Application of rifudine in preparation of preparation for promoting cell melanin synthesis

Abstract

The invention discloses application of rifudine in preparation of a preparation for promoting cell melanin synthesis, and belongs to the technical field of special drugs. The invention provides a new application of rifudine, which can promote synthesis and secretion of melanin and expression of TYR, TRP-1 and TRP-2, provides a new functional component for enhancing synthesis of melanin in vivo and preparing products for treating vitiligo and the like, and has a wide application prospect in the fields of pharmaceutical preparations, cosmetics, care products, beauty products and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of special medicines, and particularly relates to the application of rifabutin in preparing a preparation for promoting cell melanin synthesis. Background Art

[0002] Melanin is a biological pigment commonly found in the skin, hair, eyes, and nervous system of mammals. It is resistant to strong acids, insoluble in most solvents, and contains a stable free radical population. Melanin is divided into eumelanin and pheomelanin based on its chemical composition. Eumelanin absorbs all wavelengths of visible light, and its absorption spectrum extends into the ultraviolet and infrared regions. One of the biological functions of melanin is to protect animal skin from damage caused by ionizing radiation, such as ultraviolet rays. Therefore, under normal circumstances, when the human body is exposed to sunlight and ultraviolet radiation, the production of adequate melanin is necessary to resist UV damage.

[0003] Melanin is produced in melanocytes in the basal layer of the epidermis. Melanocytes contain a lysosome-like organelle called the melanosome, which is specifically responsible for the synthesis and transport of melanin. Melanin biosynthesis is tightly regulated, with the primary proteases involved in the biosynthesis process being tyrosinase (TYR), tyrosine-associated proteinase 1 (TRP 1), and tyrosine-associated proteinase 2 (TRP 2). First, L-tyrosine is converted to dopaquinone by tyrosinase. This step is the rate-limiting step in the entire process, making tyrosinase a key rate-limiting enzyme in melanin synthesis. Cysteine ​​reacts rapidly and quantitatively with dopaquinone to form a benzothiazine intermediate. Benzothiazine gradually polymerizes to form pheomelanin. When cysteine ​​is depleted in the melanosome, dopaquinone spontaneously reacts with dopachrome to produce 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Dopachrome tautomerase or certain metal ions can promote the production of DHICA. DHI and DHICA are then further oxidized to produce eumelanin, with TRP1 catalyzing the oxidation of DHICA to eumelanin.

[0004] Melanin production is crucial for good health. A moderate amount of melanin helps cells protect against UV damage, while excessive pigmentation leads to uneven skin color and poor appearance. Low levels of melanin, on the other hand, can lead to skin diseases such as vitiligo. Therefore, developing agents that can boost cellular melanin synthesis is of great significance. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a new use of rifabutin, aiming to solve the problems existing in the above-mentioned background technology.

[0006] To achieve the above objectives, the present invention adopts a technical solution that provides the use of rifabutin in the preparation of a preparation for promoting cellular melanin synthesis. The chemical name of rifabutin is 4-N-isobutyl spiperidin rifamycin S, and its structural formula is shown in Formula I: .

[0007] On the basis of the above technical solution, the present invention can also be improved as follows.

[0008] Furthermore, the preparation is a preparation that promotes the expression of TYR, TRP-1, and TRP-2.

[0009] Furthermore, the preparation also includes a melanocyte protective agent.

[0010] Furthermore, melanocyte protective agents include antioxidants.

[0011] Furthermore, the dosage form of the preparation is ointment, tincture, liniment, tablet, powder, decoction, pill or capsule.

[0012] Furthermore, the effective concentration of rifabutin acting on cells is 0.1-10 μM.

[0013] Furthermore, rifabutin acts on cells for 24 to 72 hours.

[0014] The beneficial effects of the present invention are as follows: the present invention proposes a new use of rifabutin, which can promote the synthesis and secretion of melanin and the expression of TYR, TRP-1 and TRP-2, and provides a new functional ingredient for enhancing melanin synthesis in the body and preparing products for treating vitiligo, etc., and has very broad application prospects in the fields of pharmaceutical preparations, cosmetics, nursing products, beauty products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of Rifabutin on the survival rate of B16F10 cells; Figure 2 The microscopic observations of melanin production in different groups of B16F10 cells in Example 2; Figure 3 This is a comparison chart of melanin production in different groups of B16F10 cells after lysis and centrifugation in Example 2; Figure 4 This is a graph showing the determination of melanin production in different groups of B16F10 cells in Example 2; Figure 5The microscopic observations of melanin production in B16F10 cells after treatment for different time periods in Example 3 are shown; Figure 6 This is a comparison chart of melanin production in B16F10 cells after lysis and centrifugation after treatment for different time periods in Example 3; Figure 7 This is a graph showing the determination of melanin production in B16F10 cells after treatment for different time periods in Example 3; Figure 8 The melanin production of zebrafish embryos after 24 hours of treatment with different concentrations of Rifabutin; Figure 9 The melanin production of zebrafish embryos after 48 hours of treatment with different concentrations of Rifabutin; Figure 10 The melanin production of zebrafish embryos after 72 hours of treatment with different concentrations of Rifabutin; Figure 11 The results show that the melanin content of zebrafish embryos was measured after 72 hours of treatment with different concentrations of Rifabutin. Figure 12 Rifabutin promotes the expression of TRP1, TRP-2, TYR and MITF proteins. DETAILED DESCRIPTION

[0016] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0017] Example 1: Evaluation of Rifabutin Cytotoxicity by CCK-8 Method 1. Experimental Materials and Instruments Mouse melanoma cells (B16F10), 1640 culture medium, fetal bovine serum (FBS), 96-well plates, CCK-8 reagent, and microplate reader.

[0018] 2. Experimental Procedure (1) Mouse melanoma cells B16F10 were passaged and cultured at 2×10 5 Cells were seeded into 96-well plates containing culture medium at a density of 100 cells / well and cultured in a cell culture incubator at 5% CO2 and 37°C for 24 h. The culture medium used was 1640 medium containing 10% fetal bovine serum (FBS). (2) Rifabutin at different final concentrations (0.1 μmol / L, 1 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, and 100 μmol / L) was added to the 96-well plate after cell culture and incubated with the cells at 5% CO2 and 37°C for 48 h; (3) The cell viability was determined using the CCK-8 method.

[0019] 3. Experimental Conclusion The results of cell viability determination were as follows Figure 1 As shown. Figure 1 As can be seen from the results, 0.1-50 μmol / L Rifabutin had no toxicity to B16F10 cells. This result indicates that the concentration of 1-50 μmol / L Rifabutin is within the safe range for B16F10 cells.

[0020] Example 2: Effect of Rifabutin Concentration on Intracellular Melanin Content in B16F10 Cells 1. Experimental Materials and Instruments Mouse melanoma cells (B16F10), 1640 culture medium, fetal bovine serum (FBS), 6-well plates, cell lysis buffer (RIPA), PBS, dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), centrifuge, water bath, vortexer, Rifabutin working solution, 8-methoxypsoralen (8-MOP) working solution.

[0021] 2. Experimental Procedure (1) Mouse melanoma cells B16F10 were passaged and cultured at 2×10 5 Cells were seeded into 6-well plates containing culture medium at a concentration of 100 cells / well and cultured in a cell culture incubator at 5% CO2 and 37°C for 24 h. The culture medium used was 1640 medium containing 10% fetal bovine serum (FBS). (2) The cultured cells were divided into an experimental group (Rifabutin group), a blank group (CTR group), and a positive control group (8-MOP group). The positive control group was added with 8-methoxypsoralen (8-MOP) at a final concentration of 20 μmol / L, and the experimental groups were added with different final concentrations of Rifabutin (0.1 μmol / L, 1 μmol / L, and 10 μmol / L). The cells were then incubated with Rifabutin at 5% CO2 and 37°C for 48 h. (3) After co-incubation, the cells were collected, centrifuged at 12,000 rpm for 10 min, and the supernatant was removed. 200 μL of cell lysis buffer (RIPA) was added for cell lysis. The lysis process was to place the cells in a water bath at 80°C for 1 h. (4) The absorbance was measured at 450 nm using a microplate reader. The results were repeated three times for each group, and the melanin content in the cells was calculated.

[0022] 3. Experimental Results The results of the determination of melanin content in cells are as follows Figures 2-4 As shown. Among them, Figure 2Microscopic observations of melanin production in B16F10 cells from different groups; Figure 3 This is a comparison of melanin production in different groups of B16F10 cells after lysis and centrifugation; Figure 4 The figure shows the determination of melanin production in B16F10 cells in different groups. Figure 2 、 3 As can be seen from Figures 4 and 8-MOP and Rifabutin can promote the formation of melanin in mouse melanoma cells, and as the concentration of Rifabutin increases, the cells produce more melanin. Figure 4 It can be seen that after B16F10 was treated with 0.1μM Rifabutin for 48 hours, the amount of melanin production increased slightly, but the statistical difference was not significant (P>0.05); after treatment with 1μM Rifabutin for 48 hours, a significant increase in melanin production was observed, and the effect was significant (P<0.05); after treatment with 10μM Rifabutin for 48 hours, the strongest melanin-stimulating effect was shown, and the melanin content reached the highest level (P<0.01).

[0023] It can be seen from this that Rifabutin can promote the formation of melanin in B16F10 cells, and in the B16F10 cell line, the melanin production of cells treated with Rifabutin presents a dose-dependent effect.

[0024] Example 3: Effect of 10 μmol / L Rifabutin on the Intracellular Melanin Content of B16F10 Cells at Different Incubation Times 1. Experimental Materials and Instruments Mouse melanoma cells (B16F10), 1640 culture medium, fetal bovine serum (FBS), 6-well plates, cell lysis buffer (RIPA), PBS, dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), centrifuge, water bath, vortexer, Rifabutin working solution, 8-methoxypsoralen (8-MOP) working solution.

[0025] 2. Experimental Procedure (1) Mouse melanoma cells B16F10 were passaged and cultured at 2×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate containing culture medium. Rifabutin was added at a final concentration of 10 μmol / L and cultured in a cell culture incubator at 5% CO2 and 37°C for 12 h, 24 h, and 48 h, respectively. The culture medium used was 1640 medium containing 10% fetal bovine serum (FBS). (2) After co-incubation, the cells were collected, centrifuged at 12,000 rpm for 10 min, and the supernatant was removed. 200 μL of cell lysis buffer (RIPA) was added for cell lysis. The lysis process was to place the cells in a water bath at 80°C for 1 h. (3) The absorbance was measured at 450 nm using a microplate reader. The results were repeated three times for each group, and the melanin content in the cells was calculated.

[0026] 3. Experimental Results Effect of incubation time on melanin production in B16F10 cells Figures 5-7 shown; among them, Figure 5 The microscopic observations show the melanin production in B16F10 cells after treatment for different time periods. Figure 6 This is a comparison of melanin production after lysis and centrifugation of B16F10 cells treated for different time periods; Figure 7 The figure shows the determination of melanin production in B16F10 cells after treatment for different time periods. Figure 5 and 6 It can be seen that with the extension of culture time, the content of melanin in the cells gradually increases. Figure 7 It can be seen that after 12 hours of culture, the promoting effect of 10 μmol / L Rifabutin on cell melanin production was not obvious, and the difference was not significant compared with the control group (CTR) (P>0.05); after 24 hours of culture, the promoting effect of 10 μmol / L Rifabutin on cell melanin production gradually became obvious, and the difference was significantly enhanced compared with the control group (CTR) (P<0.05); after extending the culture time to 48 hours, the promoting effect of Rifabutin on cell melanin production was significantly enhanced (P<0.001).

[0027] Therefore, in the B16F10 cell line, the melanin production of cells treated with the same concentration of Rifabutin showed a time-dependent effect.

[0028] Example 4: Effects of different concentrations of Rifabutin and different culture times on melanin production in zebrafish 1. Experimental Materials and Instruments AB zebrafish, brine shrimp, breeding tank, stereo microscope, Rifabutin working solution, 8-methoxypsoralen (8-MOP) working solution, phenylthiourea working solution, 6-well plates, 50mm culture dishes, zebrafish embryo culture medium (pac2 zebrafish embryo fibroblast culture medium).

[0029] 2. Experimental Procedure 2.1 Zebrafish spawning Healthy AB stock (male-female ratio 1:1-2:1) aged 3-12 months were selected. Females should have an enlarged abdomen with visible eggs, and males should have bright coloration. Two weeks prior to the experiment, a high-protein diet (brine shrimp) was fed to promote gonadal development. The water temperature was maintained at 28.5 ± 0.5°C, pH 7.0-7.5, and conductivity 500-1500 μS / cm. A 14-hour light / 10-hour dark cycle was used. A breeding tank with a screen was used to prevent broodstock from swallowing eggs. The night before the light cycle, the breeding fish were placed in the breeding tank, and the male and female fish were separated by a spacer to allow them to produce impulses overnight. After a 10-hour dark cycle, the spacer was removed 1 hour after the start of the light cycle. After 1-2 hours, the zebrafish spawning situation was observed, and the eggs were collected in a culture dish. The eggs were washed 2-3 times with distilled water, and high-quality fertilized eggs were selected under a stereomicroscope. Individuals with irregular morphology, damaged egg membranes or unfertilized (no blastoderm formation) were eliminated, and the selected eggs were placed in a culture medium containing KCl and cultured for 24 hours.

[0030] 2.2 Zebrafish embryo grouping and drug administration Zebrafish embryos were cultured in culture medium for 24 hours, then transferred to 6-well plates and grouped into six groups, each containing 20 eggs. These groups included a blank control group, a negative control group, a positive control group, and three experimental groups. All groups, except the blank group, were decolorized with 50 μmol / L phenylthiourea (PTU) for 24 hours, after which the PTU was removed. Following decolorization, the positive control group was treated with 8-MOP at a final concentration of 20 μmol / L, the experimental groups were treated with rifabutin at final concentrations of 0.1 μmol / L, 1 μmol / L, and 10 μmol / L, respectively, and the negative control group was treated with an equal amount of PTU. Melanin production in the zebrafish embryos was observed under a stereomicroscope 24, 48, and 72 hours after treatment.

[0031] 2.3 Determination of melanin content in zebrafish embryo samples Zebrafish embryo samples were collected in groups into 1.5 mL EP tubes. The liquid in the tubes was aspirated and 200 μL of RIPA lysis buffer was added. The samples were fully ground with a handheld grinder for one minute per group and centrifuged at 12,000 rpm in a 4°C precooled centrifuge for 10 minutes. The protein concentration of the supernatant was determined using the BCA method. 200 μL of 1N sodium hydroxide (NaOH) solution containing 10% dimethyl sulfoxide (DMSO) was added to the precipitate, and the tubes were placed in a water bath at 80°C for 1 hour. The absorbance was detected at 450 nm on a microplate reader to calculate the melanin content of the samples.

[0032] 3. Experimental Results Melanin production in zebrafish embryos Figures 8-11 shown; among them, Figure 8 The melanin production of zebrafish embryos after 24 hours of treatment with different concentrations of Rifabutin. Figure 9The melanin production of zebrafish embryos after 48 hours of treatment with different concentrations of Rifabutin. Figure 10 The melanin production of zebrafish embryos after 72 hours of treatment with different concentrations of Rifabutin. Figure 11 The results show that the melanin content of zebrafish embryos was measured after 72 hours of treatment with different concentrations of Rifabutin. Figures 8-10 It can be seen that with the extension of drug treatment time and the increase of drug concentration, the effect of 10μmol / L Rifabutin administration group on promoting melanin production gradually increased, reaching the highest level at 72h. In addition, the melanin production in the 10μmol / L Rifabutin administration group was stronger than that in the 8-MOP positive control group. Figure 11 It can be seen that compared with the positive control group, after 48 hours of treatment with 0.1μM Rifabutin, the amount of melanin production increased slightly, but the statistical difference was not significant; after 48 hours of treatment with 1μM Rifabutin, a significant increase in melanin production was observed, and the effect was significant; after 48 hours of treatment with 10μM Rifabutin, the strongest melanin-promoting effect was shown, and the melanin content reached the highest level.

[0033] It can be seen that in zebrafish embryos, the melanin production treated with Rifabutin showed dose-dependent and time-dependent effects, and the melanin-promoting effect was most significant at 10 μmol / L Rifabutin and 72 h treatment time.

[0034] Example 5: Effects of different concentrations of Rifabutin on melanin-related genes in B16F10 cells 1. Experimental Materials The materials and instruments used in the experiment are shown in the following table: cell lines Mouse melanoma cells B16F10 drug 8-Methoxypsoralen, Rifabutin Internal reference protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase) Detection protein TYR (tyrosinase), TRP-1 (tyrosinase-related protein-1), TRP-2 (tyrosinase-related protein-2), MTF (microphthalmia-related transcription factor) Total protein extraction reagent RIPA lysis buffer, BCA kit, protease inhibitors Western Blot experimental reagents and instruments Electrophoresis apparatus, transfer apparatus, electrophoresis fluid, transfer fluid, centrifuge, NC membrane, 5% skim milk, shaker 2. Experimental steps B16F10 cells were cultured in 1640 medium containing 10% FBS to 70-80% confluency, and treated with 8-MOP (20 μmol / L) and different concentrations of Rifabutin (0.1 μmol / L, 1 μmol / L, 10 μmol / L) for 48 h. Then, 200 μL of RIPA lysis buffer (containing protease inhibitors) was added to extract total protein, and the protein concentration was determined by BCA method. 10 μg of protein was loaded and subjected to SDS-PAGE electrophoresis (10% separation gel). The protein was transferred to the NC membrane by wet transfer and blocked with 5% skim milk at room temperature for 2 hours. The NC membrane was incubated with TYR, TRP-1, TRP-2, MITF, and GAPDH antibodies for 14-16 h and incubated at 4°C overnight. After 14-16 h, the NC membrane incubated with the primary antibody was incubated with the secondary antibody at room temperature for 2 h. ECL chemiluminescence detection was used, and the grayscale value of the bands was analyzed by ImageJ. The experimental results are shown in Figure 2. Figure 12 shown.

[0035] from Figure 12 As can be seen in the data, compared with the control group, Rifabutin (10 μmol / L) significantly increased the expression of TYR, TRP-1, and TRP-2 in a dose-dependent manner. 8-MOP, as a photosensitizer and melanin synthesis promoter, activated the melanogenesis pathway by upregulating the protein expression of TYR, TRP-1 / 2, and MITF, consistent with previous studies. High concentrations of Rifabutin (10 μM) significantly promoted melanin production, which may be related to the transcriptional regulation of MITF.

[0036] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. Application of rifabutin in the preparation of preparations for promoting cell melanin synthesis.

2. The use according to claim 1, characterized in that: The preparation is a preparation that promotes the expression of TYR, TRP-1, and TRP-2.

3. The use according to claim 2, characterized in that: The formulation also includes a melanocyte protective agent.

4. The use according to claim 3, characterized in that: The melanocyte protective agent includes an antioxidant.

5. The use according to claim 1, characterized in that: The dosage form of the preparation is ointment, tincture, liniment, tablet, powder, decoction, pill or capsule.

6. The use according to claim 1, characterized in that: The effective concentration of rifabutin acting on cells is 0.1~10μM.

7. The use according to claim 6, characterized in that: Rifabutin acts on cells for 24 to 72 hours.

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