A small molecule peptide from hymenoptera and application thereof

Abstract

This invention discloses a small molecule peptide derived from *Hirudo medicinalis*, with the amino acid sequence Leu-Leu-Phe-Arg. The peptide was obtained by enzymatically hydrolyzing the waste protein residue after purifying hirudin from *Hirudo medicinalis*, combined with tyrosinase-functionalized magnetic bead affinity fishing technology and mass spectrometry identification. In vitro kinetics and molecular dynamics simulations confirmed that the peptide LLFR significantly prolongs the lag time of monophenolase reactions. It penetrates the catalytic pocket of tyrosinase through an "induced fit" mechanism, forming a stable dead-end complex with the enzyme and inducing the unfolding of the enzyme's tertiary structure. Cell experiments confirmed that LLFR significantly inhibits tyrosinase activity and melanin synthesis in melanoma cells without significant cytotoxicity. This peptide exhibits high safety and good water solubility, providing a new approach for the high-value utilization of industrial waste from *Hirudo medicinalis*, and is suitable for the industrial production of whitening cosmetics and functional products.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, cosmetic raw materials and comprehensive resource utilization, specifically to a small molecule active peptide isolated from the waste residue after purifying hirudin from leeches, and the application of this small molecule peptide in the preparation of melanin-inhibiting agents. Background Technology

[0002] In the process of melanin production, tyrosinase (EC 1.14.18.1) is a binuclear copper ion-containing metalloenzyme and a key rate-limiting enzyme in the melanin biosynthesis pathway. It primarily catalyzes two key reactions: the hydroxylation of L-tyrosine to L-DOPA, and the further oxidation of L-DOPA to dopaquinone, ultimately generating melanin through a series of complex polymerization reactions. While melanin provides photoprotection against ultraviolet radiation, excessive deposition can lead to pigmentary skin conditions such as melasma, freckles, and age spots. It is also a major cause of adverse enzymatic browning in fruits, vegetables, and other foods.

[0003] Currently, while common commercially available traditional chemical tyrosinase inhibitors (such as hydroquinone, kojic acid, and arbutin) have some therapeutic effects, they generally suffer from chemical instability, insufficient skin penetration, and are prone to cytotoxicity and skin irritation with long-term use. This greatly limits their widespread and long-term application. In recent years, bioactive peptides derived from natural proteins have become a research hotspot in the field of anti-melaninization due to their mild action conditions, good biocompatibility, and high safety.

[0004] Hirudo medicinalis ( Poecilobdella manillensis Hirudo medicinalis (also known as leech) is an important medicinal natural leech in my country, widely used industrially to extract hirudin, a potent anticoagulant. However, large-scale extraction of hirudin generates a large amount of protein-rich processing residue. Currently, these byproducts are mostly discarded or treated as low-value animal feed, resulting in a significant waste of traditional Chinese medicine resources and potential environmental pollution. Exploring an efficient screening strategy to extract novel, highly active tyrosinase inhibitory peptides from Hirudo medicinalis waste residue could provide safe and effective new raw materials for the development of skin whitening and anti-browning agents, and also has significant economic and social value in promoting the extension of the traditional Chinese medicine industry chain in my country. Summary of the Invention

[0005] This invention provides a small molecule peptide isolated from the residue after extracting hirudin from Hirudin apiacea. The amino acid sequence of the small molecule peptide is Leu-Leu-Phe-Arg (LLFR). The small molecule peptide of this invention has tyrosinase inhibitory activity and can inhibit melanin synthesis. Therefore, this small molecule peptide can be used in the preparation of melanin production inhibitors.

[0006] This invention extracts protein from the residue after extracting hirudin from *Hirudo medicinalis*, followed by enzymatic hydrolysis. Activity evaluation showed that the pepsin hydrolysate exhibited excellent in vitro tyrosinase inhibitory activity and copper ion chelating ability. Subsequently, this invention constructed tyrosinase-functionalized magnetic beads using the EDC / NHS cross-linking method, which served as affinity probes to specifically "fish out" high-affinity ligands from the hydrolysate. Through high-resolution mass spectrometry identification and sequencing, combined with molecular docking virtual screening, a small peptide with the amino acid sequence Leu-Leu-Phe-Arg was ultimately identified as an inhibitor with extremely high binding potential.

[0007] The melanin production inhibitor of this invention has a peptide LLFR as its component (or active ingredient). It may also contain one or more excipients acceptable to drugs or cosmetics (skincare products) to improve the absorption effect of the drug or cosmetic (skincare product) or facilitate its use, and to make it into a suitable dosage form, such as capsules or pills, powders, tablets, granules, oral liquids, liquids, etc.

[0008] Compared with the prior art, the present invention has the following significant advantages: 1. Natural source and high safety: The small molecule peptide LLFR of this invention is derived from natural protein. Cytotoxicity experiments show that it has no significant cytotoxicity to melanoma cells within a wide effective concentration range, and has excellent biosafety. 2. Extremely strong tyrosinase inhibitory activity: In vitro experiments show that the small molecule peptide LLFR is a mixed inhibitor that can preferentially occupy the catalytic pocket of free enzymes in a competitive mode; it can significantly prolong the lag time of monophenolase reaction. Molecular dynamics simulations reveal that it induces enzyme conformational changes and forms a stable inhibitory state through an "induced fit" mechanism, using specific amino acid residues as "electrostatic anchors". 3. Excellent intracellular melanin inhibition effect: In cell models, the small molecule peptide LLFR can significantly inhibit intracellular tyrosinase activity and greatly reduce intracellular melanin accumulation, and its macroscopic depigmentation inhibition effect is better than that of the positive control kojic acid at the same concentration. 4. Easy to industrialize and transdermal absorb: The small molecule peptide LLFR consists of only 4 amino acids, with a very small molecular weight, simple structure, and good water solubility; it has excellent transmembrane transdermal absorption potential in cosmetics or topical drugs; at the same time, it is very easy to prepare on a large scale through solid-phase synthesis, and has broad commercial prospects. 5. Green and environmentally friendly: This invention provides a new way to comprehensively utilize industrial waste from leeches with high added value, and has significant economic and ecological benefits. Attached Figure Description

[0009] Figure 1The figure shows the results of in vitro tyrosinase inhibitory activity assays of five different protease hydrolysates from Hirudo medicinalis residue; Figure 2 The graph shows the results of copper ion chelating ability detection of five different protease hydrolysis products of Hirudo medicinalis residue; Figure 3 The images show scanning electron microscopy (SEM) characterization of tyrosinase-immobilized magnetic beads, where Image A shows the cross-linked tyrosinase-immobilized magnetic beads and Image B shows the blank magnetic beads before cross-linking. Figure 4 UPLC-MS / MS total ion chromatogram of ligands eluted after affinity fishing; Figure 5 The graph shows the in vitro tyrosinase inhibitory activity of the small molecule peptide LLFR at extremely low concentrations. Figure 6 A 3D visualization model of the docking of the small molecule peptide LLFR with the catalytic pocket of tyrosinase; Figure 7 The intrinsic fluorescence quenching spectrum of tyrosinase titrated with the small molecule peptide LLFR; Figure 8 Lineweaver-Burk double reciprocal plot of the small molecule peptide LLFR inhibiting diphenolase activity; Figure 9 The root mean square deviation (RMSD) plot of the LLFR-tyrosinase complex in a 100 ns molecular dynamics simulation. Figure 10 Figure showing the effect of the small molecule peptide LLFR on the viability of B16-F10 melanoma cells (CCK-8 assay). Figure 11 The figure shows the inhibition of tyrosinase activity in B16-F10 melanoma cells by the small molecule peptide LLFR. Figure 12 Comparison of macroscopic color changes in B16-F10 cell pellets after treatment with different concentrations of the small molecule peptide LLFR; Figure 13 The figure shows the inhibition of total melanin content in B16-F10 melanoma cells by the small molecule peptide LLFR. Detailed Implementation

[0010] The present invention will be further described in detail below with reference to the embodiments. However, the content described should not be regarded as a limitation of the present invention. Unless otherwise specified, the methods in this embodiment are operated in accordance with conventional methods, and the reagents used are conventional reagents or reagents prepared in accordance with conventional methods unless otherwise specified.

[0011] Example 1: Preparation of LLFR, a small molecule peptide derived from leeches. 1. Preparation and activity evaluation of enzymatic hydrolysate The industrial residue (PMR) of Hirudin extracted from leeches was pulverized and dispersed in distilled water at a ratio of 1:20 (g:mL). Hydrolysis was then performed using five proteases: pepsin (pH 2.0, 37℃), alkaline protease (pH 8.5, 50℃), trypsin (pH 8.0, 37℃), neutral protease (pH 7.0, 50℃), and papain (pH 6.0, 50℃), with an enzyme addition of 2500 U / g for each. After hydrolysis for 4 hours, the enzymes were inactivated by boiling in a water bath for 10 minutes. The supernatant was collected by centrifugation and then freeze-dried. The lyophilized powders of the above-obtained enzymatic hydrolysates were prepared into liquids with concentrations of 0.62–10 mg / mL using water. The in vitro tyrosinase inhibitory activity and copper ion chelating capacity of the hydrolysates were then determined.

[0012] (1) In vitro tyrosinase inhibitory activity assay (DOPA oxidation method): 80 μL of the above-prepared sample solution was mixed with 40 μL of tyrosinase solution (200 U / mL, dissolved in pH 6.8 phosphate buffer) and incubated at 25°C in the dark for 10 min. Then, 160 μL of L-DOPA substrate solution (2 mmol / L) was added to start the reaction. After incubation for another 15 min, the absorbance was measured at 475 nm using a microplate reader, and the inhibition rate was calculated.

[0013] (2) Determination of copper ion chelating ability (catechol violet colorimetric method): Take 10 μL of the above-prepared sample solution and mix it with 280 μL of sodium acetate buffer (50 mM, pH 6.0), then add 10 μL of CuSO4 solution (1 mg / mL) and 6 μL of catechol violet (PV) indicator solution (4 mM) sequentially. After mixing, react at room temperature for 6 min, measure the absorbance at 632 nm, and calculate the chelation rate.

[0014] See results Figure 1 , Figure 2 As shown in the figure, at a concentration of 10 mg / mL, the pepsin hydrolysate (PMR-e) exhibited the highest tyrosinase inhibition rate (77.2%) and the strongest copper ion chelating ability (70.02%), which was significantly better than other enzymatic hydrolysates (p<0.05). Its excellent dual activity may be attributed to the fact that pepsin tends to cleave at hydrophobic aromatic amino acids, thereby enriching short peptides containing targeted enzyme inhibitory pharmacophores. Therefore, PMR-Pe was selected as the substrate for subsequent affinity screening.

[0015] 2. Construction of Tyrosinase-Functionalized Magnetic Beads and Ligand "Fishing" Commercially available carboxylated magnetic beads were used, and their surfaces were activated using EDC and NHS (molar ratio 1:1). Subsequently, they were covalently cross-linked by incubation overnight at 4°C with mushroom tyrosinase PBS solution (0.1 M, pH 6.8). Electron microscopy characterization was performed. Figure 3 Figure A shows the cross-linked functionalized magnetic beads, and Figure B shows the blank magnetic beads before cross-linking. This confirms that the surface of the magnetic beads became rougher and protein aggregates appeared after coupling. A significant shift occurred in the Amide I region in the FT-IR spectrum, confirming that the enzyme was successfully loaded and maintained structural integrity. The above PMR-Pe hydrolysate (10 mg / mL) and functionalized magnetic beads were incubated at 4 °C for 60 min. After thoroughly washing with PBS containing 0.05% Tween-20 to remove impurities, the specifically bound ligands were eluted with 0.1 M Glycine-HCl buffer (pH 2.2) and immediately neutralized with 1.0 M Tris-HCl (pH 8.0).

[0016] 3. UPLC-MS / MS sequencing and molecular docking screening After desalting, the eluent was analyzed by UPLC-MS / MS high-resolution mass spectrometry. de novo Sequencing and the Hirudinea database were used to obtain highly specific total ion chromatograms. Figure 4 Twelve candidate short peptides were successfully identified. Further, the sequences were molecularly docked with mushroom tyrosinase (PDB: 2Y9X) using AutoDockVina. Considering binding energy, molecular weight, and physicochemical properties, the tetrapeptide Leu-Leu-Phe-Arg (LLFR), with an extremely strong theoretical binding energy (-7.2 kcal / mol) and a very small molecular weight (547.71 Da), was ultimately selected as the core lead compound.

[0017] Example 2: Artificial Synthesis and In Vitro High Activity Verification of the Small Molecule Peptide LLFR Based on the sequence identified in Example 1, the polypeptide LLFR (purity >95%) was synthesized using the standard Fmoc solid-phase polypeptide synthesis method. Different concentrations of the synthesized peptide were mixed with tyrosinase (200 U / mL) and incubated for 10 min, followed by the addition of substrate L-DOPA (2 mM) and reaction for 15 min. The absorbance at 475 nm was then measured.

[0018] Figure 5 The results showed that peptide LLFR exhibited excellent tyrosinase inhibitory activity, achieving complete blockade of tyrosinase activity (inhibition rate close to 100%) at an extremely low concentration of only 0.125 mg / mL.

[0019] Three-dimensional model of molecular docking between small molecule peptide LLFR and tyrosinase Figure 6The results show that the peptide LLFR can be tightly and deeply embedded in the narrow hydrophobic catalytic channel of tyrosinase. Among them, the C-terminal arginine (Arg4) plays a key role as an "electrostatic anchor," forming a dense network of hydrogen bonds and salt bridges with the negatively charged residues near the binuclear copper center; at the same time, the N-terminal leucine (Leu) and phenylalanine (Phe) provide strong hydrophobic stacking (π-π stacking), and this precise "dual anchoring" completely seals off the catalytic pocket.

[0020] Example 3: Elucidation of the kinetic mechanism by which the small molecule peptide LLFR inhibits tyrosinase 1. Induction of enzyme tertiary structure unfolding (fluorescence spectroscopy analysis) The intrinsic fluorescence spectra of tyrosinase were recorded using a fluorescence spectrophotometer (Hitachi F-7000). First, tyrosinase was dissolved in phosphate buffer (0.1 M, pH 6.8) to prepare a tyrosinase solution with a concentration of 0.2 mg / mL. Then, different concentrations of LLFR peptide solution were titrated dropwise into this tyrosinase solution to achieve final LLFR concentrations of 0, 7.0, 10.5, 17.2, 23.8, and 40.0 μg / mL, respectively. After thorough mixing, the excitation wavelength was set to 280 nm, and the emission spectra of each sample before and after drug addition were scanned and recorded in the range of 300 nm to 500 nm.

[0021] Figure 7 The results showed that as the concentration of peptide LLFR increased from 0 to 40.0 μg / mL, the intrinsic fluorescence intensity of tyrosinase exhibited a significant concentration-dependent quenching (Stern-Volmer constant Ksv = 1.04 × 10⁻⁶). 3 ·M -1 Crucially, its maximum emission wavelength redshifted from 358.8 nm to 360.8 nm (a 2.0 nm redshift). This indicates that the strong binding of the peptide LLFR leads to the partial exposure of the hydrophobic core containing tryptophan residues within the enzyme to the polar solvent, causing significant unfolding of the enzyme's tertiary structure.

[0022] 2. Significantly prolongs the lag time of monophenolase reactions. Tyrosinase catalysis involves monophenol hydroxylation and diphenol oxidation, with the lag phase of monophenolase activity being the rate-limiting step in melanin production. Different concentrations of LLFR peptide solutions were mixed with tyrosinase solution (200 U / mL, dissolved in 0.1 M, pH 6.8 phosphate buffer) to achieve final LLFR concentrations of 0, 2.5, 5.0, 10.0, and 20.0 μg / mL. The mixture was pre-incubated at 37°C in the dark for 10 min. Subsequently, the monophenol substrate L-tyrosine solution was added to initiate the reaction. The dynamic changes in absorbance at 475 nm were continuously monitored using a microplate reader. A kinetic curve of absorbance versus time was plotted, and the time span before the absorbance value began to rise significantly linearly from a plateau was recorded as the lag phase of the monophenolase reaction.

[0023] Experiments showed that the lag time was approximately 7.0 min without inhibitors; however, in the presence of peptide LLFR (20 μg / mL), this lag time was significantly prolonged to 13.5 min. This indicates that LLFR severely restricts the structural rearrangement of the enzyme from the resting state to the reactive oxygen species state, thus inhibiting melanin synthesis from the initial stage.

[0024] 3. Competitive-driven hybrid inhibition mode Using L-DOPA as a diphenolase substrate, the inhibitory type of LLFR peptide on tyrosinase was investigated. Different final concentrations (0, 0.1, 0.2, 0.4 mg / mL) of LLFR peptide solution were added to 96-well plates and thoroughly mixed with tyrosinase solution (final concentration 200 U / mL, dissolved in 0.1 M phosphate buffer at pH 6.8). After pre-incubation at 25°C in the dark for 10 min, different final concentrations (0.125, 0.25, 0.50, 1.00 mg / mL) of L-DOPA substrate solution were added to each group to initiate the reaction, and the volume was brought up with phosphate buffer. The 96-well plates were quickly placed in a microplate reader, and the linear change in absorbance at 475 nm over time was continuously monitored (the slope of the change was measured in the first 10 min) to calculate the initial reaction rate (V, ΔA475 / min). A Lineweaver-Burk double reciprocal kinetic plot was plotted with the reciprocal of different substrate concentrations ([S]) (1 / [S]) on the x-axis and the reciprocal of the initial reaction rate (V) (1 / V) on the y-axis. (Lineweaver-Burk double reciprocal plot) Figure 8The regression lines intersected precisely in the second quadrant, confirming that LLFR is a "mixed inhibitor." Secondary plotting calculations revealed that the inhibition constant (Ki) of LLFR against the free enzyme was 0.15 mg / mL, while the dissociation constant (Kis) of the enzyme-substrate complex was as high as 0.80 mg / mL. Since Ki is significantly smaller than Ki (more than a 5-fold difference), this indicates that LLFR primarily functions by preferentially occupying the catalytic pocket of the free enzyme (competitive binding).

[0025] Example 4: In-depth analysis of the molecular dynamics (MD) simulation of the LLFR “induced fit” mechanism of peptide (1) Construction of the reaction system: Molecular dynamics simulation analysis was performed using GROMACS software and the CHARMM36m all-atom molecular force field. The optimal binding conformation of LLFR-tyrosinase obtained by molecular docking screening was used as the initial coordinates. The complex was solvated using the TIP3P water molecule model and placed in a cubic water box with a boundary distance of at least 1.0 nm from the protein surface. Subsequently, an appropriate amount of Na was randomly added to the system. + and Cl - The ions not only neutralize the net charge of the system, but also bring it to a physiological salt concentration of 0.15M, thereby completing the construction of the complete MD reaction system.

[0026] (2) Simulation Experiment Parameters and Procedures: First, the steepest descent method was used to minimize energy in 50,000 steps to eliminate unreasonable steric hindrance and van der Waals overlap within the system. Subsequently, positional constraints were imposed on the heavy atoms of the protein, and ensemble equilibrium was performed sequentially for 100 ps NVT (using a V-rescale thermostat to keep the system temperature constant at 300 K) and ensemble equilibrium for 100 ps NPT (using a Parrinello-Rahman pressure regulator to keep the system pressure constant at 1 bar). After equilibrium was achieved, the positional constraints were removed, and molecular dynamics simulations of the final product were performed for 100 ns under physiological conditions of 300 K and 1 bar. The simulation step size was set to 2 fs, and the LINCS algorithm was used to constrain the lengths of all chemical bonds involving hydrogen atoms. The cutoff radii for short-range van der Waals forces and electrostatic interactions were both set to 1.2 nm, and long-range electrostatic interactions were calculated using the PME (Particle Mesh Ewald) algorithm.

[0027] (3) Trajectory data analysis: The trajectory data simulated in 100 ns were extracted, and the root mean square deviation (RMSD) and radius of gyration (Rg) were calculated using the built-in module of GROMACS. At the same time, the binding free energy between the ligand and receptor residues was calculated by combining the MM-PBSA method.

[0028] The root mean square deviation (RMSD) plot of the system ( Figure 9This revealed a significant dynamic process: the system underwent a sudden conformational change at approximately 60 ns, with the RMSD baseline jumping from ~0.15 nm and stabilizing at ~0.25 nm; the radius of gyration (Rg) also showed a slight expansion simultaneously. This time-resolved data provides conclusive physical evidence that the enzyme underwent a significant "induced-fit" structural remodeling to perfectly accommodate the large hydrophobic side chain of LLFR into the deep catalytic pocket. Further calculations using the MM-PBSA method using residue energy decomposition further elucidated that the core driving force maintaining the stability of this "induced" complex lies in electrostatic anchoring. The interaction energy between the ARG4 residue of the peptide LLFR and the enzyme remained stably at an astonishingly low level (below -130 kcal / mol). This large enthalpy change successfully overcame the entropic resistance caused by the enzyme's structural expansion, completely "locking" the complex into an energy minimum state where it could not catalyze the substrate.

[0029] Example 5: The melanin-inhibiting effect and excellent safety of the small molecule peptide LLFR 1. Cytotoxicity assessment Experimental Methods (CCK-8 assay): The CCK-8 assay was used to assess the biosafety of the small molecule peptide LLFR before its application to melanoma cells. B16-F10 melanoma cells in logarithmic growth phase were seeded into 96-well plates and cultured for 24 h to allow cell adhesion. The old culture medium was discarded, and fresh culture medium containing different concentrations (31.25, 62.5, 125, 250, 500, and 1000 μg / mL) of the LLFR peptide was added. A blank control group containing cells but without the drug was also included. The cells were incubated at 37°C in a 5% CO2 incubator for 48 h. After incubation, 10 μL of CCK-8 solution was added to each well, and incubation continued for 1–2 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0030] Results analysis: such as Figure 10 As shown, within a very wide concentration range of 31.25–1000 μg / mL, the survival rate of B16-F10 cells remained stable between 96% and 110%, with no statistically significant difference compared to the blank control group (p>0.05). This demonstrates that this natural tetrapeptide, at the concentration required to exert its whitening effect, perfectly avoids the melanocyte toxicity problem of traditional inhibitors such as hydroquinone, exhibiting extremely high safety.

[0031] 2. Effectively inhibits intracellular tyrosinase and melanin synthesis (1) Experimental methods and grouping B16-F10 melanoma cells in logarithmic growth phase were seeded into 6-well plates and cultured for 24 hours to allow cell adhesion. Then, the old culture medium was discarded, and the cells were divided into three groups (each group had replicates): ① Blank control group: fresh culture medium containing an equal volume of PBS was added; ② Positive control group: culture medium containing the commercially available skin whitening agent kojicacid was added, with a final concentration consistent with the high-dose peptide group (1.00 mg / mL); ③ Experimental groups: culture medium containing the small molecule peptide LLFR was added, with final concentrations of 0.25 mg / mL, 0.50 mg / mL, and 1.00 mg / mL, respectively. Cells in each group were incubated at 37°C and 5% CO2 for another 48 hours.

[0032] (2) Measurement of intracellular tyrosinase activity After incubation, cells in each group were washed with pre-cooled PBS, and lysis buffer containing 1% Triton X-100 was added. After lysis, the cells were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected as intracellular enzyme extraction buffer. 100 μL of the supernatant was placed in a 96-well plate, and 100 μL of L-DOPA solution (0.1%, dissolved in phosphate buffer at pH 6.8) was added. The plate was incubated at 37°C in the dark for 1 h. The absorbance was measured at 475 nm using a microplate reader, and the relative tyrosinase activity of each group was calculated.

[0033] like Figure 11 As shown, LLFR treatment exhibited a significant dose-dependent inhibitory effect. At a concentration of 1.00 mg / mL, intracellular tyrosinase activity was inhibited by 50.5%, which significantly surpassed the inhibitory effect of the positive control group kojic acid at the same concentration (inhibition rate of 40.5%).

[0034] (3) Determination of total melanin content in cells and observation of phenotype Cells were treated in the same group as described above. After 48 hours, cell slurries from each group were collected and centrifuged at 1000 rpm for 10 minutes to obtain cell pellets from each group.

[0035] Macroscopic phenotypic observation: The cell pellets collected by centrifugation from each group were directly photographed and recorded. Figure 12 It can be visually observed that as the concentration of LLFR increases, the phenotype of the cell population gradually fades from the black color of the blank control group to grayish-white, achieving almost complete decolorization. Quantitative determination of melanin content: 200 μL of 1 mol / L NaOH solution (containing 10% DMSO) was added to the above cell pellet, and the cells were incubated in an 80℃ water bath for 30 min to fully dissolve the intracellular melanin. Then, 200 μL of ultrapure water was added to each well and mixed thoroughly. The absorbance was measured at 450 nm using a microplate reader, and the relative total melanin content of each group was calculated. Quantitative results showed ( Figure 13), 1.00 mg / mL LLFR can significantly reduce total melanin accumulation by 45.0%, which is also superior to the inhibitory effect of kojic acid.

[0036] The above cellular effects fully demonstrate that LLFR, due to its extremely small molecular weight (only 547 Da), possesses superior transmembrane permeation potential, enabling it to efficiently penetrate cell membranes and melanosome membranes, and inhibit pigment production in situ, thus exhibiting enormous potential for functional cosmetic and pharmaceutical applications.

[0037] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural modifications made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A small molecule peptide, characterized in that: Derived from leeches ( Poecilobdella manillensis The amino acid sequence of the small peptide is Leu-Leu-Phe-Arg.

2. The use of the small molecule peptide according to claim 1 in the preparation of melanin production inhibitors.

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