitro 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 a 2 mL centrifuge tube, mixed well, and incubated at 37°C for 30 min. After incubation, 50 μL of L-DOPA solution (2 mM / L) was added as a substrate, mixed well, and incubated at 37°C for 20 min. After incubation, the centrifuge tube was centrifuged at 13000 r / min for 5 min in a centrifuge. 200 μL of the supernatant was taken and placed in a 96-well plate, and the absorbance at 475 nm was measured. The inhibition rate (%) relative to the control was calculated according to the following formula.

[0033] ; In the formula, T 样品 represents the absorbance of the test sample, T0 represents the absorbance of the sample background, T 对照 represents the absorbance of the control sample, and T 溶剂 represents the absorbance of the PBS buffer salt solvent.

[0034] Table 1 Reaction system for measuring the inhibitory activity of TYR inhibitors against TYR

[0035] The half-maximal inhibitory concentration (IC 50 value) of ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni against the inhibitory activity of TYR was measured, and the results are as Figure 12 shown, which were 1.67 mmol / L, 0.99 mmol / L, and 0.65 mmol / L, respectively, indicating that ZIF-8 could inhibit the activity of TYR well, and its derivatives could further enhance the inhibitory activity against TYR. The IC 50 values of ZIF-8 / Ca and ZIF-8 / Ni were reduced by 40.72% and 61.08%, respectively, compared with the IC 50 value of ZIF-8.

[0036] Example 5: Effects of ZIF-8, ZIF-8 derivatives, and common whitening agents on mouse B16F10 melanoma cells (1) Culture of mouse B16F10 melanoma cells B16F10 cells, derived from mouse skin melanoma, were cultured in Dulbecco's modified Eagle's 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, subculture was performed when the culture flask reached 80% capacity, and subculture was usually required every 2 - 3 days.

[0037] (2) Determination of the effects of ZIF-8, ZIF-8 derivatives, and common whitening agents on the activity of mouse B16F10 melanoma cells by CCK8 method When the cells grew to the logarithmic phase, they were digested to make a cell suspension. 100 μL per well, with a density of 8×10 3 cells / well, and were inoculated into a 96-well plate. After culturing for 24 h, when the cells adhered to the wall, the culture medium was discarded, and the cells were washed 2 times with PBS.

[0038] The experiment was divided into a blank group: containing only the culture medium; a control group: culturing cells normally with the culture medium; and an experimental group: culturing cells with the culture medium containing different concentrations of the sample. Eight concentrations were set for the tested samples. The administration concentrations of kojic acid, ZIF-8, ZIF-8 / Ca, and ZIF-8 / Ni were set at 15, 30, 45, 60, 75, 90, 105, and 120 μg / mL respectively, and the administration concentration of resveratrol was set at 0.2, 0.3, 0.6, 1.3, 2.5, 5.0, 10.0, and 20.0 μg / mL. Six replicate wells were set for each concentration. After culturing for 24 h, the culture medium was discarded, and then 100 μL of the culture medium containing 10% CCK-8 solution was added to each well in the dark. The mixture was incubated at 37 °C in the dark for 1 h, and the absorbance (OD value) of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The cell survival rate (%) was calculated according to the formula.

[0039] ; In the formula, OD 实验组 represents the absorbance of the experimental group, OD 空白组 represents the absorbance of the blank group, and OD 对照组 represents the absorbance of the control group.

[0040] (3) Determination of the effect of ZIF-8 on tyrosinase activity in B16F10 mouse melanoma cells by dopa oxidation method The cell culture was the same as above. When the cells grew to the logarithmic phase, they were digested to prepare a cell suspension, and inoculated into a 24-well plate at a density of 5×10 4 cells / well with a volume of 1 mL per well. After culturing for 24 h, the culture medium was discarded, and the cells were washed twice with PBS.

[0041] The experiment was divided into a blank group: without cells, adding culture medium containing samples at different concentrations; a control group: adding culture medium without samples to culture cells normally; a drug administration group: adding culture medium containing samples at different concentrations (kojic acid: 240 μg / mL, resveratrol: 10 μg / mL positive control sample, ZIF-8: 60, 120 μg / mL samples) to culture cells. After culturing for 24 h, the culture medium was discarded. After washing with PBS, 0.3 mL of trypsin cell digestive solution was added to each well for digestion, and then the cells were collected into a centrifuge tube, centrifuged at 1500 r / min for 5 min. After centrifugation, the supernatant was discarded to obtain cell pellets. After washing with PBS and centrifuging at 6400 r / min for 5 min, the supernatant was discarded. 200 μL of PBS lysis solution containing 1% Triton X-100 was added to each tube to resuspend the cells, placed on ice, and lysed thoroughly for 60 min, mixing once every 20 min. Then, it was centrifuged at 4 °C and 12000 r / min for 15 min, and the supernatant was collected for measuring TYR activity. In a 96-well plate, 150 μL of the above supernatant and 100 μL of L-DOPA solution with a concentration of 2 mmol / L were added respectively, incubated at 37 °C for 30 min, and the absorbance at 475 nm was measured. The TYR activity (%) relative to the control was calculated according to the following formula: ; In the formula, OD 给药组 represents the absorbance of the drug administration group, OD 空白组 represents the absorbance of the blank group, OD 对照组 represents the absorbance of the control group.

[0042] (4) Determination of the effect of ZIF-8 on melanin synthesis in B16F10 mouse melanoma cells by sodium hydroxide lysis method The cell culture and treatment steps were the same as (3). After discarding the culture medium, the cells were washed twice with PBS, then digested with trypsin to collect the cells, and centrifuged at 1500 r / min for 5 min. The supernatant was discarded, and the cell pellet was resuspended with 500 μL of PBS and centrifuged again. After removing the supernatant, 100 μL of 1 mol / L sodium hydroxide solution containing 10% DMSO was added to the cell pellet, incubated in an 80 °C metal bath for 1 h to fully dissolve the melanin granules, and the amount of synthesized melanin was determined by measuring the absorbance at 405 nm using an enzyme-labeled instrument. The relative intracellular melanin content (%) relative to the control was calculated according to the formula: ; In the formula, OD 给药组 represents the absorbance of the drug administration group, OD 空白组 represents the absorbance of the blank group, OD 对照组 represents the absorbance of the control group.

[0043] The CCK-8 method was used to determine the effects of kojic acid, resveratrol, ZIF-8 and its derivatives (ZIF-8 / Ca and ZIF-8 / Ni) on the viability of mouse B16F10 melanoma cells. The results are as Figures 13 - 15 shown. Kojic acid, ZIF-8, ZIF-8 / Ca and ZIF-8 / Ni, the positive controls, basically did not affect the viability of mouse B16F10 melanoma cells when the concentration was lower than 120 μg / mL, while resveratrol, the positive control, basically did not affect the viability of mouse B16F10 melanoma cells when the concentration was lower than 20 μg / mL. The above results indicate that ZIF-8 and its derivatives ZIF-8 / Ca and ZIF-8 / Ni have low cytotoxicity at a certain concentration and their biocompatibility is better than that of the positive control resveratrol.

[0044] The dopa oxidation method was used to determine the effect of ZIF-8 on the activity of TYR in B16F10 mouse melanoma cells. The results are as Figure 16 shown. Compared with the control group, the positive controls kojic acid, resveratrol and ZIF-8 could significantly reduce the relative activity of TYR ( P < 0.05), and the higher the administration concentration of ZIF-8, the stronger the effect of inhibiting the activity of TYR. The sodium hydroxide lysis method was used to determine the effect of ZIF-8 on melanin synthesis in B16F10 mouse melanoma cells. The results are as Figure 17 shown. Compared with the control group, the positive controls kojic acid and ZIF-8 could significantly reduce the relative production amount of intracellular melanin ( P < 0.05), and the higher the administration concentration of ZIF-8, the stronger the effect of inhibiting the relative production amount of intracellular melanin. The above results indicate that ZIF-8 can be used as a TYR inhibitor, can effectively reduce the activity of TYR in cells, inhibit the production of intracellular melanin, and is a potential raw material for skin whitening products.

[0045] The above-described embodiments only represent the preferred embodiments of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, according to the technical solutions and concepts described above, various other corresponding changes and deformations can also be made, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. Application of ZIF-8 and its derivatives in preparing a product for inhibiting tyrosinase activity, characterized in that, The active substance in the product is ZIF-8 and / or derivatives of ZIF-8, and the active substance can inhibit the activity of tyrosinase.

2. The application according to claim 1, wherein The product also includes pharmaceutically acceptable excipients.

3. The application according to claim 1 or 2, characterized in that The preparation method of the ZIF-8 includes the following steps: S1: Dissolve a zinc source and 2-methylimidazole in solvents respectively to form solution A and solution B; S2: Dropwise add solution A into solution B while stirring, and carry out a closed reaction to obtain a reaction product; S3: Centrifuge, wash and dry the reaction product to obtain ZIF-8 nanoparticles.

4. The application according to claim 3, wherein In step S1, the molar ratio of the 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 closed reaction is carried out for 30 - 210 min; In step S3, methanol is used for washing, and the amount of methanol used is 10 - 40 mL.

5. The application according to claim 1 or 2, characterized in that, The preparation method of the derivatives of ZIF-8 includes the following steps: a: Mix a zinc source and a metal ion in a certain molar ratio and dissolve them in methanol to form solution C; b: Dissolve 2-methylimidazole in methanol to form solution D; c: Dropwise add solution C into solution D and react for a period of time to obtain a reaction product; d: Centrifuge, wash and vacuum dry the reaction product to obtain derivatives of ZIF-8.

6. The application according to claim 5, wherein In step a, the molar ratio of the zinc source to the metal ion is 1:0.2 - 1:5; the metal ion is Ca 2+ or Ni 2+ or one or more of them; wherein the Ca 2+ source is at least one of calcium chloride and calcium carbonate; the Ni 2+ source is at least one of nickel nitrate hexahydrate, nickel nitrate, nickel chloride, nickel chloride hexahydrate, nickel bromide and nickel sulfate; In step c, the total amount of methanol used is 10 - 40 mL; the reaction time is 30 - 300 min.

7. The application according to claim 1, wherein The product includes a tyrosinase inhibitor or a whitening product.

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