Double-drug combined tyrosinase inhibitor and application thereof
By combining tyrosinase inhibitors with chalcone compounds and transfected stem cell exosomes, the problems of poor efficacy and low safety of existing tyrosinase inhibitors have been solved, achieving highly efficient melanin production inhibition and skin whitening effects.
Patent Information
- Application Number
- CN202510921661.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-04
- Publication Date
- 2025-12-12
AI Technical Summary
Existing tyrosinase inhibitors have problems with poor inhibitory effects and low safety in the field of skin whitening and skin care, and no products have been developed in clinical practice that use miR-21a-5p target to inhibit melanin production.
A dual-drug tyrosinase inhibitor is provided, consisting of a chalcone compound (E)-5,7-dihydroxy-3-(4-hydroxybenzylmethyl)chroman-4-one and exosomes derived from human adipose-derived mesenchymal stem cells transfected with AntagonomiR-21, which enhances tyrosinase inhibitory activity by inhibiting miR-21a-5p expression.
This tyrosinase inhibitor exhibits superior tyrosinase inhibitory activity and biosafety compared to the clinical drug kojic acid at specific concentrations. It shows no significant cytotoxicity to B16 cells and has a good inhibitory effect on melanin production, making it suitable for the preparation of drugs for the prevention and treatment of melanin deposition-related diseases and skin whitening products.
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Figure CN121102276A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of dermatology, in particular to a dual-drug combined tyrosinase inhibitor and its application. BACKGROUND
[0002] Tyrosinase, as a key enzyme in melanin synthesis, plays a crucial role in human pigmentation and whitening. When the content of tyrosinase is abnormal or its expression is out of control, it may lead to the occurrence of human pigmentation diseases, such as chloasma, freckle, and seborrheic keratosis, etc. These diseases not only affect the appearance of patients, but also may have a negative impact on mental health. On the other hand, insufficient or dysfunctional tyrosinase may also lead to hypopigmentation skin diseases, such as vitiligo, etc. Therefore, regulating the activity of tyrosinase has become an important means for whitening and treating pigmentation diseases.
[0003] Currently, tyrosinase inhibitors have been widely used in the field of whitening. Common tyrosinase inhibitors include kojic acid, arbutin, and nicotinamide, etc. They inhibit the activity of tyrosinase to reduce the generation of melanin, thereby achieving the effect of whitening. In recent years, flavonoids derived from natural products have attracted attention due to their high safety and significant inhibitory effect. For example, flavonoids in Sophora flavescens buds show higher inhibitory effect on tyrosinase activity than kojic acid. Researchers have also extracted flavonoids from the fruits of Juniperus communis, which also have obvious tyrosinase inhibitory capacity. These findings provide important theoretical basis for the development of new natural tyrosinase inhibitors.
[0004] Research on the mechanism of melanin generation found that extracellular vesicles (such as exosomes) can communicate between cells and express many melanin generation-related genes, such as miR-21a-5p, which plays a positive regulatory role in the process of melanin formation. Research found that miR-21a-5p targets and inhibits Sox5 gene, reduces its inhibitory effect on downstream MITF gene, thereby up-regulating MITF expression and promoting melanin production in melanocytes. However, there is no related product development and utilization of this target for melanin generation inhibition in clinical practice, and the commonly used tyrosinase inhibitors in clinical practice have problems such as low inhibitory effect and low safety. Therefore, it is of great social significance to develop new safe and efficient tyrosinase inhibitors. SUMMARY
[0005] The purpose of the present application is to provide a dual-drug combined tyrosinase inhibitor and its application to solve the above technical problems.
[0006] The present application provides a dual-drug combined tyrosinase inhibitor, which is a composition composed of chalcone compounds and stem cell exosomes.
[0007] The chalcone compound is (E)-5,7-dihydroxy-3-(4-hydroxybenzylidene) chroman-4-one (4'-Dem), and its structural formula is
[0008]
[0009] The stem cell exosome is an exosome derived from human adipose-derived mesenchymal stem cells (HADSCs-Exo) treated by AntagomiR-21 transfection.
[0010] Further, the concentration of the (E)-5,7-dihydroxy-3-(4-hydroxybenzylidene) chroman-4-one is 2-8 μg / mL, and the concentration of the exosome derived from human adipose-derived mesenchymal stem cells is 2-8 μg / mL.
[0011] Further, in the composition, the mass concentration ratio of (E)-5,7-dihydroxy-3-(4-hydroxybenzylidene) chroman-4-one and the exosome derived from human adipose-derived mesenchymal stem cells is (1-2):(1-2).
[0012] Further, in the composition, the optimal concentration of (E)-5,7-dihydroxy-3-(4-hydroxybenzylidene) chroman-4-one is 8 μg / mL, and the optimal concentration of the exosome derived from human adipose-derived mesenchymal stem cells is 4 μg / mL.
[0013] The application also provides a use of a combination of a tyrosinase inhibitor in the preparation of a medicament for preventing and / or treating a melanin deposition related disease or a skin whitening product; the tyrosinase inhibitor comprises (E)-5,7-dihydroxy-3-(4-hydroxybenzylidene) chroman-4-one and / or a stem cell exosome; the stem cell exosome is an exosome derived from human adipose-derived mesenchymal stem cells treated by AntagomiR-21 transfection.
[0014] Further, the melanin deposition related disease includes melasma or melanoma.
[0015] Further, the dosage form of the medicament is a wipeable external gel or paste, or a wettable powder, a tablet, a granule, a capsule, an oral liquid, a dripping pill, a controlled release or sustained release dosage form, or a nano preparation, an injection preparation.
[0016] The application has the following advantages:
[0017] This invention provides a dual-drug combination tyrosinase inhibitor, which is a composition of 4'-Dem combined with HADSCs-Exo. This invention uses the miR-21a-5p inhibitor AntagonomiR-21 to transfect HADSCs, obtaining HADSCs that do not express or express low levels of miR-21a-5p, and further collects the secreted exosomes HADSCs-Exo; ensuring that the obtained exosomes contain no or only a small amount of miR-21a-5p, thereby enhancing the tyrosinase inhibitor activity of this application.
[0018] The present invention provides a dual-drug combination tyrosinase inhibitor with good tyrosinase inhibitory activity and melanin production inhibitory activity, and no obvious cytotoxicity to B16 cells. In addition, at a concentration of 8 μg / mL of 4'-Dem combined with 4 μg / mL of HADSCs-Exo, the tyrosinase inhibitor exhibits superior tyrosinase inhibitory activity and biosafety compared to the clinical drug kojic acid (KA), demonstrating the application prospects of this tyrosinase inhibitor in the preparation of drugs for the prevention and / or treatment of melanin deposition-related diseases and skin whitening products. Attached Figure Description
[0019] Figure 1 This is a graph showing the inhibitory activity data of different concentrations of HADSCs-Exo, 4'-Dem, and KA on tyrosinase.
[0020] Figure 2 This is a graph showing the inhibitory activity data of different ratios of HADSCs-Exo and 4'-Dem on tyrosinase.
[0021] Figure 3 This is a graph showing the survival rate of B16 cells with different ratios of HADSCs-Exo and 4'-Dem combined.
[0022] Figure 4 This is a graph showing the effect of HADSCs-Exo combined with 4'-Dem on tyrosinase activity in B16 cells.
[0023] Figure 5 This is a graph showing the effect of HADSCs-Exo combined with 4'-Dem on melanin production in B16 cells. Detailed Implementation
[0024] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.
[0025] Example 1: Preparation of HADSCs-Exo
[0026] miR-21a-5p plays a positive regulatory role in melanin formation. Research has found that miR-21a-5p upregulates MITF expression by targeting and inhibiting the Sox5 gene, thereby reducing its inhibitory effect on the downstream MITF gene and promoting melanin production in melanocytes. Therefore, this application obtains exosomes with low or no miR-21a-5p expression by inhibiting miR-21a-5p expression in cells. Specific embodiments are as follows:
[0027] HADSCs were cultured using exosome-free HADSC-specific medium at 37°C and 5% CO2. When the cell density reached 60%, Antagonomi R-21 was mixed with the transfection reagent according to the instructions. This mixture was then added to the cell culture medium and gently mixed. The cells were cultured at 37°C and 5% CO2 for 8 hours, then the medium was replaced with fresh medium, and the cells were cultured for another 48-72 hours to ensure effective inhibition of miR-21a-5p. The supernatant from the transfected HADSCs was collected, and HADSCs-Exo was extracted according to the exosome extraction kit instructions. The collected HADSCs-Exo was stored at -80°C for later use.
[0028] Example 2: Evaluation of the tyrosinase inhibitory activity of HADSCs-Exo and 4'-Dem
[0029] This invention uses kojic acid as a positive control to determine the tyrosinase inhibitory activity of HADSCs-Exo and 4'-Dem. The experiment consisted of a sample solution group and a blank control group. The sample group concentrations were uniformly set to 1, 2, 4, 8, and 16 μg / mL, and then 40 μL of sample, 80 μL of PBS, and 40 μL of tyrosinase were added, respectively. The blank control group was treated with 120 μL of PBS and 40 μL of tyrosinase, reacted at 37°C for 10 min, and then 40 μL of tyrosine was added sequentially. After all additions were completed in the above order, the values before the reaction were measured at 492 nm; after reacting at 37°C for 25 min, the values after the reaction were measured at 492 nm and a statistical graph was plotted.
[0030] Experimental results are as follows Figure 1 As shown, at the same concentration, HADSCs-Exo exhibits lower tyrosinase inhibitory activity than the positive control drug KA, with a lower half-maximal inhibitory concentration (IC50). 50The concentration was 4.57 μg / mL, indicating that the tyrosinase inhibitory efficiency of HADSCs-Exo increased with increasing concentration, possibly due to the action of the bioactive molecules contained in HADSCs-Exo; while the tyrosinase inhibitory activity of the chalcone compound 4'-Dem was slightly higher than that of the positive control drug KA, with an IC50 value of 4'-Dem. 50 The concentration was 2.16 μg / mL, which is likely due to the binding of 4'-Dem to the active site of tyrosinase, thus inhibiting its activity. These results indicate that both HADSCs-Exo and 4'-Dem can inhibit tyrosinase activity; furthermore, 4'-Dem exhibits better tyrosinase inhibitory activity and is slightly superior to the positive control drug KA.
[0031] Example 3: Evaluation of the inhibitory activity of HADSCs-Exo combined with 4'-Dem on tyrosinase
[0032] To investigate the optimal combined concentration of the two compounds, this invention is based on the IC50 values of HADSCs-Exo and 4'-Dem. 50 The values are set as grouped as in Table 1. The experimental method for determining tyrosinase inhibitory activity described in Example 2 is used to calculate the inhibition rate of tyrosinase activity in each group and draw a statistical analysis chart.
[0033] Table 1
[0034]
[0035] Experimental results are as follows Figure 2As shown, the positive control drug kojic acid had an inhibition rate of about 90%, exhibiting good tyrosinase inhibitory activity, indicating that the experimental procedure was rigorous and error-free. Next, the tyrosinase inhibition rates of each group prepared with 4'-Dem and HADSCs-Exo in different proportions were observed. It can be seen that the AChE inhibition rates of 4'-Dem and HADSCs-Exo varied under different concentrations and ratios, with the overall trend being that the tyrosinase inhibition rate of the low-concentration groups (groups a, b, and c) was lower than that of the high-concentration groups (groups d, e, and f). Furthermore, this invention also found that, regardless of the low or high concentration groups, under the condition of the same concentrations of 4'-Dem and HADSCs-Exo, if the concentration of 4'-Dem is kept constant and the concentration of HADSCs-Exo is increased, the tyrosinase inhibition effect is enhanced, i.e., the inhibition rate of group b > the inhibition rate of group a and the inhibition rate of group e > the inhibition rate of group d. If the concentration of HADSCs-Exo is kept constant and the concentration of 4'-Dem is increased, the tyrosinase inhibition effect is stronger than the situation described above where the concentration of 4'-Dem is kept constant and the concentration of HADSCs-Exo is increased, i.e., the inhibition rate of group c > the inhibition rate of group b and the inhibition rate of group f > the inhibition rate of group e. Therefore, the above results indicate that the inhibition rate of tyrosinase is highest when the mass concentration ratio of 4'-Dem and HADSCs-Exo is 2:1, and the optimal effective concentration of the combination of 4'-Dem and HADSCs-Exo in the current test is 8 μg / mL of 4'-Dem and 4 μg / mL of HADSCs-Exo.
[0036] Example 4: Viability assay of mouse melanoma cells (B16 cells) using HADSCs-Exo combined with 4'-Dem
[0037] To evaluate the cytotoxicity of HADSCs-Exo combined with 4'-Dem, B16 cell viability was detected using the CCK8 assay. Logarithmic-phase B16 cells were harvested and subjected to a assay of 1×10⁻⁶ cells. 4 The cells were seeded at a concentration of 100 μL per well into 96-well plates and cultured normally for 24 h. Then, 100 μL of different mass concentrations of the drug solution were added according to the grouping in Table 1 of Example 3, with 3 replicates per group. The cells were cultured for another 24 h. Then, 10 μL of CCK8 solution was added to each well and the cells were cultured for another 0.5 h. The cells were then shaken for 1 min using a microplate reader and the absorbance was measured at a wavelength of 450 nm.
[0038] Experimental results are as follows Figure 3 As shown, compared with the Control group, the cell survival rate in the af group was above 85%, and no obvious cytotoxicity was observed. This indicates that the combined treatment of B16 cells with different concentration ratios of 4'-Dem and HADSCs-Exo within the concentration range of 2-8 μg / mL did not exhibit significant cytotoxicity; while KA achieved the same tyrosinase inhibitory activity as the f group ( Figure 2However, it showed significant cytotoxicity to B16 cells, demonstrating that 4'-Dem and HADSCs-Exo have better biocompatibility than KA.
[0039] Example 5: Determination of the inhibitory activity of HADSCs-Exo combined with 4'-Dem on intracellular tyrosinase in B16 cells
[0040] To further observe the tyrosinase inhibitory activity of HADSCs-Exo combined with 4'-Dem in B16 cells, this invention determined the intracellular tyrosinase activity of B16 cells using the L-DOPA oxidation method. Logarithmic-phase B16 cells were used, and 1×10⁻⁶ cells were subjected to... 4 Cells were seeded at a concentration of [number] cells / well into 96-well plates, with 100 μL of cell suspension added to each well. After normal culture for 24 h, different concentrations of drug solutions were prepared according to the Control, KA, c, and f groups in Table 1 of Example 3. Then, 100 μL of the prepared drug solution was added to the corresponding well, and the plates were cultured for another 24 h. After culture, the cell culture plates were centrifuged, the supernatant was discarded, and 150 μL of 1% Triton X-100 solution was added to each well. The plates were frozen at -80°C for 40 min, thawed at room temperature to lyse the cells, and the freeze-thaw cycle was repeated several times. The cell lysate was centrifuged, and 100 μL of the supernatant was added to a 96-well plate. 60 μL of 2 mmol / L L-DOPA solution was added, and the plates were incubated at 37°C in the dark for 20 min. The absorbance was measured at 475 nm.
[0041] Experimental results are as follows Figure 4 As shown, compared with the Control group, the tyrosinase activity in B16 cells treated with HADSCs-Exo combined with 4'-Dem was inhibited. In addition, the f group showed the best inhibitory effect and was better than the positive control drug KA group, indicating that HADSCs-Exo 4 μg / mL combined with 4'-Dem 8 μg / mL has better tyrosinase inhibitory activity than KA.
[0042] Example 6: Effect of HADSCs-Exo combined with 4'-Dem on melanin content in B16 cells
[0043] To evaluate the effect of HADSCs-Exo combined with 4'-Dem on melanin production, this invention employed the NaOH method to lyse B16 cells and measure melanin production within B16 cells. Melanin production in the logarithmic phase was measured at a concentration of 1×10⁻⁶. 5B16 cells per well were seeded into 6-well plates with 1.5 mL of cell suspension per well and cultured normally for 24 h. Different concentrations of drug solutions were prepared according to the Control, KA, c, and f groups in Table 1 of Example 3. Then, 1.5 mL of the prepared drug solution was added to the corresponding well of the plate, with 3 replicates per group. The cells were cultured for another 24 h. 1.5 mL of trypsin was added to each well for digestion. The cell slurry was collected, centrifuged, and the supernatant was discarded. 200 μL of 1 mol / L NaOH solution (containing 10% dimethyl sulfoxide) was added to each well. The plates were heated in an 80°C water bath until the cell clusters were completely lysed and the melanin granules were completely dissolved. The absorbance was measured at a wavelength of 405 nm.
[0044] Experimental results are as follows Figure 5 As shown, compared with the control group, the addition of HADSCs-Exo and 4'-Dem reduced the melanin content in B16 cells, and moderate melanin production inhibition activity was observed in the f group (HADSCs-Exo 4 μg / mL and 4'-Dem 8 μg / mL); compared with KA, the f group produced less melanin, which demonstrates that HADSCs-Exo 4 μg / mL combined with 4'-Dem 8 μg / mL has good melanin production inhibition activity.
[0045] In summary, the HADSCs-Exo combined with 4'-Dem composition of this invention exhibits good tyrosinase inhibitory activity and moderate melanin production inhibitory activity, with no significant cytotoxicity to B16 cells. Furthermore, at a concentration of 4 μg / mL HADSCs-Exo combined with 8 μg / mL 4'-Dem, this composition demonstrates superior tyrosinase inhibitory activity and biosafety compared to the clinically used drug kojic acid, indicating that the HADSCs-Exo combined with 4'-Dem composition shows promising application potential in the fields of tyrosinase inhibitors for the prevention and / or treatment of melanin deposition-related diseases or in skin-whitening cosmetics.
[0046] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.
Claims
1. A dual-drug combination tyrosinase inhibitor, characterized in that, The tyrosinase inhibitor is a composition of chalcone compounds and stem cell exosomes; The chalcone compound is (E)-5,7-dihydroxy-3-(4-hydroxyphenylmethylene)chroman-4-one, and its structural formula is: The stem cell exosomes are exosomes derived from human adipose-derived mesenchymal stem cells that have been transfected with Antagonomi R-21.
2. The tyrosinase inhibitor according to claim 1, characterized in that, The concentration of (E)-5,7-dihydroxy-3-(4-hydroxybenzyl)chroman-4-one is 2-8 μg / mL, and the concentration of exosomes derived from human adipose-derived mesenchymal stem cells is 2-8 μg / mL.
3. The tyrosinase inhibitor according to claim 1, characterized in that, In the composition, the mass concentration ratio of (E)-5,7-dihydroxy-3-(4-hydroxybenzyl)chroman-4-one to exosomes derived from human adipose-derived mesenchymal stem cells is (1-2):(1-2).
4. The tyrosinase inhibitor according to claim 1, characterized in that, The optimal concentration of (E)-5,7-dihydroxy-3-(4-hydroxybenzyl)chroman-4-one in the composition is 8 μg / mL, and the optimal concentration of exosomes derived from human adipose-derived mesenchymal stem cells is 4 μg / mL.
5. The use of the tyrosinase inhibitor according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of melanin deposition-related diseases or a skin whitening product.
6. The application according to claim 5, characterized in that, The melanin deposition-related diseases include melasma or melanoma.
7. The application according to claim 5, characterized in that, The dosage form of the drug is a wipeable topical gel or ointment, or a wettable powder, tablet, granule, capsule, oral liquid, drop pill, controlled-release or sustained-release dosage form, or nano-formulation or injectable formulation.