Short-chain melanin inhibitory peptide and application
By developing short-chain melanin-inhibiting peptides L1 and L2, the problems of long amino acid sequences, high cost, and insufficient stability in existing technologies have been solved. This has achieved effective inhibition of tyrosinase and dopaminerase, with good melanin production inhibition effect and biocompatibility, making it suitable for skin whitening and pigment regulation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-22
- Publication Date
- 2026-04-14
AI Technical Summary
Existing melanin-inhibiting peptides suffer from problems such as long amino acid sequences, high synthesis costs, insufficient stability, or limited inhibitory effects, making it difficult to balance high efficiency and safety.
The short-chain melanin-inhibiting peptides Cys-Tyr-Phe-Leu-Pro (L1) and Tyr-Gly-Phe-Leu-Pro-Cys (L2) are used to inhibit melanin production by inhibiting tyrosinase activity. They are suitable for skin administration, with an IC50 of 0.01-0.1 mg/mL. They have good tyrosinase and dopaase inhibitory capabilities, short amino acid sequences, simple preparation process, low cost, and good biocompatibility.
It achieves effective inhibition of tyrosinase and dopaminergic enzymes, significantly inhibits melanin production, and has good stability and biocompatibility. It is suitable for preparing melanin-inhibiting agents and has broad application prospects.
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Figure CN121851104A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a novel short-chain melanin-inhibiting peptide and its applications. Background Technology
[0002] Melanin is a biological macromolecule synthesized by melanocytes under the catalysis of tyrosinase, playing a crucial role in the regulation of skin pigmentation. Under normal physiological conditions, melanin has photoprotective and antioxidant functions, but its excessive synthesis and abnormal deposition can lead to pigmentary skin problems. Most existing whitening agents achieve their anti-melanin effect by inhibiting tyrosinase activity, but they generally suffer from insufficient stability, irritation, or limited biosafety.
[0003] Melanin-inhibiting peptides are considered potential active ingredients for inhibiting melanin production due to their advantages such as small molecular weight, good biocompatibility, and low toxicity. However, some reported melanin-inhibiting peptides have long amino acid sequences, high synthesis costs, and issues with stability or cellular safety; while peptides with simpler structures have limited inhibitory effects, making it difficult to balance high efficiency and safety. Therefore, there is an urgent need to develop novel melanin-inhibiting peptides with short sequences, excellent inhibitory activity, and good biocompatibility to meet the application needs in the fields of skin whitening and pigmentation regulation. Summary of the Invention
[0004] The purpose of this invention is to provide a novel short-chain melanin-inhibiting peptide, which has advantages such as a short amino acid sequence and excellent melanin-inhibiting performance. It can be used to prepare melanin-inhibiting agents and exhibits good melanin-inhibiting effects.
[0005] To achieve the purpose of this invention, the following technical solution is adopted: A short-chain melanin-inhibiting peptide, wherein the amino acid sequence of the short-chain melanin-inhibiting peptide is selected from any of the following: (1) Cys-Tyr-Phe-Leu-Pro, hereinafter referred to as L1; (2) Tyr-Gly-Phe-Leu-pro-cys, hereinafter referred to as L2.
[0006] The short-chain melanin-inhibiting peptides include, but are not limited to, linear peptide chains, cyclic peptide chains, and structures with branches containing the sequence.
[0007] The application of the short-chain melanin-inhibiting peptide in the preparation of melanin inhibitors.
[0008] The application described inhibits melanin production by suppressing tyrosinase.
[0009] The application is characterized in that: the short-chain melanin-inhibiting peptide is suitable for inhibiting the production of melanin in melanocytes through skin administration.
[0010] The aforementioned application describes the IC50 of the melanin-inhibiting peptide L1 against tyrosinase monophenolase (L-tyrosine). 50 The IC50 of L2 against tyrosinase monophenolase (L-tyrosine) is 0.01-0.1 mg / mL. 50 The concentration is 0.01-0.1 mg / mL.
[0011] The aforementioned application describes the IC50 of the melanin-inhibiting peptide L1 against tyrosinase diphenolase (L-DOPA). 50 The IC50 values for L2-dopa at concentrations of 0.01-0.1 mg / mL were respectively for tyrosinase diphenolase (L-DOPA). 50 The concentrations were 0.01-0.1 mg / mL, respectively.
[0012] In the aforementioned application, the melanin-inhibiting peptides L1 and L2 inhibit intracellular melanin production by no less than 70%.
[0013] Compared with the prior art, the advantages of the present invention are as follows: (1) The melanin-inhibiting peptide prepared by the present invention has a short amino acid sequence, a simple preparation process, low cost, and good reproducibility; (2) The melanin-inhibiting peptide prepared by the present invention has good tyrosinase inhibition and can effectively inhibit L-tyrosine and L-DOPA, thereby successfully inhibiting melanin.
[0014] (3) The melanin-inhibiting peptide prepared by the present invention has good biocompatibility.
[0015] (4) The melanin-inhibiting peptide prepared by the present invention can be used to prepare melanin-inhibiting agents and has good application prospects. Attached Figure Description
[0016] Figure 1 The IC50 of the melanin-inhibiting peptide obtained in Example 1 for L-tyrosine is... 50 ; Figure 2 The melanin-inhibiting peptide obtained in Example 1 has an IC50 response to L-DOPA. 50 ; Figure 3 This is a stability test of the melanin-inhibiting peptide obtained in Example 1; Figure 4 This is a graph showing the cell survival rate of the melanin-inhibiting peptide obtained in Example 1; Figure 5 This is a cell liveness / death diagram of the melanin-inhibiting peptide obtained in Example 1; Figure 6 This is a graph showing the hemolysis rate of the melanin-inhibiting peptide obtained in Example 1; Figure 7This is a morphological image of erythrocytes containing the melanin-inhibiting peptide obtained in Example 1. Figure 8 This is a diagram showing the L-tyrosine inhibition morphology of the melanin-inhibiting peptide obtained in Example 1 within cells; Figure 9 This is a diagram showing the L-DOPA inhibition morphology of the melanin-inhibiting peptide obtained in Example 1 within cells; Figure 10 This is a diagram showing the melanin-inhibiting morphology of the melanin-inhibiting peptide obtained in Example 1 within cells; Figure 11 This is an experiment on the prevention and control of ultraviolet-induced melanin production in mammals using the melanin-inhibiting peptide obtained in Example 1. Detailed Implementation
[0017] The present invention will be described in detail below with reference to specific embodiments.
[0018] Example 1: Preparation of melanin-inhibiting peptides (1) In this embodiment, the short-chain melanin-inhibiting peptide L1 was prepared by the Fmoc solid-phase synthesis method. Its amino acid sequence is Cys-Tyr-Phe-Leu-Pro (CYFLP). The specific steps are as follows: 5-10 ml of DCM was used to swell dichlorotriphenylmethyl chloride resin for 90-120 min. The amount used was 0.3-1.0 g, preferably 0.5 g. 0.10-0.20 g, preferably 0.126 g of proline and 100-300 μL, preferably 200 μL of DIEA (N,N-diisopropylethylamine) were added and reacted for 1-3 h, preferably 2 h. 5-15 mL, preferably 10 mL of methanol and 100-300 μL, preferably 200 μL of DIEA were added to block the unreacted groups. The Fmoc protecting group was removed using 5-15 mL, preferably 10 mL, of a 20% piperidine solution. Then, leucine (0.30-0.50 g, preferably 0.394 g), PyBoP (0.4-0.8 g, preferably 0.6 g), HoBt (0.1-0.3 g, preferably 0.16 g), and DIEA (100-300 μL, preferably 200 μL) were added sequentially. The reaction was carried out for 1-3 h, preferably 2 h. The Fmoc protecting group was removed using 10 mL of a 15%-25%, preferably 20% piperidine solution. The above steps were repeated, replacing leucine with 0.432 g phenylalanine (0.30-0.50 g, preferably 0.432 g), 0.45 g tyrosine (0.30-0.60 g, preferably 0.45 g), and cysteine (0.50-0.80 g, preferably 0.652 g) sequentially. Finally, the peptides were cleaved using a cleavage agent (TFA: water: triisopropylsilane = 95:2.5:2.5), and 10-15 mL of ice-cold ether was added to precipitate them. The peptides were then lyophilized in a freeze dryer for later use.
[0019] (2) In this embodiment, the short-chain melanin-inhibiting peptide L2 was prepared using the same Fmoc solid-phase synthesis method as in Example (1). Its amino acid sequence is Tyr-Gly-Phe-Leu-Pro-Cys (YGFLPC). The difference lies in the order of amino acid introduction, as follows: 5-10 ml of DCM was used to swell dichlorotriphenylmethyl chloride resin for 90-120 min, with an amount of 0.3-1.0 g, preferably 0.5 g. 0.15-0.30 g, preferably 0.218 g, of cysteine and 100-300 μL, preferably 200 μL, of DIEA were added and reacted for 1-3 h, preferably 2 h. Methanol and DIEA were then added to block the unreacted groups. Fmoc protection was performed using a piperidine solution, followed by the sequential addition of proline (0.30-0.50 g, preferably 0.376 g), leucine (0.30-0.50 g, preferably 0.394 g), phenylalanine (0.30-0.50 g, preferably 0.432 g), glycine (0.25-0.45 g, preferably 0.332 g), and tyrosine (0.30-0.60 g, preferably 0.45 g). Each reaction was carried out in conjunction with PyBOP, HOBt, and DIEA, and the reaction time and deprotection conditions were the same as in Example (1). Finally, the peptide was cleaved using a cleaving agent (TFA:water:triisopropylsilane = 95:2.5:2.5), and 5-15 mL of ice-cold ether was added to precipitate the peptide. The peptide was then lyophilized in a freeze dryer for later use.
[0020] Melanin inhibition, peptide tyrosinase inhibition effect (1) Tyrosinase inhibition experiment with L-tyrosine as substrate IC50 of melanin-inhibiting peptide L-tyrosine 50 Before use, melanin-inhibiting peptide and arbutin (Arb) were prepared into solutions with distilled water at concentrations ranging from 0.01 to 0.5 mg / mL, preferably 0.01, 0.025, 0.05, 0.1, 0.2, and 0.4 mg / mL. L-tyrosine was used as the substrate. In the experimental group, the test solution was mixed with PBS. After 10 minutes of incubation, 200-300 U / mL, preferably 250 U / mL, TYR solution was added to initiate the reaction. The substrate-free system served as a blank control. The control group did not contain the test solution, while the background group omitted both the substrate and the test solution. The absorbance was quantified at 475 nm (0 and 30 minutes), and the IC50 was determined. 50 The results showed that the melanin-inhibiting peptide had a good melanin-inhibiting ability.
[0021] Experimental results show that the melanin-inhibiting peptide prepared in this embodiment has a significant inhibitory effect on tyrosinase reactions based on L-tyrosine, demonstrating good potential for inhibiting melanin production.
[0022] (2) Tyrosinase inhibition experiment with L-DOPA as substrate IC50 of melanin-inhibiting peptide L-DOPA 50 Using the same method as the L-tyrosine experiment described above, the melanin-inhibiting peptide and Arb prepared in this example were prepared into solutions of 0.005-0.5 mg / mL, preferably 0.01, 0.025, 0.05, 0.1, 0.2, and 0.4 mg / mL. L-DOPA was used as the reaction substrate, and the remaining reaction conditions were consistent with the L-tyrosine experiment. The absorbance was quantified at 475 nm at 0 min and 30 min of reaction time, and the IC50 was determined. 50 value.
[0023] Figure 1 , 2 The melanin-inhibiting peptide tyrosinase inhibition effect and IC50 obtained in Example 1 are shown. 50 , Figure 1 It is a melanin-inhibiting peptide that inhibits L-tyrosine IC 50 (A) L-tyrosine inhibition; (B) L1 inhibition of L-tyrosine IC50. 50 (C) L2 inhibits the IC50 of L-tyrosine. 50 (D) Arb inhibits the IC50 of L-tyrosine. 50 ; Figure 2 It is a melanin-inhibiting peptide that inhibits L-DOPA IC50. 50 (A) L-DOPA inhibition; (B) L1 inhibition of L-DOPA's IC50. 50 (C) L2 inhibits the IC50 of L-DOPA 50 (D) Arb inhibits the IC50 of L-DOPA. 50 The results showed that the melanin-inhibiting peptide prepared in this embodiment exhibited significant tyrosinase inhibitory ability in the L-DOPA substrate system, indicating that the peptide can act on both the monophenolase and diphenolase activity stages of tyrosinase and has good potential for inhibiting melanin production. UV stability of melanin-inhibiting peptides The melanin-inhibiting peptide prepared in this example was prepared as a solution of 0.5-2 mg / mL, preferably 1 mg / mL. It was irradiated with a UV lamp for 10-120 min, preferably 15, 30, 60, 90, and 120 min, respectively. The tyrosinase inhibition assay was then performed to detect the melanin-inhibiting peptide's UV stability. The results showed that it exhibited good UV stability.
[0024] pH stability of melanin-inhibiting peptides The melanin-inhibiting peptide prepared in this example was formulated into a solution of 0.5-2 mg / mL, preferably 1 mg / mL, and treated with various pH buffers (pH = 3-11). The inhibition of tyrosinase was assessed, and finally, the UV stability of the melanin-inhibiting peptide was tested using a tyrosinase inhibition assay. The results showed that the melanin-inhibiting peptide has a wider pH adaptability, making it more convenient for use in the production of cosmetics and pharmaceuticals.
[0025] Figure 3 This is a stability diagram of the melanin-inhibiting peptide obtained in Example 1. (A,B) Effect of UV irradiation on the stability of the melanin-inhibiting peptide; (C,D) Effect of pH on the stability of the melanin-inhibiting peptide. As can be seen from the figure, the melanin-inhibiting peptide has good stability. Melanin-inhibiting peptide cytotoxicity The melanin-inhibiting peptide prepared in this example was prepared as a solution of 0.5-3 mg / mL, preferably 1 mg / mL, and its cytotoxicity was detected by MTT assay and live / dead cell staining. (1) In this example, the cytotoxic effect of the peptide chain on L929 cells and B16F10 cells was determined by MTT assay. Cells were added to each well, and 150-200 μl of cell suspension, preferably 200 μl, was added to each well. The cells were cultured for 18-30 h, preferably 24 h. Then, 150-200 μl of melanin-inhibiting peptide, preferably 200 μl, normal culture medium, and ultrapure water were added to the plate. The cells were cultured for 18-30 h, preferably 24 h. MTT was then added to the plate, and the reaction was carried out in the dark for 4 h. DMSO was then added, and the absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader to indirectly reflect the cell viability. The results showed that the melanin-inhibiting peptide prepared in this example had no cytotoxicity and a high cell viability, indicating that it had good biocompatibility.
[0026] (2) In this example, the toxic effects of the peptide chain on L929 cells and B16F10 cells were determined using the live-dead cell staining method. Cells were added to confocal dishes, and 0.5-1 mg / mL (preferably 1 mL) of cell suspension was added to each well. The cells were cultured for 18-30 h, preferably 24 h. Then, 0.5-1 mg / mL (preferably 1 mL) of melanin-inhibiting peptide was added to the plates. After AM / PI staining, the cell viability was observed under a laser confocal microscope.
[0027] Figure 4 , 5 These are the cytotoxicity data for the melanin-inhibiting peptide obtained in Example 1. Figure 4 Cell survival rate data; Figure 5 The figure shows cell viability data for melanin-inhibiting peptides. As can be seen from the figure, the cell survival rate was good and there was no cytotoxicity during incubation with the melanin-inhibiting peptides. Melanin-inhibiting peptide hemolysis Before use, melanin-inhibiting peptide solutions with concentrations ranging from 0.1 to 5 mg / mL were prepared using distilled water, preferably 0.25, 0.5, 1, 2, and 4 mg / mL. The melanin-inhibiting peptide solutions of different concentrations were incubated with fresh blood at room temperature, centrifuged, and the supernatant was used to test the hemolysis rate using an ELISA reader. The morphology of red blood cells was observed under a scanning electron microscope.
[0028] Figure 6 , 7 These are the hemolytic data of the melanin-inhibiting peptide obtained in Example 1. Figure 6 Data on hemolysis rate; Figure 7 The image shows the morphology of red blood cells. As can be seen from the image, the hemolysis rate of this melanin-inhibiting peptide is low, the red blood cell morphology is good, and there is no hemolytic effect.
[0029] Intracellular tyrosinase inhibition effect of melanin-inhibiting peptides Mouse melanoma cells B16F10 were seeded in 96-well plates and incubated for 18-30 h, preferably 24 h, in RPMI 1640 medium containing different concentrations of melanin-inhibiting peptides or Arb. DMEM medium and substrate solution (L-tyrosine or L-DOPA) were added to each well, and the reaction was carried out at 37 °C for 2-6 h, preferably 4 h. After the reaction, unreacted substrate solution was removed, and intracellular melanin formation was observed using an inverted microscope.
[0030] For further quantitative analysis, B16F10 cells were treated with the melanin-inhibiting peptide or Arb described in this invention for 18-30 h, preferably 24 h, and then the cells were collected. After cell collection, the cells were lysed and centrifuged at 10,000-14,000 r / min at 4 °C, preferably 12,000 r / min, and the supernatant was collected for later use. The supernatant was mixed with L-tyrosine or L-DOPA substrate solution and reacted under specified conditions for 20-40 min, preferably 30 min, using ultrapure water as a blank control. The absorbance was measured at 475 nm to characterize the tyrosinase activity level.
[0031] Figure 8 , 9 This is a morphological diagram of intracellular tyrosinase inhibition of the melanin-inhibiting peptide obtained in Example 1. Figure 8 A morphological diagram of intracellular L-tyrosine inhibition; Figure 9 The image shows the intracellular L-tyrosine inhibition morphology. The results indicate that the B16F10 cells treated with the melanin-inhibiting peptide prepared in this example showed a significant reduction in intracellular tyrosinase activity, suggesting that the melanin-inhibiting peptide can effectively inhibit intracellular tyrosinase activity. Melanin-inhibiting peptides inhibit intracellular melanin production. Mouse melanoma cells B16F10 were seeded in six-well plates and cultured for 18-30 h. Experimental and control groups were then established. Cells in the experimental group were treated with RPMI 1640 medium containing the melanin-inhibiting peptide or Arb described in this invention, while cells in the control group were cultured in RPMI 1640 medium only. To induce melanin production, the melanin-inducing agent α-MSH was added to all the culture systems.
[0032] After the specified treatment time, cells were digested with trypsin and collected in centrifuge tubes. An alkaline solution was added to dissolve intracellular melanin; preferably, a 40 mg / mL sodium hydroxide solution was used. The reaction was carried out at 37 °C for 8–16 h. After the reaction, ultrapure water was used as a blank control, and the absorbance of the samples was measured at 475 nm to characterize the intracellular melanin content. Based on the changes in absorbance, the inhibitory effect of different samples on melanin production was calculated.
[0033] Figure 10 This is a morphological diagram of intracellular melanin inhibition of the melanin-inhibiting peptide obtained in Example 1. The experimental results show that, under α-MSH induction conditions, the melanin-inhibiting peptide prepared in this example can effectively reduce the melanin content in B16F10 cells, indicating that the melanin-inhibiting peptide has a good inhibitory effect on intracellular melanin production.
[0034] Melanin-inhibiting peptide mammalian UV protection model experiment Disinfected PBS, melanin-inhibiting peptide solution, and arbutin solution were used to treat ultraviolet-induced pigmentation in mice. The ultraviolet light used was preferably UVB, with an irradiation frequency of 2-4 times per week, preferably 3 times per week, and an irradiation dose of 80-120 mJ / cm², preferably 100 mJ / cm². Skin pigmentation in mice was observed 1-5 hours, preferably 3 hours, before each ultraviolet irradiation session, and the results were observed after three weeks.
[0035] Figure 11 This is an experiment on the prevention and control of ultraviolet-induced melanin in mammals using the melanin-inhibiting peptide obtained in Example 1. As shown in the figure, the pigmentation in the group with added melanin-inhibiting peptide was significantly reduced, indicating that the melanin-inhibiting peptide can inhibit melanin production and has a good melanin-inhibiting function.
[0036] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.
Claims
1. A short-chain melanin-inhibiting peptide, characterized in that, The amino acid sequence of the short-chain melanin-inhibiting peptide is selected from any of the following: (1) Cys-Tyr-Phe-Leu-Pro, hereinafter referred to as L1; (2) Tyr-Gly-Phe-Leu-pro-cys, hereinafter referred to as L2.
2. The short-chain melanin-inhibiting peptide as described in claim 1, characterized in that: This includes, but is not limited to, linear peptide chains, cyclic peptide chains, and structures in which the branched strands contain the sequence.
3. The use of the short-chain melanin-inhibiting peptide according to claim 1 or 2 in the preparation of melanin inhibitors.
4. The application according to claim 2, characterized in that: It inhibits melanin production by inhibiting tyrosinase.
5. The application according to claim 2, characterized in that: The short-chain melanin-inhibiting peptide is suitable for inhibiting melanin production in melanocytes via skin administration.
6. The application according to claim 2, characterized in that: The melanin-inhibiting peptide L1 has an IC50 effect on tyrosinase monophenolase (L-tyrosine). 50 The IC50 of L2 against tyrosinase monophenolase (L-tyrosine) is 0.01-0.1 mg / mL. 50 The concentration is 0.01-0.1 mg / mL.
7. The application according to claim 2, characterized in that: The melanin-inhibiting peptide L1 has an IC50 effect on tyrosinase diphenolase (L-DOPA). 50 The IC50 values for L2-L2 tyrosinase diphenolase (L-DOPA) were 0.01-0.1 mg / mL, respectively. 50 The concentrations were 0.01-0.1 mg / mL, respectively.
8. The application according to claim 2, characterized in that: The melanin-inhibiting peptides L1 and L2 inhibit intracellular melanin production by no less than 70%.