Amino whitening polypeptide and preparation method thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly discloses an amino acid whitening polypeptide and a preparation method thereof, the structural general formula of the polypeptide is R-A-B-NH2, R is a modified acyl serving as an N terminal and having a carbon number range of 6-10, A is D-proline and a derivative thereof, and B is D-arginine and a derivative thereof. The amino whitening polypeptide disclosed by the invention is prepared by adopting an Fmoc solid-phase synthesis method. The whitening polypeptide provided by the invention has excellent melanin synthesis inhibition activity, plays a role through targeted inhibition of TRP-1, can enable the melanin synthesis inhibition rate of B16 cells to reach the standard under the concentration of 1 mu M, has statistical significance, and is obviously superior to a traditional whitening agent which can achieve a similar inhibition effect only when the concentration is high; and the effective use concentration of the whitening product is greatly reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to an amino whitening polypeptide and a preparation method thereof. BACKGROUND

[0002] There are many whitening components in the prior art, which generally include plant extract, vitamin derivative, traditional polypeptide, etc. These whitening components have the following problems: The plant extract whitening agent, such as arbutin, has a limited mechanism of action, only through reversible inhibition of tyrosinase activity, no regulation of TRP-1 / TRP-2 pathway, and unstable chemical structure, hydrolyzed to hydroquinone in pH>6.0 environment, has certain cytotoxicity, and the effectiveness depends on high concentration, generally more than 100uM to achieve 15% inhibition rate.

[0003] The vitamin derivative, such as nicotinamide, mainly works by inhibiting melanosome transfer, has no direct inhibition on melanin synthesis, and has low transdermal efficiency, and its LogP=-0.37 leads to less than 10% of the keratin layer retention rate; there is also dose-dependent stimulation.

[0004] The traditional polypeptide has high enzymatic risk, and the L-type polypeptide has a half-life of less than 2 hours on the skin surface; and there is a molecular weight barrier, generally the molecular weight of such polypeptide is more than 500Da, and the transdermal rate of such polypeptide with a molecular weight of less than 1.0x10 -5 cm / h, and the product prepared by the traditional polypeptide preparation method has low purity, which is also the reason for the high concentration requirement. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides an amino whitening polypeptide and a preparation method thereof, aiming to provide a polypeptide short chain structure formed by double D-type amino acids, which has good target inhibition of melanin synthesis, thereby reducing the required concentration, and has good transdermal efficiency and anti-inflammatory effect, and has high purity through an optimized preparation method.

[0006] The technical scheme adopted by the present application is as follows: In a first aspect, the present application provides an amino whitening polypeptide, and the structure general formula of the polypeptide is as follows: R-A-B-NH2 Wherein, R is a modified acyl group as N-terminal and the number of carbons ranges from 6 to 10, A is D-proline and its derivative, and B is D-arginine and its derivative.

[0007] In combination with the first aspect, the present application provides a first embodiment of the first aspect, wherein the R is octanoyl.

[0008] In combination with the first aspect, the present application provides a second implementation of the first aspect, wherein the polypeptide has a molecular weight of 400-420.

[0009] In combination with the first aspect, the present application provides a third implementation of the first aspect, wherein the polypeptide has a molecular weight of 412-413.

[0010] In combination with the first aspect, the present application provides a fourth implementation of the first aspect, wherein the A is 4-hydroxy-D-proline.

[0011] In combination with the first aspect, the present application provides a fourth implementation of the first aspect, wherein the A is 4-hydroxy-D-proline.

[0012] In combination with the second aspect, the present application provides a first implementation of the second aspect, wherein a D-arginine with a protecting group is first immobilized on a trityl resin as a carrier, and then a D-proline with a protecting group is immobilized after deprotection, and then N-terminal modification is performed, and then the polypeptide is cut and separated from the resin carrier after immobilization, and then the polypeptide is preliminarily purified through ice-ether precipitation, and then the polypeptide is further purified through high-performance liquid chromatography gradient elution to obtain a whitening polypeptide product.

[0013] In combination with the second aspect, the present application provides a second implementation of the second aspect, wherein the whitening polypeptide product after further purification has a purity of not less than 99%.

[0014] In combination with the second aspect, the present application provides a third implementation of the second aspect, wherein the cutting liquid used for cutting and separation comprises trifluoroacetic acid, water, triisopropylsilane and 1,2-ethanedithiol in a volume ratio of 87.5:5:5:2.5.

[0015] In combination with the second aspect, the present application provides a fourth implementation of the second aspect, wherein the whitening polypeptide product obtained after purification has a molecular weight of 412.5.

[0016] The present application has the following beneficial effects: (1) The whitening polypeptide provided by the present application has excellent melanin synthesis inhibition activity, and plays a role by targeting inhibition of TRP-1, and can make the melanin synthesis inhibition rate of B16 cells reach a level with statistical significance at a concentration of 1 μM, which is significantly better than the case that a traditional whitening agent needs a high concentration to achieve a similar inhibition effect, and greatly reduces the effective use concentration of a whitening product; (2) The present application realizes efficient skin transdermal performance by optimizing the polypeptide structure, and breaks through the skin transdermal threshold by controlling the molecular weight of the polypeptide, and adjusts the lipid-water partition coefficient by modifying the octanoyl group at the N-terminal, which not only ensures that the polypeptide can effectively penetrate the stratum corneum to reach the target point in the epidermis, but also avoids the retention of the polypeptide in the stratum corneum, and meets the transdermal demand of whitening of the whole skin; (3) The application adopts a double D-amino acid structure design, so that the whitening polypeptide has excellent anti-degradation ability, can resist the degradation of skin surface protease, significantly prolongs the half-life of the polypeptide on the skin surface layer, solves the problem of short half-life and easy inactivation of traditional L-type polypeptide on the skin surface, ensures that the polypeptide can continuously exert the whitening effect, and reduces the product use frequency.

[0017] (4) The whitening polypeptide has high purity and good biocompatibility, the polypeptide HPLC purity is not less than 99% through a specific preparation process, the impurity content is extremely low, the skin irritation risk can be reduced, the survival rate of the skin beneficial bacteria lactobacillus has no significant influence, the skin microecological balance can be compatible, and the whitening demand of sensitive skin parts is met. (5) The whitening polypeptide has the synergistic effects of whitening and potential anti-inflammatory, the process of targeting inhibition of TRP-1 can indirectly reduce the release of skin inflammatory factors, the double D-amino acid structure reduces the immunogenicity, avoids the irritation problem of high concentration use of traditional whitening agents, realizes the dual effects of “whitening + mild care”, and improves the product use experience. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the synthesis information of the amino whitening polypeptide product in the embodiment of the application; Figure 2 is the HPLC information of the amino whitening polypeptide product in the embodiment of the application; Figure 3 is the MS information of the amino whitening polypeptide product in the embodiment of the application; Figure 4 is the solubility information of the amino whitening polypeptide product in the embodiment of the application; Figure 5 is the B16 melanocyte inhibition experiment result graph between the amino whitening polypeptide sample and other test reagents in the embodiment of the application; Figure 6 is the microscopic graph of the B16 melanocyte inhibition experiment between the amino whitening polypeptide sample and other test reagents in the embodiment of the application. DETAILED DESCRIPTION

[0019] The application will be further explained in conjunction with the drawings and specific embodiments.

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application will be clearly and completely described below in conjunction with the drawings in the embodiments of the application. Obviously, the described embodiments are part of the embodiments of the application, not all the embodiments.

[0021] The following detailed description of embodiments of the application provided in the accompanying drawings is not intended to limit the scope of the application claimed, but merely represents selected embodiments of the application. Based upon a review of the embodiments presented in this application, all other embodiments falling within the scope of the application are intended to be reserved to those skilled in the art with the benefit of this application without necessitating creative steps.

[0022] Embodiment 1 The present embodiment discloses an amino whitening polypeptide product, the general structure is R-A-B-NH2.

[0023] Wherein, R is N-terminal modified acyl, the number of carbons ranges from 6 to 10, which can be selected from hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl; A is D-proline and its derivatives, which can be selected from D-proline, 4-methyl-D-proline, 4-hydroxy-D-proline; B is D-arginine and its derivatives, which can be selected from D-arginine, D-arginine hydrochloride, D-arginine methanesulfonate.

[0024] The present embodiment provides a preparation method for the polypeptide product, which is specifically as follows: Resin pretreatment: 100 mg of Trityl resin with a loading capacity of 0.2 mmol / g is taken and soaked in dichloromethane for 5 min. After the resin is fully swollen, the dichloromethane is discarded.

[0025] B-site amino acid coupling: 3 equivalents of B with a protection group (the protection group is selected from Fmoc-D-Arg(Pbf)-OH) are dissolved in 5 mL of N,N-dimethylformamide, 3 equivalents of N,N-diisopropylethylamine are added, and after stirring and dissolving, the solution is poured into the resin container, and nitrogen is bubbled and stirred for 60 min.

[0026] Resin washing: the resin is washed with 10 mL of N,N-dimethylformamide for 5 times, and the filtrate is discarded each time to remove unreacted B and excess reagents.

[0027] Deprotection: 5 mL of 20% piperidine-N,N-dimethylformamide mixture is added to the resin container, nitrogen is bubbled and stirred for 20 min to remove the Fmoc protection group of B, and then the resin is washed with 10 mL of N,N-dimethylformamide for 5 times.

[0028] A-site amino acid coupling: 3 equivalents of A with a protection group (the protection group is selected from Fmoc-D-Pro-OH), 3 equivalents of O-benzotriazol-tetramethyluronium hexafluorophosphate, and 3 equivalents of N,N-diisopropylethylamine are dissolved in 5 mL of N,N-dimethylformamide, poured into the resin container, and stirred for 60 min with nitrogen bubbling; the reaction endpoint is verified by the Kjeldahl method, and if the resin is colorless, the reaction is complete, otherwise continue to stir.

[0029] Washing and deprotection, the resin was washed with 10 mL of N,N-dimethylformamide for 5 times, 5 mL of 20% piperidine-N,N-dimethylformamide mixture was added for deprotection for 20 min, and then washed with 10 mL of N,N-dimethylformamide for 5 times.

[0030] N-terminal acyl modification, 3 equivalents of R (acyl chloride or acyl anhydride corresponding to the number of carbons) were dissolved in 5 mL of dichloromethane, 3 equivalents of N,N-diisopropylethylamine were added, poured into the resin container, stirred with nitrogen bubbling for 40 min, and the N-terminal modification was completed; the resin was washed with 10 mL of dichloromethane for 5 times, and the resin was dried by nitrogen blowing.

[0031] Polypeptide cleavage, 5 mL of cleavage solution (trifluoroacetic acid: water: triisopropylsilane: 1,2-ethanedithiol = 87.5:5:5:2.5) was added to the resin, stirred with nitrogen bubbling for 50 min, and the filtrate was collected by filtration.

[0032] Preliminary purification, the filtrate was slowly dropped into 40 mL of ice ethyl ether, white solid was precipitated, the supernatant was discarded by centrifugation, and the solid was collected.

[0033] High performance liquid chromatography purification, the solid was dissolved in water / acetonitrile (10:90, containing 0.1% trifluoroacetic acid), filtered through a 0.22 μm filter membrane, then injected into a high performance liquid chromatograph, the chromatographic column was Inertsil ODS-SP 4.6x250mm, the mobile phase A was water containing 0.1% trifluoroacetic acid, the mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid, 0-25 min B phase 5% to 90%, detection wavelength 220 nm, the main peak eluent was collected, and the white powder polypeptide product was obtained by freeze-drying, and the purity of the obtained polypeptide product was not less than 99%.

[0034] Example 2: The polypeptide structure of this example is octanoyl-A-B-NH2, wherein A is D-proline and its derivatives, and B is D-arginine and its derivatives.

[0035] Compared with other carbon number acyl groups in example 1, octanoyl group can increase the conformational freedom of A by 30%, and enhance the binding ability of polypeptide to TRP-1.

[0036] At the same time, the octanoyl group has a lipid-water partition coefficient LogP=2.1, which can change the skin mucosa permeability, and the transdermal rate is increased by 40% compared with hexanoyl modified polypeptide and 25% compared with decanoyl modified polypeptide, and the B16 cell melanin synthesis inhibition rate at 1 μM concentration is increased by an average of 18% compared with other carbon number acyl group polypeptides.

[0037] Example 3: The polypeptide structure of this example is octanoyl-A-B-NH2, wherein A is D-proline and its derivatives, and B is D-arginine and its derivatives, and the molecular weight is controlled at 400-420 Da, preferably 412.5 Da.

[0038] The optimization principle is that the skin transdermal threshold is generally 500 Da, and the molecular weight of 400-420 Da can break through the transdermal barrier, avoiding the problem that the transdermal rate of traditional polypeptides is less than 1%.

[0039] Among them, 412.5 Da is the optimal value, at which the spatial structure of the polypeptide can perfectly adapt to the active site of TRP-1, and the binding energy can reach -8.2 kcal / mol, which is more stable than that of 400 Da polypeptide (binding energy -6.5 kcal / mol) and 420 Da polypeptide (binding energy -7.1 kcal / mol), and the melanin synthesis inhibition rate of B16 cells at 1 μM concentration is increased by 12%-15% compared with other molecular weight interval polypeptides.

[0040] Example 4: 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.

[0041] The preferred principle is that the side chain hydroxyl of 4-hydroxy-D-proline can form a coordination bond with Cu 2+ in the active center of TRP-1, competitively blocking the combination of Cu 2+ with the substrate L-dopa, enhancing the targeted inhibition effect. Compared with D-proline and 4-methyl-D-proline, the melanin synthesis inhibition rate of B16 cells at 1 μM concentration is increased by 20%-23%, the TRP-1 enzyme activity inhibition rate is increased by 18%-21%, and the anti-enzymatic half-life is prolonged to 8.5 h, which is more than 50% higher than other A group polypeptides.

[0042] For this product, a detailed preparation method is provided: (1) Resin pretreatment Take 100 mg of Trityl resin (loading capacity 0.2 mmol / g), add dichloromethane and soak for 5 min, gently shake the container during the soaking to make the resin swell uniformly, pour out the dichloromethane after 5 min, and reserve the resin.

[0043] (2) Fixing C-terminal amino acid B (D-arginine, ensuring that its carboxyl group is connected to the resin) Fmoc-D-Arg(Pbf)-OH (the amino group of B is protected by Fmoc, and the carboxyl group is free, which is convenient for connection with the resin) is selected, 3 equivalents of Fmoc-D-Arg(Pbf)-OH are dissolved in 5 mL of N,N-dimethylformamide, 3 equivalents of N,N-diisopropyl ethylamine (activating carboxyl group) are added, and the resin container is poured. Bubble stirring for 60 min under nitrogen to form an ester bond between the carboxyl group of B and the -OH of the resin; wash the resin with 10 mL of N,N-dimethylformamide for 5 times to remove the unbound B.

[0044] (3) Removing the amino protecting group of B This stage exposes the N-terminal of B, preparing for the connection of A. Specifically, 5 mL of 20% piperidine-N,N-dimethylformamide mixture is added to the resin, and nitrogen is bubbled to stir for 20 min to remove the Fmoc protecting group of B, obtaining H2N-B-resin (the N-terminal of B is free); 10 mL of N,N-dimethylformamide is used to wash 5 times to remove residual piperidine.

[0045] (4) Coupling intermediate amino acid A Fmoc-D-Hyp(tBu)-OH (the amino group of A is protected by Fmoc, and the carboxyl group is free) is selected; 3 equivalents of Fmoc-D-Hyp(tBu)-OH, 3 equivalents of O-benzotriazole-tetramethyluronium hexafluorophosphate, and 3 equivalents of N,N-diisopropyl ethylamine are dissolved in 5 mL of N,N-dimethylformamide and poured into the resin container.

[0046] Nitrogen is bubbled to stir for 60 min to form a peptide bond between the carboxyl group of A and the free amino group of B, obtaining Fmoc-A-B-resin; Kjeldahl detection shows no color; 10 mL of N,N-dimethylformamide is used to wash 5 times.

[0047] (5) Removing the amino protecting group of A 5 mL of 20% piperidine-N,N-dimethylformamide mixture is added to the resin, and nitrogen is bubbled to stir for 20 min to remove the Fmoc protecting group of A, obtaining H2N-A-B-resin (the N-terminal of A is free); 10 mL of N,N-dimethylformamide is used to wash 5 times, and then 10 mL of dichloromethane is used to wash 5 times, and the resin is dried.

[0048] (6) N-terminal modification R 3 equivalents of octanoyl chloride are dissolved in 5 mL of dichloromethane, 3 equivalents of N,N-diisopropyl ethylamine (to activate the acyl chloride) are added, and the mixture is poured into the resin container; nitrogen is bubbled to stir for 40 min to form an amide bond between the octanoyl group and the free amino group of A, obtaining octanoyl-A-B-resin; 10 mL of dichloromethane is used to wash 5 times, and the resin is dried.

[0049] (7) Polypeptide cleavage 5 mL of cleavage solution (trifluoroacetic acid 4.375 mL + water 0.25 mL + triisopropylsilane 0.25 mL + 1,2-ethanedithiol 0.125 mL) is prepared according to the volume ratio of 87.5:5:5:2.5, and stirred uniformly; the cleavage solution is poured into the reaction container, nitrogen is bubbled to stir for 50 min, and the stirring speed is controlled during the process to avoid resin precipitation; after the cleavage is completed, the filtrate (containing free polypeptide) is collected by filtering with filter paper, and the resin residue is discarded.

[0050] (9) Preliminary purification The collected filtrate was slowly dripped into pre-cooled ice ether at -20°C, the dripping speed was controlled at 1 drop per second, and the mixture was stirred at low speed (100 rpm) with a magnetic stirrer; after the dripping was completed, the mixture was stirred for 5 min to allow the polypeptide to precipitate as a white solid; then the mixture was centrifuged at 4000 rpm for 10 min, and the upper ether waste liquid was poured out, and the white solid at the bottom was retained.

[0051] (10) Sample processing 2 mL of a water / acetonitrile mixture (volume ratio 10:90, containing 0.1% trifluoroacetic acid) was added to the white solid, and the solid was completely dissolved by gently shaking; the solution was filtered through a 0.22 μm organic phase filter membrane to remove small particle impurities, and the filtrate was collected.

[0052] (11) High performance liquid chromatography purification: The chromatographic conditions were set as follows: the chromatographic column was Inertsil ODS-SP 4.6 x 250 mm, the mobile phase A was water (containing 0.1% trifluoroacetic acid), the mobile phase B was acetonitrile (containing 0.1% trifluoroacetic acid), the elution gradient was 0-25 min B phase 5% to 90%, the flow rate was 1 mL / min, the detection wavelength was 220 nm, and the column temperature was 30°C; The filtrate was injected into the high performance liquid chromatograph, and after the instrument was stabilized, the elution was started, and the chromatogram was recorded; the main peak eluent with a retention time of about 12.3 min was collected, and the impurity peak eluent was discarded.

[0053] (12) Freeze-drying and characterization: the main peak eluent was placed in a freeze dryer, the temperature was set to -50°C, the vacuum degree was 10 Pa, and the freeze-drying was carried out for 24 h until complete drying, and a white powdery polypeptide product was obtained; mass spectrometry detection confirmed that the molecular weight was 412.5 Da, high performance liquid chromatography detection confirmed that the purity was ≥99.2%, and solubility detection confirmed that it was soluble in ultrapure water, phosphate buffer, and dimethyl sulfoxide.

[0054] Reference Figures 1-4 The detection information of the product in this embodiment is shown in the table.

[0055] Experimental verification 1. TRP-1 target effectiveness experiment (1) Molecular docking verification TRP-1 receptor source: human TRP-1 crystal structure with PDB database number 6H3S, removing crystal water and ligand, the structure is suitable for skin melanocyte TRP-1 research; Docking tool and parameters: AutoDockVina software, AMBERff14SB force field, taking Cu²⁺ in the active center of TRP-1 as the origin, setting a 20 x 20 x 20 Å grid (interval 0.375 Å), and 30 independent docking; (2) TRP-1 enzyme activity inhibition experiment 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 the general skin surface: 4.5-6.0); Group setting: Blank control (enzyme + substrate only); Polypeptide group (1 μM, 5 μM, 10 μM, the core effective concentration is 1 μM); Positive control (100 μM arbutin); Reaction conditions: incubate at 37°C for 60 min, detect the absorbance at 475 nm (the characteristic absorption peak of dopaquinone) by a microplate reader, and calculate the enzyme inhibition rate = (blank absorbance - sample absorbance) / blank absorbance x 100%.

[0056] Experimental data Table 1 (3) Conclusion As shown in Table 1, the polypeptide can specifically target and inhibit TRP-1 activity, with an enzyme inhibition rate of 16.13% at a concentration of 1 μM (p < 0.05), and an IC50 of about 9.8 μM, which is significantly better than 100 μM arbutin (inhibition rate 14.52%, IC50 about 344 μM).

[0057] The lowest binding energy of molecular docking is -8.2 kcal / mol, which is highly consistent with the enzyme activity inhibition data, proving that the polypeptide can stably bind to the active center of TRP-1, inhibit the enzyme catalytic function by competitive binding of Cu²⁺ (hydroxyl coordination of 4-hydroxy-D-proline) and electrostatic interaction (guanidino of D-arginine binding to negative sites). The enzyme inhibition rate of 1 μM polypeptide is 16.13%, which is almost the same as the melanin inhibition rate of 15.38% found in the later experiment, indicating that the inhibition of polypeptide on melanin synthesis is completely derived from the inhibition of TRP-1 target, without other non-specific interference, and the target effectiveness is clear.

[0058] 2. Skin penetration experiment (1) Experimental content Donor concentration: 1 mM polypeptide (octanoyl-4-hydroxy-D-proline-D-arginine-NH2, molecular weight 412.5 Da); Donor volume: 2 mL of donor volume in the Franz diffusion cell supply chamber, total polypeptide amount 820.4 μg; Skin model: fresh pig ear skin, remove subcutaneous fat, thickness 300 μm, TEWL (trans-epidermal water loss) = 12.3 g / m²・h before experiment (≤15 g / m²・h, confirm the integrity of the skin barrier); Receiving pool parameters: volume 5 mL (pH 7.4 PBS), constant temperature 37℃, magnetic stirring 300 rpm; each time 1 mL (1 mL of fresh receiving liquid is supplemented to maintain constant volume), sampling time 0.5, 1, 2, 4, 6, 8, 12, 24 h; Detection method: HPLC measures the polypeptide concentration in the receiving liquid (chromatographic column Inertsil ODS-SP 4.6x250mm, mobile phase A: water containing 0.1% TFA, mobile phase B: acetonitrile containing 0.1% TFA, 0-25 min B phase 5%→90%, detection wavelength 220 nm).

[0059] Blank control: 0.9% physiological saline + 0.1% Tween 80 (no polypeptide) is added to the supply chamber to detect whether the receiving liquid is contaminated; Positive control: the positive control is 20mM nicotinamide with a molecular weight of 123Da, the concentration of the donor is verified by HPLC to be 19.8mM with a deviation of <1%, and the remaining experimental conditions are consistent with the polypeptide, which is used to verify the effectiveness of the experimental system.

[0060] Experimental data Table 2 Based on the calculation of the conventional parameters of skin anatomy, the concentration of polypeptide in the dermis layer after 24h is about 58.8μM, which ensures that the target site can be reached after transdermal penetration and active. The total amount of nicotinamide penetration is 3280.6μg, and the penetration efficiency is about 66.7%, and the Kp value is 1380x10 - 5 cm / h, which proves that the experimental system has no deviation.

[0061] (2) Conclusion At a concentration of 1mM, the optimal polypeptide (octanoyl-4-hydroxy-D-proline-D-arginine-NH2) has excellent transdermal properties on the pig ear skin model, with a 24h cumulative penetration amount Q 24 h=242.09μg / cm², transdermal rate Kp=1000x10 -5 cm / h, which is significantly better than traditional whitening agents. Although the transdermal rate of nicotinamide is slightly higher, the polypeptide can achieve melanin inhibition effect at a concentration of 1μM, while nicotinamide requires 1000μM or more, highlighting the low concentration advantage of polypeptide.

[0062] Octanoyl modification (LogP=2.1) and molecular weight optimization (412.5Da) are the keys to excellent transdermal properties, which are completely consistent with the technical design of enhancing mucosal penetration by N-terminal acyl and breaking through the transdermal threshold by molecular weight optimization.

[0063] As shown in Table 2, the polypeptide molecular weight is 412.5 Da, which is less than 500 Da, and the N-terminal octanoyl modification makes LogP = 2.1, which ensures fat solubility to penetrate the stratum corneum of the skin, and avoids the stratum corneum retention caused by too strong fat solubility, and realizes efficient transdermal penetration. The polypeptide concentration in the dermis layer corresponding to the 24h cumulative penetration amount is about 58.8μM, which proves that the polypeptide can penetrate the ordinary skin barrier and reach the effective concentration, meeting the demand of transdermal effect of whitening products.

[0064] 3. Anti-inflammatory effect experiment of ordinary skin (1) Experimental method Cell model: human immortalized keratinocytes (HaCaT cells, main functional cells of ordinary skin epidermis); Inflammation induction: 10 ng / mL TNF-a (tumor necrosis factor-a, key factor of ordinary skin inflammation) treated cells for 24h to establish inflammation model; Group setting: Normal control (without inflammation induction); Inflammation model control (TNF-a induction without drug); Polypeptide group (1μM, 5μM, effective concentration range); Positive control (1mM of glycyrrhizic acid disodium, concentration about 0.04%, common anti-inflammatory ingredient of ordinary skin); Detection index: after 48h incubation, ELISA method was used to detect the concentration of IL-6 (interleukin-6) and IL-8 (interleukin-8) in cell supernatant (core factor of ordinary skin inflammation).

[0065] Table 3 of experimental data (2) Conclusion As shown in Table 3, the polypeptide has significant anti-inflammatory effect on ordinary skin, and at a concentration of 1μM, it can reduce the IL-6 and IL-8 concentrations of TNF-a induced HaCaT cells by 45.0% and 42.9% respectively (p<0.05), which is equivalent to the effect of 0.04% glycyrrhizic acid disodium.

[0066] The anti-inflammatory mechanism is directly related to the design, and the double D-amino acid structure can significantly reduce the recognition of immune cells to exogenous polypeptide (ordinary L-amino acid polypeptide is easy to trigger immune inflammation), and the TRP-1 inhibition can indirectly reduce the release of inflammatory factors (excessive activity of TRP-1 will activate the skin inflammation pathway); The anti-inflammatory effect of 1μM polypeptide is consistent with the effective concentration of whitening, which means that it can have whitening and anti-inflammatory synergistic effect in the context of ordinary skin whitening, solving the problem of irritation of traditional whitening agents (such as niacinamide >5% concentration irritation rate ≥22%).

[0067] 4. Anti-degradation experiment of ordinary skin (1) Experimental method 1) In vitro enzymatic experiment Protease system: aminopeptidase (1 U / mL) + neutral peptidase (0.5 U / mL, the main polypeptide degradation enzyme on the surface of ordinary skin), solvent: PBS (pH 5.5, the pH of ordinary skin); Group setting: Polypeptide group of the present technology (1 μM, double D-amino acids); Control polypeptide group (1 μM, L-proline-L-arginine, traditional L-type polypeptide); Detection: incubation at 37°C, sampling at 0, 1, 2, 4, 6, and 8 h, respectively, HPLC measurement of the remaining amount of polypeptide, and calculation of the half-life (t1 / 2).

[0068] 2) In vivo retention experiment Animal model: female ICR mice (6-8 weeks old), back depilation (exposure of ordinary skin); Administration method: back application of 1 μM polypeptide solution (0.2 mL, simulating external use on ordinary skin); Sampling and detection: mice were sacrificed at 1, 2, 4, and 6 h after administration, and the back skin tissue (500 μm thick) was taken, homogenized, and then the polypeptide concentration was measured by HPLC to evaluate the retention amount in the tissue.

[0069] Table 4 of in vitro experimental data Table 5 of in vivo experimental data (3) Conclusion The anti-degradation ability of the polypeptide in the ordinary skin environment is significant, with an in vitro half-life t1 / 2=8.6 h (traditional L-type polypeptide only 1.9 h), and 18.7 ng / g of polypeptide can still be detected in the ordinary skin tissue after 6 h in vivo (corresponding to a concentration of about 0.45 μM, close to 50% of the effective concentration of 1 μM).

[0070] The anti-degradation core is derived from the double D-amino acid design: ordinary skin proteases (aminopeptidase, neutral peptidase) have stereospecificity, which can only recognize the spatial configuration of L-amino acids and cut the peptide bond, while the mirror image structure of D-amino acids cannot be recognized by the enzyme, so the polypeptide peptide bond (D-AA-D-AA) is not degraded, and the half-life is greatly prolonged; The in vivo retention data prove that the polypeptide can maintain a long action time in ordinary skin, and does not need to be frequently applied to continuously exert the whitening and anti-inflammatory effects, solving the short-acting problem of traditional L-type polypeptide with a skin half-life <2 h, and improving the product use convenience.

[0071] 5. Melanin production inhibition test (1) Experimental method B16 cells in logarithmic growth phase were selected, and the cell suspension density was adjusted to 9X105 cells per well, 2mL of the cell suspension was inoculated into a 6-well plate, and the plate was placed in a 37°C, 5% CO2 incubator. The medium was changed after 24h. After 72h of drug action, the culture solution was discarded, the cells were washed twice with PBS, 500uL of 1mol / L NaOH was added to each well, the cells were transferred to a 1.5mL centrifuge tube, and incubated in a 80°C water bath for 30min to completely dissolve the cell clumps. Then, ultrapure water was added to dilute the final concentration of NaOH to 0.2mol / L. After mixing, 100uL of each group of solution was taken and placed in a 96-well plate, and three duplicate wells were set. The OD value was measured at 475nm by using an enzyme-labeled instrument. The melanin content (%) = (OD experimental group / OD control group) x 100%.

[0072] Test object: Negative control group: complete culture medium.

[0073] Positive control group: 1000uM nicotinamide, 100uM arbutin, 1000uM nicotinamide + 100uM arbutin, 7037uM kojic acid.

[0074] Sample group: 1uM of the second peptide.

[0075] Experimental data Table 6 Referring to Table 6, under the experimental method, the sample solution with a concentration of 1uM can significantly inhibit the synthesis of melanin in B16 cells, and has a whitening effect.

[0076] The present application is not limited to the above-mentioned optional embodiments, and anyone can derive other various forms of products under the inspiration of the present application. The above-mentioned specific embodiments should not be understood as limiting the protection scope of the present application, and the protection scope of the present application should be defined by the claims, and the specification can be used to explain the claims.

Claims

1. An amino acid-based skin-whitening polypeptide, characterized in that: The general structural formula of a polypeptide is as follows: RAB-NH2 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.

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. The amino-based skin-whitening polypeptide according to claim 1, characterized in that: A is 4-hydroxy-D-proline.

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

7. The preparation method according to claim 6, characterized in that: Using triphenylmethyl resin as a carrier, D-arginine with a protecting group was first immobilized, then D-proline with a protecting group was immobilized after deprotection, and then N-terminal modification was performed. After immobilization, the peptide was cleaved and separated from the resin carrier. After preliminary purification by precipitation with ice-cold ether, the whitening peptide product was further purified by gradient elution of high performance liquid chromatography.

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

9. The preparation method according to claim 7, 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.

10. The preparation method according to claim 7, characterized in that: The molecular weight of the purified whitening polypeptide product was 412.5.

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