Amino whitening polypeptide and preparation method thereof

Abstract

The application belongs to the technical field of biological materials, and specifically discloses an amino acid whitening polypeptide and a preparation method thereof. The structural general formula of the polypeptide is as follows: R-A-B-NH2, wherein R is a modified acyl group serving as an N terminal and having a carbon number ranging from 6 to 10, A is D-proline and a derivative thereof, and B is D-arginine and a derivative thereof. The amino whitening polypeptide is prepared by using an Fmoc solid-phase synthesis method. The whitening polypeptide has excellent melanin synthesis inhibition activity, and plays a role by targeting inhibition of TRP-1. At a concentration of 1 μM, the melanin synthesis inhibition rate of B16 cells reaches a statistically significant standard, which is significantly better than a conventional whitening agent that can only achieve a similar inhibition effect at a high concentration, and greatly reduces the effective use concentration of a whitening product.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to an amino-based skin-whitening polypeptide and its preparation method. Background Technology

[0002] There are many whitening ingredients in existing technologies, which can be broadly categorized into plant extracts, vitamin derivatives, and traditional peptides. These whitening ingredients have the following problems:

[0003] Plant-derived skin whitening agents, such as arbutin, have limited mechanisms of action. They only work by reversibly inhibiting tyrosinase activity and have no regulatory effect on the TRP-1 / TRP-2 pathway. Furthermore, their chemical structure is unstable and hydrolyzes into hydroquinone in environments with pH > 6.0, exhibiting certain cytotoxicity. Moreover, their effectiveness depends on high concentrations, generally requiring concentrations greater than 100 μM to achieve a 15% inhibition rate.

[0004] Vitamin derivatives, such as nicotinamide, mainly work by inhibiting melanosome transfer, but do not directly inhibit melanin synthesis. They have low transdermal efficiency, with a LogP of -0.37 resulting in a stratum corneum retention rate of less than 10%. They also exhibit dose-dependent irritation.

[0005] Traditional enzymatic hydrolysis of peptides carries a high risk; L-type peptides have a half-life of less than 2 hours on the skin surface. Furthermore, a molecular weight barrier exists; generally, these peptides have a molecular weight greater than 500 Da, while their transdermal permeability is less than 1.0 × 10⁻⁶. -5 The concentration requirement is high because traditional peptide preparation methods result in products with low purity. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an amino-based skin-whitening polypeptide and its preparation method. The aim is to provide a short-chain polypeptide structure formed by two D-type amino acids, which has good targeting for inhibiting melanin synthesis, thereby reducing the required concentration. At the same time, it has good transdermal efficiency and anti-inflammatory effects, and the optimized preparation method achieves high purity.

[0007] The technical solution adopted in this invention is as follows:

[0008] In a first aspect, the present invention provides an amino-based skin-whitening polypeptide, the general structural formula of which is as follows:

[0009] RAB-NH2

[0010] Wherein, R is a modified acyl group at the N-terminus with a carbon number ranging from 6 to 10, A is D-proline and its derivatives, and B is D-arginine and its derivatives.

[0011] In conjunction with the first aspect, the present invention provides a first embodiment of the first aspect, wherein R is octanoyl.

[0012] In conjunction with the first aspect, the present invention provides a second embodiment of the first aspect, wherein the polypeptide has a molecular weight of 400-420.

[0013] In conjunction with the first aspect, the present invention provides a third embodiment of the first aspect, wherein the polypeptide has a molecular weight of 412-413.

[0014] In conjunction with the first aspect, the present invention provides a fourth embodiment of the first aspect, wherein A is 4-hydroxy-D-proline.

[0015] Secondly, the present invention provides a preparation method for preparing the amino-whitening polypeptide described in any of the above claims, which is prepared by Fmoc solid-phase synthesis.

[0016] In conjunction with the second aspect, the present invention provides a first embodiment of the second aspect, using triphenylmethyl resin as a carrier, first immobilizing D-arginine with a protecting group, then deprotecting and immobilizing D-proline with a protecting group, then performing N-terminal modification, and after immobilization, cutting and separating the polypeptide from the resin carrier, after preliminary purification by precipitation with ice-cold ether, and further purification by gradient elution by high performance liquid chromatography to obtain the whitening polypeptide product.

[0017] In conjunction with the second aspect, the present invention provides a second embodiment of the second aspect, wherein the purity of the further purified whitening polypeptide product is not less than 99%.

[0018] In conjunction with the second aspect, the present invention provides a third embodiment of the second aspect, wherein the cutting fluid used for cutting and separation comprises trifluoroacetic acid, water, triisopropylsilane and 1,2-ethylenedithiol in a volume ratio of 87.5:5:5:2.5.

[0019] In conjunction with the second aspect, the present invention provides a fourth embodiment of the second aspect, wherein the molecular weight of the purified whitening polypeptide product is 412.5.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) The whitening polypeptide provided by the present invention has excellent melanin synthesis inhibition activity. It works by targeting and inhibiting TRP-1. At a concentration of 1 μM, it can achieve a statistically significant level of inhibition of melanin synthesis in B16 cells. This is significantly better than the traditional whitening agents that require high concentrations to achieve similar inhibition effects, and greatly reduces the effective concentration of whitening products.

[0022] (2) This invention achieves efficient transdermal skin performance by optimizing the peptide structure. It breaks through the transdermal skin threshold by controlling the molecular weight of the peptide. At the same time, it modifies the N-terminus with an octanoyl group to adjust the lipid-water partition coefficient, which ensures that the peptide can effectively penetrate the stratum corneum to reach the target point of the epidermis, while avoiding the peptide from being retained in the stratum corneum, thus meeting the transdermal skin whitening needs of all skin areas.

[0023] (3) The present invention adopts a double D-amino acid structure design, which enables the whitening peptide to have excellent anti-degradation ability, resist the degradation effect of skin surface proteases, significantly prolong the half-life of the peptide on the skin surface, solve the problem of short half-life and easy inactivation of traditional L-type peptides on the skin surface, ensure that the peptide can continue to exert whitening effect and reduce the frequency of product use.

[0024] (4) The whitening polypeptide of the present invention has high purity and good biocompatibility. Through a specific preparation process, the HPLC purity of the polypeptide is not less than 99%, and the impurity content is extremely low, which can reduce the risk of skin irritation. At the same time, the polypeptide has no significant effect on the survival rate of beneficial skin bacteria lactobacillus, can be compatible with the skin microecological balance, and is suitable for the whitening needs of sensitive skin areas.

[0025] (5) The whitening polypeptide of the present invention has the synergistic effect of whitening and potential anti-inflammatory. Its targeted inhibition of TRP-1 can indirectly reduce the release of skin inflammatory factors. At the same time, the double D-amino acid structure reduces immunogenicity, avoids the irritation problem when using traditional whitening agents at high concentrations, and achieves the dual effect of "whitening + gentle care" to improve the product user experience. Attached Figure Description

[0026] Figure 1 This refers to the synthesis information of the amino-based whitening polypeptide product in the embodiments of this invention;

[0027] Figure 2 This refers to the HPLC information of the amino whitening polypeptide product in the embodiments of this invention;

[0028] Figure 3 This refers to the MS information of the amino whitening polypeptide product in the embodiments of this invention;

[0029] Figure 4 This is the solubility information of the amino whitening polypeptide product in the embodiments of the present invention;

[0030] Figure 5 This is a graph showing the results of the B16 melanocyte inhibition experiment between the amino whitening peptide sample and other test reagents in this embodiment of the invention.

[0031] Figure 6 This is a micrograph of the B16 melanocyte inhibition experiment between the amino-whitening peptide sample and other test reagents in the embodiments of the present invention. Detailed Implementation

[0032] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0033] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0034] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0035] Example 1:

[0036] This embodiment discloses an amino-based skin-whitening polypeptide product with the general structural formula RAB-NH2.

[0037] Wherein, R is an N-terminal modified acyl group with a carbon number ranging from 6 to 10, and can be selected from hexanoyl, heptanoyl, octanoyl, nonanoyl, and decanoyl.

[0038] A is D-proline and its derivatives, which may include D-proline, 4-methyl-D-proline, or 4-hydroxy-D-proline.

[0039] B represents D-arginine and its derivatives, such as D-arginine, D-arginine hydrochloride, or D-arginine methanesulfonate.

[0040] This embodiment provides a preparation method for the polypeptide product, as detailed below:

[0041] For resin pretreatment, take 100 mg of Trityl resin with a loading of 0.2 mmol / g, add dichloromethane and soak for 5 min to allow the resin to fully swell, then discard the dichloromethane.

[0042] For B-site amino acid coupling, 3 equivalents of B with a protecting group (Fmoc-D-Arg(Pbf)-OH) were dissolved in 5 mL of N,N-dimethylformamide, and 3 equivalents of N,N-diisopropylethylamine were added. After stirring and dissolving, the mixture was poured into a resin container and stirred under nitrogen for 60 min.

[0043] The resin was washed five times with 10 mL of N,N-dimethylformamide, and the filtrate was discarded each time to remove unreacted B and excess reagent.

[0044] To remove the protection, add 5 mL of a 20% piperidine-N,N-dimethylformamide mixture to the resin container, bubble and stir with nitrogen for 20 min to remove the Fmoc protecting group of B, and then wash 5 times with 10 mL of N,N-dimethylformamide.

[0045] For A-position amino acid coupling, 3 equivalents of protected A (using Fmoc-D-Pro-OH as the protecting group), 3 equivalents of O-benzotriazole-tetramethylurea hexafluorophosphate, and 3 equivalents of N,N-diisopropylethylamine were dissolved in 5 mL of N,N-dimethylformamide, poured into a resin container, and stirred under nitrogen for 60 min. The reaction endpoint was verified using the Kjeldahl method; if the resin turned colorless, the reaction was complete; otherwise, stirring was continued.

[0046] The resin was washed and deprotected again. It was washed 5 times with 10 mL of N,N-dimethylformamide, and then deprotected for 20 min with 5 mL of 20% piperidine-N,N-dimethylformamide mixture. Finally, it was washed 5 times with 10 mL of N,N-dimethylformamide.

[0047] N-terminal acyl group modification: Dissolve 3 equivalents of R (acyl chloride or acyl anhydride corresponding to the number of carbon atoms) in 5 mL of dichloromethane, add 3 equivalents of N,N-diisopropylethylamine, pour into a resin container, and bubble and stir under nitrogen for 40 min to complete the N-terminal modification; wash the resin 5 times with 10 mL of dichloromethane, and dry the resin under nitrogen.

[0048] For peptide cleavage, add 5 mL of cleavage solution (trifluoroacetic acid: water: triisopropylsilane: 1,2-ethylenedithiol = 87.5:5:5:2.5) to the resin, bubble and stir with nitrogen for 50 min, and then filter and collect the filtrate.

[0049] For initial purification, the filtrate was slowly added dropwise to 40 mL of ice-cold ether, resulting in the precipitation of a white solid. The supernatant was discarded by centrifugation, and the solid was collected.

[0050] Purification was performed by high-performance liquid chromatography (HPLC). The solid was dissolved in water / acetonitrile (10:90, containing 0.1% trifluoroacetic acid), filtered through a 0.22 μm filter membrane, and injected into the HPLC system. The column was an Inertsil ODS-SP 4.6 × 250 mm. Mobile phase A was water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. The mobile phase was 5% to 90% in phase B for 0-25 min. The detection wavelength was 220 nm. The eluent of the main peak was collected and lyophilized to obtain a white powdery peptide product. The purity of the obtained peptide product was not less than 99%.

[0051] Example 2:

[0052] The polypeptide structure in this embodiment is octanoyl-AB-NH2, where A is D-proline and its derivatives, and B is D-arginine and its derivatives.

[0053] Compared to other acyl groups with different carbon numbers in Example 1, the octanoyl group can increase the conformational freedom of A by 30%, thereby enhancing the binding ability of the peptide to TRP-1.

[0054] Meanwhile, the lipid-water partition coefficient LogP=2.1 of the octanoyl group can achieve a change in skin and mucous membrane permeability, with a transdermal rate that is 40% higher than that of hexanoyl-modified peptides and 25% higher than that of decanoyl-modified peptides. Furthermore, at a concentration of 1 μM, the inhibition rate of melanin synthesis in B16 cells is on average 18% higher than that of other carbon number acyl peptides.

[0055] Example 3:

[0056] The polypeptide structure of this embodiment is octanoyl-AB-NH2, where A is D-proline and its derivatives, B is D-arginine and its derivatives, and the molecular weight is controlled between 400-420 Da, preferably 412.5 Da.

[0057] The optimization principle is that the skin transdermal threshold is usually 500 Da, and a molecular weight of 400-420 Da can break through the transdermal barrier, avoiding the problem of traditional peptides having a transdermal rate of less than 50%.

[0058] The optimal molecular weight is 412.5 Da. At this molecular weight, the spatial structure of the peptide can perfectly match the active site of TRP-1, and the binding energy reaches -8.2 kcal / mol. This is more stable than the 400 Da peptide (binding energy -6.5 kcal / mol) and the 420 Da peptide (binding energy -7.1 kcal / mol). Moreover, at a concentration of 1 μM, the inhibition rate of melanin synthesis in B16 cells is 12%-15% higher than that of peptides in other molecular weight ranges.

[0059] Example 4:

[0060] The polypeptide structure of this embodiment is octanoyl-4-hydroxy-D-proline-D-arginine-NH2, with a molecular weight of 412.5 Da, which is the optimal product.

[0061] The preferred principle is that the side chain hydroxyl group of 4-hydroxy-D-proline can react with Cu in the active center of TRP-1. 2+ Formation of coordinate bonds, competitively blocking Cu 2+ Binding to the substrate L-DOPA enhances the targeted inhibition effect. Compared with D-proline and 4-methyl-D-proline, at a concentration of 1 μM, the inhibition rate of melanin synthesis in B16 cells is increased by 20%-23%, the inhibition rate of TRP-1 enzyme activity is increased by 18%-21%, and the anti-enzymatic half-life is extended to 8.5 h, which is more than 50% higher than other A-group peptides.

[0062] For this product, a detailed preparation method is provided:

[0063] (1) Resin pretreatment

[0064] Take 100 mg of Trityl resin (loading capacity 0.2 mmol / g), add dichloromethane and soak for 5 min, gently shaking the container during this time to allow the resin to swell evenly. After 5 min, pour out the dichloromethane and retain the resin.

[0065] (2) Fix the C-terminal amino acid B (D-arginine, to ensure that its carboxyl group is connected to the resin)

[0066] Fmoc-D-Arg(Pbf)-OH (where the amino group of B is protected by Fmoc, and the carboxyl group is free, facilitating its bonding with the resin) was selected. Three equivalents of Fmoc-D-Arg(Pbf)-OH were dissolved in 5 mL of N,N-dimethylformamide, and three equivalents of N,N-diisopropylethylamine (to activate the carboxyl group) were added. The mixture was then poured into a resin container. Nitrogen gas was bubbled and stirred for 60 min to allow the carboxyl group of B to form an ester bond with the -OH group of the resin. The resin was then washed five times with 10 mL of N,N-dimethylformamide to remove unbound B.

[0067] (3) Remove the amino protecting group of B

[0068] This stage exposes the N-terminus of B, preparing for the connection of A. Specifically, 5 mL of a 20% piperidine-N,N-dimethylformamide mixture is added to the resin, and the mixture is bubbled and stirred under nitrogen for 20 min to remove the Fmoc protecting group of B, resulting in H2N-B- resin (with the N-terminus of B free). The resin is then washed five times with 10 mL of N,N-dimethylformamide to remove residual piperidine.

[0069] (4) Coupling of intermediate amino acid A

[0070] Fmoc-D-Hyp(tBu)-OH (the amino group of A is protected by Fmoc, and the carboxyl group is free) was selected; 3 equivalents of Fmoc-D-Hyp(tBu)-OH, 3 equivalents of O-benzotriazole-tetramethylurea hexafluorophosphate, and 3 equivalents of N,N-diisopropylethylamine were dissolved in 5 mL of N,N-dimethylformamide and poured into a resin container.

[0071] Nitrogen gas was bubbled and stirred for 60 min to allow the carboxyl group of A to form a peptide bond with the free amino group of B, resulting in Fmoc-AB-resin; Kjeldahl test showed no color; washed 5 times with 10 mL of N,N-dimethylformamide.

[0072] (5) Remove the amino protecting group of A

[0073] Add 5 mL of 20% piperidine-N,N-dimethylformamide mixture to the resin, bubble and stir with nitrogen for 20 min to remove the Fmoc protecting group of A, and obtain H2N-AB- resin (the N-terminus of A is free); wash 5 times with 10 mL of N,N-dimethylformamide, then wash 5 times with 10 mL of dichloromethane, and dry the resin.

[0074] (6) N-terminal modification of R

[0075] Dissolve 3 equivalents of octanoyl chloride in 5 mL of dichloromethane, add 3 equivalents of N,N-diisopropylethylamine (activating acyl chloride), and pour into a resin container; bubble and stir with nitrogen for 40 min to form an amide bond between the octanoyl group and the free amino group of A, to obtain octanoyl-AB- resin; wash 5 times with 10 mL of dichloromethane and dry the resin.

[0076] (7) Peptide cleavage

[0077] Prepare 5 mL of cutting solution (4.375 mL trifluoroacetic acid + 0.25 mL water + 0.25 mL triisopropylsilane + 0.125 mL 1,2-ethylenedithiol) at a volume ratio of 87.5:5:5:2.5 and stir until homogeneous. Pour the cutting solution into a reaction vessel, purge with nitrogen and stir for 50 min, controlling the stirring rate to prevent resin precipitation. After cutting, filter with filter paper, collect the filtrate (containing free peptides), and discard the resin residue.

[0078] (9) Preliminary purification

[0079] The collected filtrate was slowly added dropwise to ice-cold ether pre-cooled to -20°C at a rate of 1 drop per second, while stirring at a low speed (100 rpm) with a magnetic stirrer. After the addition was complete, stirring was continued for 5 minutes to allow the peptides to fully separate into a white solid. The mixture was then placed in a centrifuge and centrifuged at 4000 rpm for 10 minutes. The upper layer of ether waste liquid was poured off, and the white solid at the bottom was retained.

[0080] (10) Sample processing

[0081] Add 2 mL of water / acetonitrile mixture (volume ratio 10:90, containing 0.1% trifluoroacetic acid) to the white solid, and gently shake to completely dissolve the solid; filter the solution through a 0.22 μm organic phase filter membrane to remove small particulate impurities, and collect the filtrate.

[0082] (11) High performance liquid chromatography purification:

[0083] Chromatographic conditions were set as follows: Inertsil ODS-SP4.6×250mm column, mobile phase A was water (containing 0.1% trifluoroacetic acid), mobile phase B was acetonitrile (containing 0.1% trifluoroacetic acid), elution gradient was 0-25min B phase 5% to 90%, flow rate was 1mL / min, detection wavelength was 220nm, and column temperature was 30℃.

[0084] Inject the filtrate into the high-performance liquid chromatograph, and start elution after the instrument stabilizes. Record the chromatogram. Collect the main peak eluent with a retention time of about 12.3 min, and discard the impurity peak eluent.

[0085] (12) Freeze-drying and characterization: The main peak eluent was placed in a freeze dryer, the temperature was set to -50℃ and the vacuum degree to 10Pa, and the freeze-drying was carried out for 24 hours until completely dry, resulting in a white powdery polypeptide product. The molecular weight was confirmed to be 412.5 Da by mass spectrometry, the purity was confirmed to be ≥99.2% by high performance liquid chromatography, and the solubility was confirmed to be soluble in ultrapure water, phosphate buffer, and dimethyl sulfoxide.

[0086] Reference Figures 1-4 The document shows the testing information for the product in this embodiment.

[0087] Experimental verification

[0088] 1. Effectiveness experiment targeting TRP-1

[0089] (1) Molecular docking verification

[0090] TRP-1 receptor source: The human TRP-1 crystal structure with PDB database number 6H3S was used, and the water of crystallization and ligands were removed. This structure is suitable for TRP-1 research in skin melanocytes.

[0091] Docking tools and parameters: AutoDockVina software, AMBERff14SB force field, with the TRP-1 active center Cu²⁺ as the origin, a 20×20×20Å grid (0.375Å spacing), 30 independent dockings;

[0092] (2) TRP-1 enzyme activity inhibition experiment

[0093] Experimental system: recombinant human TRP-1 enzyme (10 nM) + L-DOPA substrate (500 μM, TRP-1 natural substrate), solvent is PBS at pH 5.5 (simulating the pH of normal skin surface: 4.5-6.0).

[0094] Group settings:

[0095] Blank control (enzyme + substrate only);

[0096] Peptide groups (1μM, 5μM, 10μM, with a core effective concentration of 1μM);

[0097] Positive control (100 μM arbutin);

[0098] Reaction conditions: Incubate at 37℃ for 60 min, detect absorbance at 475 nm (characteristic absorption peak of dopaquinone) using an ELISA reader, and calculate enzyme inhibition rate = (blank absorbance - sample absorbance) / blank absorbance × 100%.

[0099] Experimental Data Table 1

[0100]

[0101] (3) Conclusion

[0102] As shown in Table 1, the peptide can specifically target and inhibit TRP-1 activity. At a concentration of 1 μM, the enzyme inhibition rate reached 16.13% (p<0.05), and the IC50 was about 9.8 μM, which was significantly better than 100 μM arbutin (inhibition rate 14.52%, IC50 about 344 μM).

[0103] The minimum binding energy of molecular docking, -8.2 kcal / mol, is in high agreement with the enzyme activity inhibition data, proving that the peptide can stably bind to the TRP-1 active site and directly inhibit the enzyme's catalytic function through competitive binding to Cu²⁺ (hydroxyl coordination of 4-hydroxy-D-proline) and electrostatic interaction (D-arginine guanidinium binding to the negative potential site).

[0104] The enzyme inhibition rate of 16.13% of the 1μM peptide was almost identical to the 15.38% melanin inhibition rate of B16 cells found in the later experiments, indicating that the inhibitory effect of the peptide on melanin synthesis is entirely due to the inhibition of the TRP-1 target, without other non-specific interference, and the target effectiveness is clear.

[0105] 2. Skin permeability test

[0106] (1) Experimental content

[0107] Donor concentration: 1 mM peptide (octanoyl-4-hydroxy-D-proline-D-arginine-NH2, molecular weight 412.5 Da).

[0108] Donor volume: The supply chamber volume of the Franz diffusion cell is 2 mL, and the total donor peptide amount is 820.4 μg;

[0109] Skin model: Fresh pig ear skin, subcutaneous fat removed, thickness 300μm, TEWL (transdermal water loss) measured before experiment = 12.3g / m²・h (≤15g / m²・h, confirming skin barrier integrity).

[0110] Receiver parameters: volume 5mL (pH7.4 PBS), constant temperature 37℃, magnetic stirring 300rpm; 1mL sample taken each time (1mL fresh receiver solution added to maintain constant volume), sampling time 0.5, 1, 2, 4, 6, 8, 12, 24h;

[0111] Detection method: HPLC was used to determine the peptide concentration in the receiving solution (Inertsil ODS-SP4.6×250mm column, mobile phase A: water containing 0.1% TFA, mobile phase B: acetonitrile containing 0.1% TFA, 0-25min B phase 5%→90%, detection wavelength 220nm).

[0112] Blank control: Add 0.9% physiological saline + 0.1% Tween 80 (without peptides) to the supply room and test whether the receiving fluid is contaminated;

[0113] Positive control: The positive control was 20 mM nicotinamide with a molecular weight of 123 Da. The donor concentration was verified by HPLC to be 19.8 mM with a deviation of <1%. The remaining experimental conditions were the same as those for the peptide and were used to verify the effectiveness of the experimental system.

[0114] Experimental Data Table 2

[0115]

[0116] Based on standard skin anatomy parameters, the dermal peptide concentration was approximately 58.8 μM after 24 hours, ensuring that it could reach the target site and exert its activity after transdermal penetration. The total amount of nicotinamide permeated after 24 hours was 3280.6 μg, with a penetration efficiency of approximately 66.7% and a Kp value of 1380 × 10⁻⁶. - 5 cm / h proves that the experimental system is without bias.

[0117] (2) Conclusion

[0118] At a concentration of 1 mM, the optimal peptide (octanoyl-4-hydroxy-D-proline-D-arginine-NH2) exhibited excellent transdermal permeability in a porcine ear skin model, with a cumulative permeation of Q over 24 hours. 24 h = 242.09 μg / cm², transdermal transdermal rate Kp = 1000 × 10⁻⁶ -5 The transdermal penetration rate is significantly better than traditional whitening agents. Although niacinamide has a slightly higher transdermal penetration rate, peptides can achieve melanin inhibition at a concentration of 1 μM, while niacinamide requires concentrations above 1000 μM, highlighting the advantage of peptides at low concentrations.

[0119] Octyl modification (LogP=2.1) and molecular weight optimization (412.5Da) are key to excellent transdermal performance, which is completely consistent with the technical design of enhancing mucosal penetration with N-terminal acyl groups and breaking through the transdermal threshold with molecular weight.

[0120] As shown in Table 2, the polypeptide's molecular weight is 412.5 Da, less than 500 Da, and the N-terminal octanoyl modification results in a LogP of 2.1. This ensures both lipid solubility for penetration into the stratum corneum and avoids excessive lipid solubility leading to stratum corneum retention, thus achieving highly efficient transdermal absorption. The cumulative penetration over 24 hours corresponds to a polypeptide concentration of approximately 58.8 μM in the dermis, demonstrating that the polypeptide can penetrate the ordinary skin barrier and reach an effective concentration, meeting the requirements for transdermal efficacy of whitening products.

[0121] 3. Experiment on the anti-inflammatory effect on ordinary skin

[0122] (1) Experimental methods

[0123] Cell model: human immortalized keratinocytes (HaCaT cells, the main functional cells of normal skin epidermis).

[0124] Inflammation induction: Cells were treated with 10 ng / mL TNF-α (tumor necrosis factor-α, a key factor in common skin inflammation) for 24 h to establish an inflammation model;

[0125] Group settings:

[0126] Normal control (no inflammation induced);

[0127] Inflammation model control (TNF-α induction, no drug);

[0128] Peptide groups (1μM, 5μM, effective concentration range);

[0129] Positive control (1mM dipotassium glycyrrhizate, concentration approximately 0.04%, a commonly used anti-inflammatory ingredient for ordinary skin).

[0130] Detection indicators: After 48 hours of incubation, the concentrations of IL-6 (interleukin-6) and IL-8 (interleukin-8) in the cell supernatant were detected by ELISA (core factors of common skin inflammation).

[0131] Experimental Data Table 3

[0132]

[0133] (2) Conclusion

[0134] As shown in Table 3, the polypeptide has a significant anti-inflammatory effect on the skin. At a concentration of 1 μM, it can reduce the concentrations of IL-6 and IL-8 in TNF-α-induced HaCaT cells by 45.0% and 42.9%, respectively (p<0.05), which is comparable to that of 0.04% dipotassium glycyrrhizate.

[0135] The anti-inflammatory mechanism is directly related to the design. The double D-amino acid structure can significantly reduce the recognition of exogenous peptides by immune cells (ordinary L-amino acid peptides are prone to triggering immune inflammation). At the same time, TRP-1 inhibition can indirectly reduce the release of inflammatory factors (excessive TRP-1 activity will activate the skin inflammation pathway).

[0136] The anti-inflammatory effect of 1μM peptide is consistent with the effective whitening concentration, indicating that it can have a synergistic effect of whitening and anti-inflammation in ordinary skin whitening scenarios, solving the irritation problem of traditional whitening agents (such as niacinamide with an irritation rate of ≥22% at a concentration of >5%).

[0137] 4. Anti-degradation effect experiment on ordinary skin

[0138] (1) Experimental methods

[0139] 1) In vitro enzymatic digestion experiment

[0140] Protease system: aminopeptidase (1U / mL) + neutral peptidase (0.5U / mL, the main polypeptide-degrading enzyme on the surface of normal skin), solvent is PBS at pH 5.5 (pH of normal skin);

[0141] Group settings:

[0142] This technology contains a polypeptide group (1μM, bis-D-amino acid).

[0143] Control peptide group (1 μM, L-proline-L-arginine, traditional L-type peptide).

[0144] Detection: Incubate at 37℃, and take samples at 0, 1, 2, 4, 6 and 8 h respectively. HPLC is used to determine the remaining amount of peptide and calculate the half-life (t1 / 2).

[0145] 2) In vivo retention experiment

[0146] Animal model: Female ICR mice (6-8 weeks old), with hair removed from the back (exposing normal skin);

[0147] Administration method: Apply 1 μM peptide solution (0.2 mL, simulating topical application to normal skin) to the back;

[0148] Sampling and detection: Mice were sacrificed at 1, 2, 4 and 6 h after drug administration, and skin tissue (500 μm thick) was taken from the back. After homogenization, the peptide concentration was measured by HPLC to assess the amount retained in the tissue.

[0149] In vitro experimental data table 4

[0150]

[0151] In vivo experimental data table 5

[0152]

[0153] (3) Conclusion

[0154] The peptide exhibits significant resistance to degradation in normal skin environments, with an in vitro half-life of t1 / 2 = 8.6 h (compared to only 1.9 h for traditional L-type peptides). After 6 h in vivo, 18.7 ng / g of the peptide can still be detected in normal skin tissue (corresponding to a concentration of approximately 0.45 μM, close to 50% of the effective concentration of 1 μM).

[0155] The core of its anti-degradation effect stems from the dual D-amino acid design: ordinary skin proteases (aminopeptidase, neutral peptidase) have stereospecificity and can only recognize the spatial configuration of L-amino acids and cleave peptide bonds, while the mirror image structure of D-amino acids cannot be recognized by enzymes. Therefore, the peptide bond (D-AA-D-AA) is not degraded, and the half-life is greatly extended.

[0156] In vivo data proves that peptides can maintain their effects for a longer period of time in normal skin, and can continuously exert whitening and anti-inflammatory effects without frequent reapplication, solving the problem of short-acting effects of traditional L-type peptides with a skin half-life of less than 2 hours, thus improving the convenience of product use.

[0157] 5. Melanin Production Inhibition Test

[0158] (1) Experimental methods

[0159] B16 cells in logarithmic growth phase were selected, and the cell suspension density was adjusted to 9 x 10⁵ cells / well. 2 mL of cell suspension was seeded into each well of a 6-well plate and incubated at 37°C in a 5% CO₂ incubator. The medium was changed after 24 h. After 72 h of drug treatment, the culture medium was discarded, and the cells were washed twice with PBS. 500 μL of 1 mol / L NaOH was added to each well, and the cells were transferred to 1.5 mL centrifuge tubes and incubated in an 80°C water bath for 30 min to completely dissolve the cell clumps. Ultrapure water was then added to dilute the NaOH to a final concentration of 0.2 mol / L. The solutions were mixed thoroughly, and 100 μL of each solution was transferred to each well of a 96-well plate, with three duplicate wells. The OD value was measured at 475 nm using a microplate reader. The melanin content (%) was calculated as (OD experimental group / OD control group) × 100%.

[0160] Test substance:

[0161] Negative control group: complete culture medium.

[0162] Positive control group: 1000 μM nicotinamide, 100 μM arbutin, 1000 μM nicotinamide + 100 μM arbutin, 7037 μM kojic acid.

[0163] Sample group: 1μM second type of peptide.

[0164] Experimental Data Table 6

[0165]

[0166] Referring to Table 6, under the experimental method, the 1 μM sample solution can significantly inhibit melanin synthesis in B16 cells, thus exhibiting a whitening effect.

[0167] This invention is not limited to the optional embodiments described above, and anyone can derive other various forms of products based on the inspiration of this invention. The specific embodiments described above should not be construed as limiting the scope of protection of this invention; the scope of protection of this invention should be determined by the claims, and the specification can be used to interpret the claims.

Claims

1. An amino-based skin-whitening polypeptide, characterized in that: The general structural formula of a polypeptide is as follows: RAB-NH2, where, R is a modifying acyl group at the N-terminus with 6-10 carbon atoms, A is 4-hydroxy-D-proline, and B is D-arginine.

2. The amino-based skin-whitening polypeptide according to claim 1, characterized in that: R is octanoyl.

3. The amino-based skin-whitening polypeptide according to claim 1, characterized in that: The polypeptide has a molecular weight of 400-420.

4. The amino-based skin-whitening polypeptide according to claim 1, characterized in that: The polypeptide has a molecular weight of 412-413.

5. A preparation method, characterized in that: The amino-whitening polypeptide described in any one of claims 1-4 is prepared and synthesized using the Fmoc solid-phase synthesis method.

6. The preparation method according to claim 5, characterized in that: The process involves using triphenylmethyl resin as a carrier, first immobilizing D-arginine with a protecting group, then deprotecting and immobilizing 4-hydroxy-D-proline with a protecting group. After immobilization, the peptide is cleaved and separated from the resin carrier, initially purified by precipitation with ice-cold ether, and then further purified by gradient elution with high performance liquid chromatography to obtain the whitening peptide product.

7. The preparation method according to claim 6, characterized in that: The purity of the further purified whitening polypeptide product is not less than 99%.

8. The preparation method according to claim 6, characterized in that: The cutting fluid used for the cutting and separation process includes trifluoroacetic acid, water, triisopropylsilane, and 1,2-ethylenedithiol in a volume ratio of 87.5:5:5:2.

5.

9. The preparation method according to claim 8, characterized in that: The molecular weight of the purified whitening polypeptide product was 412.5.

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