Tyrosinase inhibitor and whitening application
Through artificial intelligence-assisted drug design methods, compounds such as tannin-3-propionic acid and cytidine-5'-phosphate were identified and developed, which solved the shortcomings of existing tyrosinase inhibitors in terms of inhibitory effect, safety, transdermality and stability, and achieved a more efficient and safer skin whitening effect.
Patent Information
- Application Number
- CN202510375763.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2025-05-13
AI Technical Summary
Existing tyrosinase inhibitors have shortcomings in inhibitory effects, safety, transdermality, stability and ease of use, and it is difficult to effectively solve the problem of dull skin, especially in the needs of stubborn spots and rapid whitening.
Using artificial intelligence-assisted drug design method, the compound structure data, biological activity data and computational chemical algorithms were integrated to identify and develop safe and reliable compounds with tyrosinase inhibitory activity, such as tangin-3-propionic acid and cytidine-5'-phosphate, and their effectiveness was verified in combination with experiments.
It has achieved significant tyrosinase inhibitory activity and good transdermal performance, overcome the potential toxic side effects of traditional inhibitors, the accessibility of raw materials, molecular stability and insufficient transdermal properties, and provides better raw material selection for whitening products, achieving more efficient and safer skin whitening effects.
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Figure CN119970775A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the fields of cosmetics and biomedicine, and in particular to a tyrosinase inhibitor and application thereof in a whitening cosmetic composition or a whitening pharmaceutical composition. Background Art
[0002] In today's cosmetics field, especially in the field of whitening products, the problem of dull skin caused by excessive melanin production has always been a difficult problem to be solved. Tyrosinase plays a key role in melanin synthesis, making it a common target in skin whitening products. Tyrosinase can gradually convert tyrosine into dopa and dopaquinone, and finally produce melanin. Inhibiting tyrosinase activity can reduce the formation of melanin, so it has a significant whitening effect.
[0003] There are many tyrosinase inhibitors on the market, which can be divided into natural extracts and chemically synthesized products according to their sources. Natural extracts include arbutin and niacinamide, among which arbutin is mainly extracted from bearberry leaves, which achieves whitening by competitively inhibiting the activity of tyrosinase, and its application concentration in whitening cosmetics is usually 2%-10%; niacinamide is widely present in food and organisms, and can reduce the production of melanin by blocking the activity of tyrosinase, inhibit the transfer of melanin from melanocytes to keratinocytes, and reduce skin pigmentation. It is commonly found in various whitening essences, lotions and other products, and its concentration is mostly 2%-5%; vitamin C derivatives are representatives of chemically synthesized products, such as ascorbic acid-2-glucoside (AA2G), magnesium ascorbyl phosphate (MAP), sodium ascorbyl phosphate (SAP), etc., which can achieve whitening by reducing melanin intermediates. Existing tyrosinase inhibitors generally have some side effects or poor effects. Taking arbutin as an example, its ability to inhibit tyrosinase activity is limited, and it is not effective in improving stubborn pigmentation, uneven skin tone and other problems. It will be irritating and may produce cytotoxicity at high concentrations, which limits its addition concentration in whitening products, thereby affecting the whitening effect. Niacinamide has no obvious direct inhibitory effect on tyrosinase, and may also cause skin intolerance in some people, such as redness, itching, tingling and other allergic reactions, and its whitening effect is relatively slow, requiring long-term use to see obvious results, and it is difficult to meet consumers' demand for rapid whitening. Vitamin C and its derivatives have poor transdermal permeability, resulting in a low effective concentration in the skin and limited whitening effect. They are easily decomposed at high temperatures or improper pH conditions, have poor stability, and have high requirements for formulation technology, which increases the difficulty and cost of product research and development and production.
[0004] In addition, most existing whitening ingredients face the problem of transdermal absorption, and it is difficult to effectively reach the melanocytes deep in the skin to exert their effects, which limits their full whitening effect. In order to solve the problem of insufficient transdermal penetration of active ingredients and easy restriction by the skin barrier, some existing technologies use nanoemulsification or liposome encapsulation to enable active substances to better penetrate the stratum corneum, but such methods have high requirements for formulation technology, professional technology and complex production processes, which not only increases production costs, but also may lead to unstable product quality; and the release control, stability and cost of active substances after encapsulation are still difficult, affecting the sustainability and stability of the whitening effect. In the field of medical beauty, the stratum corneum barrier can be punctured by microneedles or the skin can be opened instantaneously by ion electrophoresis to assist the penetration of whitening ingredients. Although this method has significantly improved the transdermal effect, in actual daily skin care scenarios, the threshold for consumers to use is high, and there is a risk of skin damage or infection, and safety is difficult to guarantee. It is not suitable for most consumers who pursue daily safe whitening. Summary of the invention
[0005] In response to the above problems, the present invention adopts an artificial intelligence-assisted drug design (AIDD) method, integrates a large amount of compound structure data, biological activity data and computational chemistry algorithms, performs virtual screening on a large number of compounds, identifies compounds with potential tyrosinase inhibitory activity, and combines experimental verification to develop new compounds that have tyrosinase inhibitory activity, are safe and reliable, and have good transdermal performance. In terms of tyrosinase inhibition and whitening uses, it is expected to overcome the shortcomings of the above-mentioned prior art in terms of inhibitory effect, safety, transdermal properties, stability and ease of use due to its unique molecular structure, provide better raw material selection for the research and development of whitening products, and achieve more efficient and safer skin whitening effects.
[0006] The purpose of the present invention is achieved by the following technical solutions:
[0007] The first aspect of the present invention is to provide a tyrosinase inhibitor, which comprises compound A and / or compound B, wherein compound A is rhodanine-3-propionic acid or a cosmetically acceptable salt thereof; and compound B is cytidine-5'-phosphate or a cosmetically or pharmaceutically acceptable salt thereof.
[0008] Rhodanine-3-propionic acid has natural antibacterial properties and outstanding inhibitory activity against tyrosinase. It can be extracted from hawthorn. It is natural and safe for consumption. Hawthorn, as a common edible plant, has a wide source and low cost, and can be used as a high-quality extraction raw material.
[0009] Cytidine 5'-(dihydrogen phosphate) is a nucleotide composed of cytosine base, ribose and phosphate groups. It is involved in RNA construction and energy conversion and is widely present in the human body. It is a mild, safe and cost-effective ingredient. Its phosphate group enhances binding affinity, increases molecular polarity, and improves solubility and bioavailability, which makes it have good potential in whitening applications and can better play the role of inhibiting tyrosinase.
[0010] The second aspect of the present invention is to provide an application of the tyrosinase inhibitor in the preparation of a product for inhibiting tyrosinase activity.
[0011] In some specific embodiments, the tyrosinase inhibitor is used to prepare a drug for preventing and / or treating human pigmentation diseases and melanoma caused by abnormal melanin;
[0012] In some specific embodiments, the tyrosinase inhibitor is used for preparing a topical skin preparation for whitening;
[0013] The third aspect of the present invention is to provide a cosmetic composition or a pharmaceutical composition, which comprises a cosmetically or pharmaceutically effective amount of the tyrosinase inhibitor.
[0014] In some specific embodiments, in order to enable the inhibitor to better act on the skin and exert a whitening effect, the inhibitor can be prepared into various dosage forms as needed, and the dosage form of the composition can be an anhydrous composition, an aqueous dispersion, a solution, a cream, a gel, a film, a mist, a pellet or a foam.
[0015] In some specific embodiments, the composition may contain other cosmetically or pharmaceutically acceptable auxiliary ingredients, such as excipients or adjuvants, excipients such as carriers or diluents, adjuvants such as other agents that inhibit tyrosinase activity, moisturizers, emulsifiers, preservatives, antioxidants, emollients, wetting agents, thickeners, surfactants, antibacterial agents, fragrances, inorganic salts, etc.; each auxiliary ingredient plays an independent or synergistic role to achieve optimal whitening and product stability and ensure product quality.
[0016] The beneficial effects of the present invention are:
[0017] The present invention adopts artificial intelligence-assisted drug design (AIDD) method, integrates a large amount of compound structure data, biological activity data and computational chemistry algorithms, performs virtual screening on a large number of compounds, identifies compounds with potential tyrosinase inhibitory activity, and combines experimental verification to confirm that two compounds, rhodanine-3-propionic acid and cytidine-5'-phosphate, have significant tyrosinase inhibitory activity. Compared with traditional tyrosinase inhibitors, they have certain advantages in terms of potential toxic and side effects, raw material accessibility, and molecular stability. At the same time, the two compounds also show good transdermal performance, which is an advantage that many existing whitening ingredients do not have. It can ensure that the compound effectively penetrates the skin to reach the target site, improve the whitening effect, and is expected to overcome the shortcomings of existing inhibitors in terms of inhibitory effect, safety, transdermal property, stability, and ease of use, and provide a better raw material selection for the research and development of whitening products, and achieve a more efficient and safer skin whitening effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The present invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative work.
[0019] Figure 1 is a chemical structure diagram of rhodanine-3-propionic acid and cytidine-5'-phosphate according to the present invention;
[0020] Figure 2 is the enzyme activity curve of rhodanine-3-propionic acid in Example 1;
[0021] Figure 3 is the enzyme activity curve of cytidine-5'-phosphate in Example 1;
[0022] Figure 4 is the cumulative permeation curve of rhodanine-3-propionic acid in Example 1;
[0023] Figure 5 is the transdermal flux curve of rhodanine-3-propionic acid in Example 1;
[0024] Figure 6 is the cumulative permeation curve of cytidine-5'-phosphate in Example 1;
[0025] Figure 7 This is the transdermal flux curve of cytidine-5'-phosphate in Example 1. DETAILED DESCRIPTION
[0026] The present invention is further described in conjunction with the following examples.
[0027] Example 1
[0028] The embodiment of the present invention relates to a tyrosinase inhibitor, which includes rhodanine-3-propionic acid or its cosmetically or pharmaceutically acceptable salt, and / or cytidine-5'-phosphate or its cosmetically or pharmaceutically acceptable salt, and the tyrosinase inhibitor has significant tyrosinase inhibitory activity and good transdermal performance. This embodiment takes rhodanine-3-propionic acid and cytidine-5'-phosphate as examples to experimentally determine their in vitro tyrosinase inhibitory activity and transdermal performance.
[0029] 1. In vitro tyrosinase inhibitory activity
[0030] (1) Experimental Materials
[0031] Tyrosinase lyophilized powder was from mushrooms, Rhodanine-3-propionic acid, Cytidine 5'-(dihydrogenphosphate), and Hydroquinone (positive control) were all analytically pure with a purity of ≥98%, phosphate buffer (PBS, pH=6.8), and DMSO (used to dissolve the compounds).
[0032] (2) Reaction system configuration:
[0033] Rhodanine-3-propionic acid: prepared at concentrations of 10mM, 5mM, 2mM, 1mM, 0.5mM, and 0.1mM.
[0034] Cytidine 5'-phosphate: prepared at concentrations of 20mM, 10mM, 5mM, 2mM, and 1mM.
[0035] Hydroquinone: prepared at concentrations of 20mM, 10mM, 5mM, 2mM, and 1mM.
[0036] The tyrosinase freeze-dried powder was dissolved in PBS at pH 6.8 to prepare an enzyme solution of 1000 U / mL.
[0037] L-DOPA was dissolved in PBS at pH 6.8 to prepare a 1 mM substrate solution.
[0038] (3) Experimental operation:
[0039] In a 96-well plate, 50 μL of pH 6.8 PBS buffer, 40 μL of different concentrations of compound (containing 10% DMSO) solution and 10 μL of mushroom tyrosinase solution (1000 U / mL, dissolved in pH 6.8 PBS) were added in sequence, and incubated at 25°C for 10 min to allow the compound to fully bind to tyrosinase. 100 μL of L-DOPA solution (1 mM, dissolved in pH 6.8 PBS) was added to each well, and the reaction was started by incubating for another 10 min.
[0040] The blank control group was treated with 40 μL of pH 6.8 PBS buffer instead of the compound solution.
[0041] (4) Result detection:
[0042] The absorbance (OD) value of each well was measured at a wavelength of 490 nm using a MultiskanTM FC microplate reader. The OD at 0 min and 20 min was recorded respectively. 490nm Kojic acid, hydroquinone and arbutin were used as positive control reagents.
[0043] The tyrosinase inhibition rate was calculated as follows:
[0044] %inhibition=[(AB)-(CD)] / (AB)×100
[0045] Where A is the OD of the blank control group at 20 minutes 490nm value; B is the OD of the blank control group at 0 min 490nm Value; C is the OD of the compound group at 20 min 490nm Value; D is the OD of the test compound group at 0 min 490nm The experiment was repeated 3 times and the average value was taken to evaluate the enzyme activity of each concentration of the compound on tyrosinase. The enzyme activity curve was drawn in Graphpad Prism 10 and the IC value of the compound was calculated. 50 value.
[0046] (5) Experimental results
[0047] The enzyme activity curve of rhodanine-3-propionate and cytidine-5'-phosphate is as follows Figure 2-3 As shown, the IC of rhodanine-3-propionic acid can be obtained. 50 The value was 0.7349 mM, with a 95% CI of 0.6450 mM to 0.8230 mM, and the IC of cytidine-5'-phosphate 50 The value was 6.289mM, and the 95% CI was 6.042mM to 6.549mM. Taking hydroquinone as the positive control, its IC 50 The value was 10.15 mM, with a 95% CI of 8.023 mM to 13.06 mM.
[0048] 2. Transdermal performance
[0049] (1) Experimental Materials
[0050] A Franz diffusion cell was selected with an exposed area diameter of 1 cm and an effective diffusion area of approximately 0.785 cm 2 , volume is 15mL.
[0051] Obtain Panamanian suckling pig skin for the experiment, remove subcutaneous fat, rinse repeatedly with physiological saline until the rinse fluid is clear and free of impurities, absorb surface moisture with filter paper, and cut into a size suitable for Franz diffusion cell.
[0052] Rhodanine-3-propionic acid and cytidine-5'-phosphate were dissolved in sterile saline containing 30% ethanol to prepare a 10 mM solution. Sterile saline containing 30% ethanol was used as the receiving solution.
[0053] (2) Experimental operation
[0054] Place the diffusion cell in a constant temperature water bath at 32°C and stir at 600r / min using a magnetic stirrer. Pipette 400μL of 10mM rhodanine-3-propionic acid solution or cytidine-5'-phosphate solution into the sample pool and cover with a layer of plastic wrap to prevent the solution from evaporating. At 30min, 1h, 2h, 3h, 4h, 8h and 24h, take out 500μL of receiving solution from the receiving pool and immediately add an equal amount of sterile saline containing 30% ethanol to keep the volume of the receiving solution constant.
[0055] (3) Result detection
[0056] Preparation and detection of standard products: Prepare standard solutions of rhodanine-3-propionic acid and cytidine-5'-phosphate respectively, with concentrations of 10mM, 1mM, 0.1mM, 0.01mM, and 0.001mM. Use an ultraviolet-visible spectrophotometer (uv vis) to scan the standard solutions of each concentration and find the maximum absorption wavelength λ within 200nm to 1000nm. max . With absorbance as the ordinate and concentration as the abscissa, a standard curve was established. The sample of the receiving solution taken out each time was measured by UV-visible spectrophotometer at λ max The absorbance was measured at a wavelength (260 nm), and the concentrations of rhodanine-3-propionic acid and cytidine-5'-phosphate in the receiving solution were calculated according to the regression equation of the standard curve. The experiment was repeated 3 times and the average value was taken to improve the reliability and accuracy of the experimental results.
[0057] (4) Data calculation and analysis
[0058] According to the concentration of the compound in the receiving solution, the volume of the receiving solution and the effective diffusion area of the diffusion cell, the cumulative permeation amount (μmol / cm 2 ) and transdermal flux (μmol / cm 2 h).
[0059] Calculate the maximum absorption rate J max :By analyzing the transdermal flux data at different time points, find the maximum value of transdermal flux, i.e. J max .
[0060] Calculate the total permeability A total The cumulative permeation at the end of the experiment (24 h) is taken as the total permeation A total .
[0061] Calculation of skin retention: Calculate the skin retention based on the total amount of compounds initially added to the supply pool and the cumulative permeation at different time points. The calculation formula is skin retention = total amount of compounds initially added to the supply pool - cumulative permeation.
[0062] Calculate the 1h transdermal absorption rate, 8h transdermal absorption rate, and 24h transdermal absorption rate: calculate based on the ratio of the cumulative permeation amount in 1h, 8h, and 24h to the total amount of compound initially added to the supply pool. The formula is Absorption rate (%) = (cumulative permeation amount at the corresponding time / total amount of compound initially added to the supply pool) × 100%.
[0063] Calculation of cumulative permeability and retention rate: The cumulative permeability is calculated in the same way as the absorption rate at each time point; the retention rate formula is retention rate (%) = (skin retention amount / total amount of compound initially added to the supply pool) × 100%.
[0064] With time as the horizontal axis, the cumulative permeation (μmol / cm 2 ) as the ordinate, and a curve is drawn to show the cumulative permeation of the compound at different times. 2 h) is the vertical axis, and a curve is drawn to show the change trend of transdermal flux over time.
[0065] (5) Experimental results
[0066] The results of the transdermal experiments of rhodanine-3-propionic acid and cytidine-5'-phosphate are shown in Table 1. The cumulative permeation curve and transdermal flux curve are shown in Figure 4-7 shown.
[0067] Table 1 Transdermal test results of rhodanine-3-propionic acid and cytidine-5'-phosphate
[0068] index Rhodanine-3-propionic acid Cytidine-5'-phosphate <![CDATA[Maximum absorption rate J max (μmol / cm 2 ·h)]]> 0.303±0.005 0.473±0.042 <![CDATA[Skin retention amount (μmol / cm 2 )]]> 1.270±0.074 1.046±0.161 1h transdermal absorption rate (%) 0.044±0.014 0.323±0.045 8h transdermal absorption rate (%) 0.920±0.054 1.780±0.197 24h transdermal absorption rate (%) 2.309±0.229 2.309±0.229 Cumulative transmittance (%) 29.92％±1.49％ 45.34％±4.50％ Retention rate (%) 24.94％±1.44％ 20.54％±3.15％
[0069] In comparison, the 24-hour cumulative transdermal permeation rate of α-arbutin (5% commercial cream) was 12.66%, that of rhodanine-3-propionic acid was 29.92%, which was 2.36 times that of arbutin, and that of cytidine-5'-phosphate was 45.34%, which was 3.58 times that of arbutin. It is proved that the compound can penetrate the skin barrier more efficiently and can be used as a preferred ingredient in transdermal delivery systems, exceeding the active ingredients in traditional whitening products. The above data refer to: Aung, NN, Ngawhirunpat, T., Rojanarata, T., Patrojanasophon, P., Pamornpathomkul, B., & Opanasopit, P. (2020). Fabrication, characterization and comparison of α-arbutin loaded dissolving and hydrogelforming microneedles. International journal of pharmaceutics, 2020, 586, 119508. DOI: 10.1016 / j.ijpharm.2020.119508.
[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.
Claims
1. A tyrosinase inhibitor, characterized in that The invention comprises compound A and / or compound B, wherein compound A is rhodanine-3-propionic acid or a cosmetically acceptable salt thereof; and compound B is cytidine-5'-phosphate or a cosmetically or pharmaceutically acceptable salt thereof.
2. Use of the tyrosinase inhibitor according to claim 1 in the preparation of a product for inhibiting tyrosinase activity.
3. The use according to claim 2, characterized in that: Used for preparing drugs for preventing and / or treating human pigmentation diseases and melanoma caused by abnormal melanin.
4. The use according to claim 2, characterized in that: Application in preparing external skin preparations for whitening.
5. A cosmetic composition or a pharmaceutical composition, characterized in that: The composition comprises a cosmetically or pharmaceutically effective amount of the tyrosinase inhibitor according to claim 1.
6. The composition according to claim 5, characterized in that The dosage form of the composition is an anhydrous composition, an aqueous dispersion, a solution, a cream, a gel, a film, a mist, a pellet or a foam.
7. The composition according to claim 5, characterized in that The composition comprises at least one cosmetically or pharmaceutically acceptable excipient or adjuvant.
8. The composition according to claim 7, characterized in that The adjuvant is one or more of other agents that inhibit tyrosinase activity, moisturizers, emulsifiers, preservatives, antioxidants, emollients, wetting agents, thickeners, surfactants, antibacterial agents, fragrances, and inorganic salts.