CN115505068B

CN115505068B - Preparation method and application of tyrosinase inhibitor for inhibiting melanin generation

Info

Publication number: CN115505068B
Application number: CN202110692752.9A
Authority: CN
Inventors: 吕永琴; 张童
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2023-10-20
Anticipated expiration: 2041-06-22
Also published as: CN115505068A

Abstract

The invention discloses a preparation method and application of a tyrosinase inhibitor for inhibiting melanin generation. The invention carries out high-throughput screening on a tyrosinase inhibitor synthesis formula in a 96-well plate, prepares a reaction solution by adjusting the composition and the proportion of N-isopropyl acrylamide, a functional monomer, a cross-linking agent and a surfactant, fills nitrogen, and then adds an initiator to carry out precipitation polymerization or inverse emulsion polymerization to screen out the tyrosinase inhibitor formula. The selected hydrogel polymer is capable of inhibiting tyrosinase activity by binding to an enzyme active site or to a key glycosylation site on the enzyme surface. The semi-inhibition concentration IC50 of the tyrosinase inhibitor prepared by the invention to tyrosinase is 1.92 mu M, which is superior to most natural tyrosinase inhibitors. The tyrosinase inhibitor has good inhibition effect on tyrosinase in a solution system or in cells and animal bodies, and reduces melanin generation.

Description

Preparation method and application of tyrosinase inhibitor for inhibiting melanin generation

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of a tyrosinase inhibitor for inhibiting melanin generation.

Background

Melanin is the primary pigment responsible primarily for pigmentation of human skin, hair and eyes, and is produced by melanocytes through melanogenesis. The melanin synthesis and accumulation of these pigments increases, occurring in many types of skin diseases, including acanthosis nigricans, cervical cancer, chloasma, neurodegeneration associated with parkinson's disease and the risk of skin cancer. Although melanin production is a complex process, represented by many enzymes and chemical reactions, enzymes such as tyrosinase and other tyrosinase-related proteins (TYRP 1 and TYRP 2) play a vital role in melanin synthesis. Tyrosinase is a multifunctional copper-containing metalloenzyme with binuclear copper ions, and plays a role of a rate-limiting enzyme in melanin synthesis. Tyrosinase is the most important and successful target for melanin production inhibitors that directly inhibit the catalytic activity of tyrosinase, since it is a key enzyme for melanin synthesis by melanin production. Most of the cosmetics or skin lightening agents on the market are tyrosinase inhibitors. The tyrosinase inhibitors are gradually and deeply studied at the present stage, but a plurality of tyrosinase inhibitors have defects such as unstable physicochemical properties, obvious side effects, poor solubility and the like, and the effect of inhibiting melanin generation is not satisfactory.

Disclosure of Invention

The invention provides a preparation method and application of a tyrosinase inhibitor for inhibiting melanin generation.

The preparation method of the tyrosinase inhibitor for inhibiting melanin generation comprises the following steps: the synthesis formula of the tyrosinase inhibitor is subjected to high-throughput screening in a 96-well plate, a reaction solution is prepared by adjusting the composition and the proportion of N-isopropyl acrylamide, functional monomers, a cross-linking agent and a surfactant, and an initiator is added after nitrogen is filled for precipitation polymerization or inverse emulsion polymerization, so that the formula of the tyrosinase inhibitor is screened.

The precipitation polymerization or inverse emulsion polymerization is carried out at 40-80 ℃ for 3-24 hours.

After the reaction of precipitation polymerization or inverse emulsion polymerization is finished, the hydrogel nano particles are obtained through dialysis purification and freeze drying.

The functional monomer in the formula of the tyrosinase inhibitor is selected from the following components: 4-vinylphenylboronic acid, acrylamide, acrylic acid, N-tert-butyl acrylamide, N-phenyl acrylamide, 4-vinylimidazole, 4-vinylphenol and (3-acrylamidopropyl) trimethylammonium chloride.

The cross-linking agent in the formula of the tyrosinase inhibitor is selected from the following components: one or two of N, N' -methylenebisacrylamide, 1, 4-bis (acryloyl) piperazine and ethylene glycol dimethacrylate.

The initiator is ammonium persulfate, and the addition amount is 5-80mg/mL.

The mol ratio of the N-isopropyl acrylamide, the functional monomer and the cross-linking agent in the formula of the tyrosinase inhibitor is 5-98:5-40:0.5-15.

The invention has the following beneficial effects:

the invention synthesizes the hydrogel polymer in a 96-well plate, then carries out high-throughput screening on the inhibition effect of tyrosinase, and regulates the inhibition effect of the hydrogel polymer on tyrosinase by changing the types and the proportion of functional monomers. The selected hydrogel polymer is capable of inhibiting tyrosinase activity by binding to an enzyme active site or to a key glycosylation site on the enzyme surface. The functional monomer used in the preparation and synthesis process is low in price, the screening and preparation process is simple, the prepared hydrogel nanoparticle is uniform in particle size, controllable in size and shape and good in physical and chemical stability. The screened tyrosinase inhibitor has semi-inhibition concentration IC50 of 1.92 mu M to tyrosinase, is superior to most natural tyrosinase inhibitors, and has good inhibition effect to tyrosinase. The tyrosinase inhibitor has good inhibition effect on tyrosinase in a solution system or in cells and animal bodies, and reduces melanin generation. The tyrosinase inhibitor is hydrogel nano particles, has very low biotoxicity, and has important application value and wide application prospect in the field of medical whitening.

Drawings

FIG. 1 inhibition of tyrosinase by polymers in 96-well plates in example 1.

FIG. 2 is a scanning electron microscope characterization of tyrosinase inhibitors prepared in examples 2-5.

FIG. 3 shows the tyrosinase inhibitory effect of the tyrosinase inhibitors prepared in examples 2-5 on tyrosinase in solution.

FIG. 4 is an IC50 of tyrosinase inhibitor NP4 versus tyrosinase.

FIG. 5 is a graph showing inhibition of tyrosinase inhibitor NP4 by linewiver-Burk.

FIG. 6 is a cytotoxicity assay of the tyrosinase inhibitor NP4 on B16F10 cells.

FIG. 7 shows that tyrosinase inhibitors NP1-4 inhibited tyrosinase activity in B16F10 cells.

FIG. 8 shows that tyrosinase inhibitors NP1-4 inhibited melanogenesis in B16F10 cells.

FIG. 9 is a graph showing that tyrosinase inhibitor NP4 inhibited melanogenesis in mouse skin.

FIG. 10 shows the measurement of melanin content in the skin of mice by the Masson-Fontana Ammonia silver method.

FIG. 11 is a section of visceral tissue from mice stained with hematoxylin-eosin.

Detailed Description

In order to enable those skilled in the art to better understand the technical scheme of the invention, the preparation method and the application of the tyrosinase inhibitor for inhibiting melanin generation provided by the invention are described in detail below with reference to specific examples.

Example 1: synthetic formula for high-throughput screening tyrosinase inhibitor in 96-well plate

mu.L of a pre-reaction solution was prepared at a total concentration of each component of 80 mM. Wherein the mole percentage of N, N' -methylene bisacrylamide is 10%, the mole percentage of acrylamide is 5%, 10%, 20% and 40%, and the rest is N-isopropyl acrylamide. After the preparation of the pre-reaction solution, N is used ₂ Air was blown for 15 minutes. Adding the pre-reaction liquid with different functional monomer contents into 96In the well plate, 3mg of ammonium persulfate was added as an initiator per well, and the reaction was sealed at 65℃for 8 hours. After the completion of the reaction, unreacted monomers were removed by washing with pure water to obtain polymers deposited in 96-well plates. mu.L of mushroom tyrosinase (300U/mL) was added to each well, after 10 minutes of pre-incubation at room temperature, 100. Mu. L L-DOPA (3, 4-dihydroxyphenylalanine) (2.5 mM) was added and the assay plate was further incubated at 25℃for 20 minutes. After the incubation was completed, absorbance at 475nm was measured and percent inhibition relative to the blank was calculated. The tyrosinase activity inhibition effect of the polymer in the 96-well plate is shown in fig. 1.

Example 2: preparation of tyrosinase inhibitor 1 (NP 1)

Preparing 50mL of reaction solution with the total molar concentration of each component of 100mM, wherein the molar percentage of N-isopropyl acrylamide is 85mol%, the molar percentage of 4-vinylphenylboronic acid is 5mol%, the molar percentage of 4-vinylimidazole is 5mol%, and the molar percentage of cross-linking agent N, N' -methylenebisacrylamide is 5mol%; 10mg of sodium dodecyl sulfate was further added, 30mg of ammonium persulfate as an initiator was added under nitrogen atmosphere, and the polymerization was carried out at 65℃for 4 hours using a magnetic stirrer. The polymerized solution was purified by dialysis against an excess of pure water, and the polymer nanoparticles were obtained after lyophilization. The scanning electron microscope characterization is shown in fig. 2 a.

Example 3: preparation of tyrosinase inhibitor 2 (NP 2)

Preparing 50mL of reaction solution with the total molar concentration of each component being 80mM, wherein the molar percentage of N-isopropyl acrylamide is 73mol%, the molar percentage of 4-vinylphenylboronic acid is 10mol%, the molar percentage of N-tertiary butyl acrylamide is 15mol%, and the molar percentage of cross-linking agent N, N' -methylene bisacrylamide is 2mol%; 10mg of sodium dodecyl sulfate was further added, 30mg of ammonium persulfate as an initiator was added under nitrogen atmosphere, and the polymerization was carried out at 80℃for 3 hours using a magnetic stirrer. The polymerized solution was purified by dialysis against an excess of pure water, and the polymer nanoparticles were obtained after lyophilization. The scanning electron microscope characterization is shown in fig. 2 b.

Example 4: preparation of tyrosinase inhibitor 3 (NP 3)

Preparing 50mL of reaction solution with the total molar concentration of each component of 20mM, wherein the molar percentage of N-isopropyl acrylamide is 55mol%, the molar percentage of 4-vinylphenylboronic acid is 30mol%, the molar percentage of acrylic acid is 5mol%, and the molar percentage of crosslinking agent N, N' -methylenebisacrylamide is 10mol%; 10mg of sodium dodecyl sulfate was further added, 10mg of ammonium persulfate as an initiator was added under nitrogen atmosphere, and the polymerization was carried out at 65℃for 6 hours using a magnetic stirrer. The polymerized solution was purified by dialysis against an excess of pure water, and the polymer nanoparticles were obtained after lyophilization. The scanning electron microscope characterization is shown in fig. 2 c.

Example 5: preparation of tyrosinase inhibitor 4 (NP 4)

Preparing 50mL of reaction solution with the total molar concentration of each component of 20mM, wherein the molar percentage of N-isopropylacrylamide is 40mol%, the molar percentage of 4-vinylphenylboronic acid is 40mol%, the molar percentage of 4-vinylphenol is 5mol%, and the molar percentage of cross-linking agent N, N' -methylenebisacrylamide is 15mol%; 10mg of sodium dodecyl sulfate was further added, 30mg of ammonium persulfate as an initiator was added under nitrogen atmosphere, and the polymerization was carried out at 40℃for 24 hours using a magnetic stirrer. The polymerized solution was purified by dialysis against an excess of pure water, and the polymer nanoparticles were obtained after lyophilization. The scanning electron microscope characterization is shown in fig. 2 d.

Application example 1: determination of the inhibition effect of tyrosinase inhibitor on tyrosinase activity

mu.L of mushroom tyrosinase (300U/mL) was mixed with 50. Mu.L of a solution of tyrosinase inhibitor NP1-4 at a concentration of 1mg/mL, respectively. After pre-incubation for 10min at room temperature, 100 μ L L-DOPA (3, 4-dihydroxyphenylalanine) (2.5 mM) was added and the assay plates were further incubated for 20 min at 25 ℃. After the incubation was completed, absorbance was measured at 475nm and percent inhibition was calculated relative to the control. The tyrosinase inhibitor NP1-4 showed inhibitory effect on tyrosinase activity as shown in FIG. 3.

Application example 2: analysis of mechanism of inhibition of tyrosinase activity by tyrosinase inhibitors

And selecting NP4 with optimal tyrosinase inhibition effect to analyze inhibition mechanism. mu.L of mushroom tyrosinase (300U/mL) and 50. Mu.L of NP4 solutions of different concentrations were mixed. After pre-incubation for 10min at room temperature, 100 μ L L-DOPA (3, 4-dihydroxyphenylalanine) (2.5 mM) was added and the assay plates were further incubated for 20 min at 25 ℃. After the incubation was completed, absorbance at 475nm was measured and percent inhibition relative to control was calculated. The half-maximal inhibitory amount IC50 was calculated as shown in fig. 4.

50. Mu.L of mushroom tyrosinase at different concentrations and 50. Mu.L of NP4 solution at different concentrations were mixed. After pre-incubation for 10 minutes at room temperature, 100. Mu. L L-DOPA (3, 4-dihydroxyphenylalanine) (2.5 mM) was added, absorbance was measured at 475nm every 1 minute for 20 minutes. Percent inhibition relative to control was calculated. Calculation of K by means of a double reciprocal curve _m ，V _m As shown in FIG. 5 and Table 1, K was increased when NP4 concentration was increased _m Rise to V _m The decrease indicates that the mechanism of inhibition of tyrosinase by NP4 is mixed inhibition.

TABLE 1

	K _m (mM)	V _m (μM/s)
			TYR	0.16	0.37
NP4 0.05mg/mL	0.34	0.19
			NP4 0.125mg/mL	0.49	0.12
NP4 0.25mg/mL	0.67	0.10

Application example 3: tyrosinase inhibitors inhibit intracellular tyrosinase activity

After incubation of different concentrations of the autoclaved NP4 solution with B16F10 mouse melanoma cells for 24 hours, the supernatant was aspirated, and 20. Mu.L MTT solution (5 mg/mL, i.e., 0.5% MTT) was added to each well and incubation was continued for 4 hours. After 4 hours, the culture was terminated and the culture solution in the wells was carefully aspirated. 100 mu L of dimethyl sulfoxide was added to each well, and the mixture was placed on a shaking table and shaken at a low speed for 10 minutes to sufficiently dissolve the crystals. Absorbance was measured for each well at OD 490nm in an enzyme-linked immunosorbent assay. Cytotoxicity of NP4 against B16F10 cells was calculated and the experimental results are shown in FIG. 6.

Tyrosinase inhibitors NP1-4 were incubated with B16F10 cells for 24 hours, respectively, and the tyrosinase activity and melanin content in the B16F10 cells were determined. To measure intracellular tyrosinase activity, B16F10 cells were seeded in 96-well plates (1X 10) ⁴ Cells/well), for 2 hours. The medium was replaced with DMEM in solution (100. Mu.g/mL) of the tyrosinase inhibitor NP1-4, respectively, and the cells were incubated for 24 hours. The medium was then removed and the cells were washed with PBS. Then 100. Mu.L of PBS containing 1% Triton X-100 was added to each well and the wells were shaken on a shaker for 10min at low speed. Next, 100. Mu.L of 2.5mmol/L L-DOPA was added and the cells were incubated at 37℃for 60 minutes. Tyrosinase activity was assessed by measuring absorbance at 475nm with an ELISA reader. The test results are shown in fig. 7.

NP1-4 was incubated with B16F10 cells for 24 hours and tyrosinase activity and melanin content in B16F10 cells was determined. To measure intracellular tyrosinase activity, B16F10 cells were seeded in 96-well plates (1X 10) ⁴ Cells/well), for 2 hours. The medium was replaced with DMEM in solution (100. Mu.g/mL) of the tyrosinase inhibitor NP1-4, and the cells were incubated for 24 hours. The medium was then removed and the cells were washed with PBS. mu.L of 1M NaOH containing 1% Triton X-100 was then added to each well and the cell pellet was lysed at 80℃for 1 hour, and the amount of melanin was determined by measuring absorbance at 405nm using ELISA reader. The test results are shown in fig. 8.

Application example 4: tyrosinase inhibitor for inhibiting melanogenesis in mice

Female C57 mice of eight weeks of age were selected, subcutaneously injected on the backs of the mice with NP4 (0.2 mg), and irradiated on alternate days with an ultraviolet lamp (UV intensity 200mW/cm each time) ² ) The photo record mice back skin tone change (fig. 9). After 8 days, mice were sacrificed, the skin and viscera of the mice were drawn, the melanin content in the skin of the mice was detected by the Masson-Fontana ammonia silver method (fig. 10), and the viscera of the mice were tested by hematoxylin-eosin staining method (fig. 11), and no obvious viscera damage was found, indicating that NP4 had lower biotoxicity.

Claims

1. A method for preparing a tyrosinase inhibitor for inhibiting melanin formation, which is characterized by comprising the following steps: high-throughput screening is carried out on a tyrosinase inhibitor synthesis formula in a 96-well plate, a reaction solution is prepared by adjusting the composition and the proportion of N-isopropyl acrylamide, functional monomers, a cross-linking agent and a surfactant, and an initiator is added after nitrogen is filled for precipitation polymerization or inverse emulsion polymerization, so that the tyrosinase inhibitor formula is screened;

the functional monomer is selected from the following components: one or more of 4-vinylphenylboronic acid, acrylamide, acrylic acid, N-tert-butyl acrylamide, N-phenyl acrylamide, 4-vinylimidazole, 4-vinylphenol and (3-acrylamidopropyl) trimethylammonium chloride;

the cross-linking agent is selected from the group consisting of: one or two of N, N' -methylenebisacrylamide, 1, 4-bis (acryloyl) piperazine and ethylene glycol dimethacrylate;

the molar ratio of the N-isopropyl acrylamide to the functional monomer to the cross-linking agent is 5-98:5-40:0.5-15.

2. The process according to claim 1, wherein the precipitation polymerization or inverse emulsion polymerization is carried out at 40 to 80℃for 3 to 24 hours.

3. The preparation method of claim 1, wherein the hydrogel nanoparticles are obtained by dialysis purification and freeze-drying after the reaction of precipitation polymerization or inverse emulsion polymerization.

4. The preparation method according to claim 1, wherein the initiator is ammonium persulfate and the addition amount is 5-80mg/mL.