A short peptide compound, a preparation method thereof, a cosmetic composition, and application thereof

Abstract

The application belongs to the technical field of skin care product raw materials, and discloses a kind of short peptide compounds and preparation method, cosmetic composition and application.The short peptide compound has structural formula: the short peptide compound provided in the application contains peptide segment of tyrosine (Tyr), beta-alanine (beta-Ala), cysteine (Cys) and proline (Pro) and other antioxidant amino acids, these amino acids can directly participate in the removal of free radicals or enhance the antioxidant effect by forming a stable peptide structure, so as to obtain a short peptide compound with antioxidant and physiological repair effect.Meanwhile, the mild Fmoc route is used in the preparation method, the condensation reaction time of each step is short, the production cycle is greatly shortened, the raw material source is convenient, the process is stable, the quality is controllable, the product yield and purity are high, and the production can be scaled up.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of skin care raw materials, in particular to a short peptide compound, a preparation method, a cosmetic composition and application thereof. BACKGROUND

[0002] The application of peptides in cosmetics is mainly due to their high biological activity, easy absorption and low toxicity, they can improve and repair skin problems, especially in terms of moisturizing, whitening, anti-wrinkle and anti-aging, etc. Cosmetic peptides can be divided into several types according to their mechanism of action, such as signal peptides, neurotransmitter inhibiting peptides, carrier peptides, enzyme inhibiting peptides, etc. In addition, some specific types of peptides also have applications in cosmetics, such as the application of antibacterial peptides in acne-removing, anti-dandruff and oral care products, and the effect of composite bioactive peptides in improving skin barrier function and increasing water content in stratum corneum.

[0003] Peptides are compounds formed by condensation of two or more amino acids, and the active groups and sequences on the amino acid residues create the diversity of their structure and function, and show great prospects in the fields of biological medicine and materials. The complex relationship between the efficacy of peptide compounds and their molecular structure mainly includes the influence of amino acid composition, molecular weight, conformation and electronic properties, as well as enzyme digestion and simulated digestion conditions. Therefore, it is necessary to design and screen reasonable structures to explore ideal functional peptide raw materials, so as to provide more choices for the development and application of skin care products. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a short peptide compound, a preparation method, a cosmetic composition and application thereof, aiming to solve the problem that the prior art lacks ideal functional peptide raw materials for the development and application of skin care products.

[0005] The technical scheme of the present application is as follows:

[0006] In a first aspect of the present application, a short peptide compound is provided, which has the following structural formula:

[0007]

[0008] In a second aspect of the present application, a preparation method of the short peptide compound is provided, comprising the following steps:

[0009] (1) Preparation of Lys: Fmoc-L-Lys(Boc)-Wang Resin is taken into a reactor, and a dimethylformamide solution of piperidine is added for Fmoc removal, and solid-liquid separation is performed to obtain L-Lys(Boc)-Wang Resin;

[0010] (2) Coupling of Pro: Fmoc-L-Pro-OH, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing L-Lys(Boc)-Wang Resin, after reaction, de-Fmoc was carried out with piperidine in dimethylformamide solution, solid-liquid separation was carried out, and L-Pro-L-Lys(Boc)-Wang Resin was obtained;

[0011] (3) Coupling of Cys: Fmoc-L-Cys(Dpm)-OH, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing L-Pro-L-Lys(Boc)-Wang Resin, after reaction, de-Fmoc was carried out with piperidine in dimethylformamide solution, solid-liquid separation was carried out, and L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin was obtained;

[0012] (4) Coupling of Arg: Fmoc-L-Arg(Pbf)-OH, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, after reaction, de-Fmoc was carried out with piperidine in dimethylformamide solution, solid-liquid separation was carried out, and L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin was obtained;

[0013] (5) Coupling of Ile: Fmoc-L-Ile-OH, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, after reaction, de-Fmoc was carried out with piperidine in dimethylformamide solution, solid-liquid separation was carried out, and L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin was obtained;

[0014] (6), coupling of Thr: Fmoc-L-Thr(tBu)-OH, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, after reaction, de-Fmoc was carried out with piperidine in dimethylformamide solution, solid-liquid separation was carried out, and L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin was obtained;

[0015] (7), coupling of Lys: Fmoc-L-Lys(Boc)-OH, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, after reaction, de-Fmoc was carried out with piperidine in dimethylformamide solution, solid-liquid separation was carried out, and L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin was obtained;

[0016] (8), coupling of Phe: Fmoc-L-Phe-OH, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, after reaction, de-Fmoc was carried out with piperidine in dimethylformamide solution, solid-liquid separation was carried out, and L-Phe-L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin was obtained;

[0017] (9) Coupling of Fmoc-β-Ala: Fmoc-β-Ala, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing L-Phe-L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, after reaction, de-Fmoc was carried out with piperidine in dimethylformamide solution, solid-liquid separation was carried out, and β-Ala-L-Phe-L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin was obtained;

[0018] (10) Coupling of nicotinic acid: nicotinic acid, 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide in dimethylformamide solution were added to the reactor containing the β-Ala-L-Phe-L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and the reaction was carried out for 2-3 h, and after the reaction product was washed and dried, a solid-phase resin was obtained;

[0019] (11) The solid-phase resin in step (10) was cleaved with a cleavage solution, and after rotary evaporation, a reaction residue was obtained, the reaction residue was precipitated with isopropyl ether, and after separation and purification, the short peptide compound was obtained.

[0020] Optionally, in steps (1)-(9), the mass concentration of the piperidine in dimethylformamide solution is 10-20 wt%.

[0021] Optionally, in steps (1)-(9), the solid-liquid separation step comprises: separating a solid-phase material by suction filtration, and repeatedly washing the solid-phase material with dimethylformamide, methanol and dichloromethane.

[0022] Optionally, in step (10), the molar ratio of nicotinic acid, 1-hydroxybenzotriazole, N, N'-diisopropylcarbodiimide to Fmoc-L-Lys(Boc)-Wang Resin is (1.8-2.5):(1.8-2.5):(1.8-2.5):1.

[0023] Optionally, in step (11), the cleavage solution is trifluoroacetic acid cleavage solution, and the cleavage time is 2-3 h.

[0024] In a third aspect, the present application provides a cosmetic composition, characterized in that it comprises the short peptide compound of claim 1 and a cosmetically acceptable carrier, wherein the cosmetically acceptable carrier optionally comprises one or more of water, glycerol, alcohol, vegetable oil, and petrolatum.

[0025] Optionally, the short peptide compound has an antioxidant effective concentration of 100-4000 ppm in the cosmetic composition.

[0026] Optionally, the short peptide compound has a repair effective concentration of 100-1000 ppm in the cosmetic composition.

[0027] In a fourth aspect, the present application provides use of the cosmetic composition in skin care products and cosmetics.

[0028] Advantages:

[0029] The short peptide compound provided by the present application has significant antioxidant and repair physiological effects by using a peptide segment containing antioxidant amino acids such as tyrosine (Tyr), beta-alanine (β-Ala), cysteine (Cys), and proline (Pro). These amino acids can directly participate in the removal of free radicals or enhance the antioxidant effect by forming a stable peptide structure, thereby obtaining a short peptide compound with antioxidant and repair physiological effects. Meanwhile, the mild Fmoc route is used in the preparation method, the condensation reaction time of each step is short, the production cycle is greatly shortened, the raw material source is convenient, the process is stable, the quality is controllable, the product yield and purity are high, and the product can be produced on a large scale. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A mass spectrum of the short peptide compound K9 prepared in Example 1 of the present application.

[0031] Figure 2 A high performance liquid chromatography (HPLC) detection spectrum of the short peptide compound K9 prepared in Example 1 of the present application.

[0032] Figure 3 A result graph of the effect of the short peptide compound K9 prepared in Example 1 of the present application on cell proliferation in a cytotoxicity test.

[0033] Figure 4 A result graph of the effect of the short peptide compound K9 prepared in Example 1 of the present application on cell apoptosis in a cytotoxicity test.

[0034] Figure 5 Gene expression of keratin 1 (KRT-1) in a cell repair test of the short peptide compound K9 prepared in Example 1 of the present application.

[0035] Figure 6 Gene expression of keratin 5 (KRT-5) in a cell repair test of the short peptide compound K9 prepared in Example 1 of the present application.

[0036] Figure 7 Gene expression of keratin 10 (KRT-10) in a cell repair test of the short peptide compound K9 prepared in Example 1 of the present application.

[0037] Figure 8 Gene expression of keratin 14 (KRT-14) in a cell repair test of the short peptide compound K9 prepared in Example 1 of the present application.

[0038] Figure 9 Gene expression of hyaluronan synthase 3 (HAS3) in a cell repair test of the short peptide compound K9 prepared in Example 1 of the present application.

[0039] Figure 10 Gene expression of aquaporin 3 (AQP3) in a cell repair test of the short peptide compound K9 prepared in Example 1 of the present application.

[0040] Figure 11 ABTS radical scavenging rate result graph in the antioxidant efficacy test of the control group VC.

[0041] Figure 12 ABTS radical scavenging rate result graph in the antioxidant efficacy test of the short peptide compound K9 prepared in Example 1 of the present application.

[0042] Figure 13 DPPH radical scavenging rate result graph in the antioxidant efficacy test of the control group VC.

[0043] Figure 14 DPPH radical scavenging rate result graph in the antioxidant efficacy test of the short peptide compound K9 prepared in Example 1 of the present application. DETAILED DESCRIPTION

[0044] The present application provides a short peptide compound, a preparation method thereof, a cosmetic composition and an application. In order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0046] The following is described in detail through specific examples.

[0047] Example 1

[0048] Preparation of short peptide compound K9

[0049] The reagent materials used in the examples are as follows:

[0050] Fmoc-L-Lys(Boc)-Wang Resin (9-fluorenylmethoxycarbonyl-L-lysine-γ-tert- butyloxycarbonyl-Wang Resin), Fmoc-L-Pro-OH (9-fluorenylmethoxycarbonyl-L- proline), Fmoc-L-Cys(Dpm)-OH (9-fluorenylmethoxycarbonyl-L-cysteine (diphenylmethyl-protected thiol group)), Fmoc-L-Arg(Pbf)-OH (9-fluorenylmethoxycarbonyl-L-arginine (2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)), Fmoc-L-Ile(Boc)-OH (9-fluorenylmethoxycarbonyl-L-isoleucine-ε-tert-butyloxycarbonyl), Fmoc-L-Thr(tBu)-OH (9-fluorenylmethoxycarbonyl-L-threonine (tert-butyl-protected hydroxyl group)), Fmoc-L-Lys(Boc)-OH (9-fluorenylmethoxycarbonyl-L-lysine-ε-tert-butyloxycarbonyl), Fmoc-Phe-OH (9-fluorenylmethoxycarbonyl-L-phenylalanine), Fmoc-β-Ala-OH (9-fluorenylmethoxycarbonyl-β-alanine), nicotinic acid, HOBT (1-hydroxybenzotriazole), DIC (N,N'-diisopropylcarbodiimide), piperidine;

[0051] Solvents: DMF (N,N-dimethylformamide), dichloromethane, TFA (trifluoroacetic acid), ACN (acetonitrile), ice isopropyl ether.

[0052] The specific operation procedure for solid-phase synthesis of K9 using Fmoc (fluorenylmethoxycarbonyl) protection is as follows:

[0053] (1) Preparation of the first Lys: 0.67 mmol / g of Fmoc-L-Lys(Boc)-Wang Resin (12 g, 8 mmol, 1.0 equivalent) was subjected to Fmoc deprotection with 20% piperidine in DMF (150 mL), and the reaction was allowed to proceed for about 30 min. A small amount of the resin was taken for ninhydrin detection, and the result was positive, indicating that the reaction was complete. After stopping the reaction, the reaction liquid was suctioned dry, and the obtained resin was washed with 200 mL of DMF twice, each time for 2 min; and washed with dichloromethane once, each time for 2 min.

[0054] (2) Coupling of the second Pro: The raw material Fmoc-L-Pro-OH (5.4 g, 16 mmol, 2.0 eq), HOBT (2.7 g, 20 mmol, 2.5 eq), DIC (2.52 g, 20 mmol, 2.5 eq) were added into the reactor, DMF (100 mL), and the reaction was allowed to proceed for 2-3 h. A small amount of resin was taken for ninhydrin test, and the result was negative, indicating that the reaction was complete. After stopping the reaction, the reaction solution was drained, and the obtained resin was washed with 200 mL of DMF twice, each time for 2 min. The Fmoc protecting group was removed using 20% piperidine in DMF (150 mL), and the reaction was allowed to proceed for about 30 min. A small amount of resin was taken for ninhydrin test, and the result was positive, indicating that the reaction was complete. After stopping the reaction, the reaction solution was drained, and the obtained resin was washed with 200 mL of DMF twice, each time for 2 min; and 100 mL of dichloromethane once, each time for 2 min.

[0055] (3) Coupling of the third Cys: The raw material Fmoc-L-Cys(Dpm)-OH (8.15 g, 16 mmol, 2.0 eq), HOBT (2.7 g, 20 mmol, 2.5 eq), DIC (2.52 g, 20 mmol, 2.5 eq) were added into the reactor, DMF (100 mL), and the reaction was allowed to proceed for 2-3 h. A small amount of resin was taken for ninhydrin test, and the result was negative, indicating that the reaction was complete. After stopping the reaction, the reaction solution was drained, and the obtained resin was washed with 200 mL of DMF twice, each time for 2 min. The Fmoc protecting group was removed using 20% piperidine in DMF (150 mL), and the reaction was allowed to proceed for about 30 min. A small amount of resin was taken for ninhydrin test, and the result was positive, indicating that the reaction was complete. After stopping the reaction, the reaction solution was drained, and the obtained resin was washed with 200 mL of DMF twice, each time for 2 min; and 100 mL of dichloromethane once, each time for 2 min.

[0056] (4) Coupling of the fourth Arg: The raw material Fmoc-L-Arg(Pbf)-OH (10.38 g, 16 mmol, 2.0 eq), HOBT (2.7 g, 20 mmol, 2.5 eq), DIC (2.52 g, 20 mmol, 2.5 eq) were added into the reactor, DMF (100 mL), and the reaction was allowed to proceed for 2-3 h. A small amount of resin was taken for ninhydrin test, and the result was negative, indicating that the reaction was complete. After stopping the reaction, the reaction solution was drained, and the obtained resin was washed with 200 mL of DMF twice, each time for 2 min. The Fmoc protecting group was removed using 20% piperidine in DMF (150 mL), and the reaction was allowed to proceed for about 30 min. A small amount of resin was taken for ninhydrin test, and the result was positive, indicating that the reaction was complete. After stopping the reaction, the reaction solution was drained, and the obtained resin was washed with 200 mL of DMF twice, each time for 2 min; and 100 mL of dichloromethane once, each time for 2 min.

[0057] (5), the coupling of the fifth lie: to the reactor is added raw material Fmoc-L-Ile-OH (5.65 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), DMF (100 mL), and the reaction is allowed to proceed for 2-3 h, and a small amount of resin is taken for ninhydrin detection, which is negative, indicating that the reaction is complete. After stopping the reaction, the reaction solution is drained, and the resulting resin is washed twice with 200 mL of DMF, each time for 2 min. The Fmoc protecting group is removed using 20% piperidine in DMF (150 mL), and the reaction is allowed to proceed for about 30 min. A small amount of resin is taken for ninhydrin detection, which is positive, indicating that the reaction is complete. After stopping the reaction, the reaction solution is drained, and the resulting resin is washed twice with 200 mL of DMF, each time for 2 min; and once with 100 mL of dichloromethane, each time for 2 min.

[0058] (6), the coupling of the sixth Thr: to the reactor is added raw material Fmoc-L-Thr(tBu)-OH (6.36 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), DMF (100 mL), and the reaction is allowed to proceed for 2-3 h, and a small amount of resin is taken for ninhydrin detection, which is negative, indicating that the reaction is complete. After stopping the reaction, the reaction solution is drained, and the resulting resin is washed twice with 200 mL of DMF, each time for 2 min. The Fmoc protecting group is removed using 20% piperidine in DMF (150 mL), and the reaction is allowed to proceed for about 30 min. A small amount of resin is taken for ninhydrin detection, which is positive, indicating that the reaction is complete. After stopping the reaction, the reaction solution is drained, and the resulting resin is washed twice with 200 mL of DMF, each time for 2 min; and once with 100 mL of dichloromethane, each time for 2 min.

[0059] (7), the coupling of the seventh Lys: to the reactor is added raw material Fmoc-L-Lys(Boc)-OH (7.50 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), DMF (100 mL), and the reaction is allowed to proceed for 2-3 h, and a small amount of resin is taken for ninhydrin detection, which is negative, indicating that the reaction is complete. After stopping the reaction, the reaction solution is drained, and the resulting resin is washed twice with 200 mL of DMF, each time for 2 min. The Fmoc protecting group is removed using 20% piperidine in DMF (150 mL), and the reaction is allowed to proceed for about 30 min. A small amount of resin is taken for ninhydrin detection, which is positive, indicating that the reaction is complete. After stopping the reaction, the reaction solution is drained, and the resulting resin is washed twice with 200 mL of DMF, each time for 2 min; and once with 100 mL of dichloromethane, each time for 2 min.

[0060] (8) Coupling of the eighth Phe: To the reactor was added the starting material Fmoc-Phe-OH (6.20 g, 16 mmol, 2.0 equiv), HOBT (2.7 g, 20 mmol, 2.5 equiv), DIC (2.52 g, 20 mmol, 2.5 equiv), DMF (100 mL), and the reaction was allowed to proceed for 2-3 h. A small amount of resin was removed and tested by the ninhydrin test, which was negative, indicating that the reaction was complete. The reaction was stopped and the reaction mixture was drained. The resulting resin was washed twice with 200 mL of DMF for 2 min each time. The Fmoc protecting group was removed using 20% piperidine in DMF (150 mL) for about 30 min. A small amount of resin was removed and tested by the ninhydrin test, which was positive, indicating that the reaction was complete. The reaction was stopped and the reaction mixture was drained. The resulting resin was washed twice with 200 mL of DMF for 2 min each time; once with 100 mL of dichloromethane for 2 min; and once with 100 mL of methanol for 2 min.

[0061] (9) Coupling of the ninth Fmoc-β-Ala-OH: To the reactor was added the starting material Fmoc-β-alanine (5.0 g, 16 mmol, 2.0 equiv), HOBT (2.7 g, 20 mmol, 2.5 equiv), DIC (2.52 g, 20 mmol, 2.5 equiv), DMF (100 mL), and the reaction was allowed to proceed for 2-3 h. A small amount of resin was removed and tested by the ninhydrin test, which was negative, indicating that the reaction was complete. The reaction was stopped and the reaction mixture was drained. The resulting resin was washed twice with 200 mL of DMF for 2 min each time. The Fmoc protecting group was removed using 20% piperidine in DMF (150 mL) for about 30 min. A small amount of resin was removed and tested by the ninhydrin test, which was positive, indicating that the reaction was complete. The reaction was stopped and the reaction mixture was drained. The resulting resin was washed twice with 200 mL of DMF for 2 min each time; once with 100 mL of dichloromethane for 2 min each time.

[0062] (10) Coupling of the tenth nicotinic acid: To the reactor was added the starting material nicotinic acid (2.0 g, 16 mmol, 2.0 equiv), HOBT (2.7 g, 20 mmol, 2.5 equiv), DIC (2.52 g, 20 mmol, 2.5 equiv), DMF (100 mL), and the reaction was allowed to proceed for 2-3 h. A small amount of resin was removed and tested by the ninhydrin test, which was negative, indicating that the reaction was complete. The reaction was stopped and the reaction mixture was drained. The resulting resin was washed twice with 200 mL of DMF for 2 min each time; once with 100 mL of dichloromethane for 2 min each time; once with 100 mL of methanol for 2 min each time; once with 100 mL of dichloromethane for 2 min each time; and once with 100 mL of methanol for 2 min each time. The resin was dried in a vacuum oven at 40 °C overnight.

[0063] (11) The resin was cleaved using a 94% trifluoroacetic acid solution at 0°C as a classic cleavage solution for approximately 3 hours. After the reaction, the filtrate was concentrated by rotary evaporation to remove most of the trifluoroacetic acid. The resulting reaction residue was precipitated with ice-cold isopropyl ether, centrifuged to precipitate the crude peptide, and finally vacuum dried.

[0064] 3. Purification steps

[0065] (1) First, the crude peptide obtained above was subjected to sample purity analysis. After determining the retention time of the main peak, the sample was analyzed by linear gradient analysis;

[0066] (2) Weigh 50 mg of crude peptide and dissolve it in mobile phase to dilute it to 3 mL. After filtering with a 0.45 μm organic filter membrane, perform gradient separation using a high-performance liquid chromatograph. Collect the mobile phase corresponding to the retention time of the target absorption peak and freeze-dry to obtain the target peptide, i.e., a short peptide compound, which is recorded as K9.

[0067] Chromatographic column: C18 column (4.6×250mm, 5μl)

[0068] Mobile phase A: 0.1% trifluoroacetic acid / water solution

[0069] Mobile B: Acetonitrile

[0070] Flow rate: 1 mL / min

[0071] Column temperature: 30°C

[0072] Detection wavelength: 250nm, 210nm

[0073] Injection volume: 5 μl

[0074] The time-mobile phase gradient for separation of the short peptide compound K9 peptide by high performance liquid chromatography is shown in Table 1 below:

[0075] Table 1

[0076]

[0077]

[0078] (3) 200 mg of sample was placed in a 10 mL dissolution bottle, deionized water was added, and after ultrasonic dissolution, it was filtered with a 0.45 μm organic phase filter and the supernatant was collected. This step was used to mass-produce the short peptide compound K9;

[0079] (4) Put it into high performance liquid chromatography, collect the peak by linear gradient preparation, and confirm whether the MS is correct;

[0080] (5) In order to meet the required purity of the liquid volume, the purity of the collected liquid should be tracked and fed back;

[0081] (6), the collected solution was concentrated to 40-50 mL at 40°C;

[0082] (7), the concentrated solution was placed in a 100 mL beaker and frozen in a freezer;

[0083] (8), after HPLC purification, freeze-drying was performed to obtain the short peptide compound K9 of the present application.

[0084] Test Example 1

[0085] Mass spectrum of K9

[0086] The short peptide compound prepared in Example 1 was subjected to mass spectrometry test, and the test results are shown in Figure 1 , which proves that the short peptide compound K9 is successfully synthesized in the present application.

[0087] Test Example 2

[0088] Purity test of K9

[0089] The short peptide compound prepared in Example 1 was subjected to high performance liquid chromatography (HPLC) detection, and the detection results are shown in Figure 2 , according to the peak area in the figure, the purity of K9 was determined to be 96.11%.

[0090] Test Example 3

[0091] Cytotoxicity test of short peptide compound K9

[0092] Test 1: CCK8 detection kit was used to detect the effect on cell proliferation. The specific operation is as follows: human immortalized epidermal cells (HaCaT cells) and mouse monocyte macrophage leukemia cells (RAW264.7 cells) were cultured in a 37°C, 5% CO2 cell incubator with DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin & streptomycin complete medium (hereinafter referred to as complete medium)

[0093] HaCaT cells and RAW264.7 cells were respectively plated in a 96-well plate at 10000 cells per well for 24 hours, and 100 microliters of the above complete medium containing short peptide compound K9 (0-2000 ppm) was added, 6 replicates per group. After incubation for 24 hours, the old culture medium was removed, and 110 microliters of culture medium containing CCK8 solution (complete medium:CCK8 = 10:1) was added. After incubation in a 37°C, 5% CO2 cell incubator for 2 hours, the absorbance of each well was measured by a microplate reader at 450 nm, and the cell survival rate was calculated.

[0094] The experimental results are shown in Figure 3As shown in the results, it is shown that the short peptide compound K9 has no effect on the proliferation of HaCaT cells and RAW264.7 cells in the range of 0-1000 ppm.

[0095] Test 2: The effect on apoptosis was detected by flow cytometry, and the specific operation was as follows: human immortalized epidermal cells (HaCaT cells) were cultured in a 37°C, 5% CO2 cell incubator with DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin & streptomycin complete medium (hereinafter referred to as complete medium). HaCaT cells were plated in a 6-well plate at 200,000 cells per well for 24 hours, and 2 mL of complete medium containing K9 was added. The experiment was divided into three concentrations of 0, 100 and 300 ppm, with 3 replicate holes in each group. After 48 hours of incubation, the cells were collected and the apoptosis was detected by flow cytometry.

[0096] The experimental results are shown in Figure 4 As shown in the results, the apoptosis rate is below 1% and the cell survival rate is above 99% as in the blank control group, indicating that the short peptide compound K9 prepared in the embodiment has high safety.

[0097] Test Example 5

[0098] Effect of short peptide compound K9 on cell repair

[0099] Human immortalized epidermal cells (HaCaT cells) were cultured in a 37°C, 5% CO2 cell incubator with DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin & streptomycin complete medium (hereinafter referred to as complete medium). HaCaT cells were plated in a 6-well plate at 200,000 cells per well for 24 hours, and 2 mL of complete medium containing K9 was added. The experiment was divided into three concentrations of 0, 100 and 300 ppm, with 3 replicate holes in each group. After 48 hours of incubation, the cells were collected and the expression of keratin 1 (KRT-1), keratin 5 (KRT-5), keratin 10 (KRT-10), keratin 14 (KRT-14), hyaluronic acid synthase 3 (HAS3), and aquaporin 3 (AQP3) genes was detected by fluorescence quantitative PCR method. The results are shown in Figures 5-10 As shown in the results, compared with the blank control group, the short peptide compound K9 significantly promoted the expression of related genes, indicating that the short peptide compound prepared in the embodiment has good repair effect.

[0100] Test Example 6

[0101] Antioxidant efficacy test of short peptide compound K9

[0102] The standard GB / T 39100-2020 was referred to, and the DPPH and ABTS method was used for polypeptide antioxidant test.

[0103] The experimental results are shown inFigures 11-14 As a result, the short peptide compound K9 has an ABTS free radical scavenging rate of 80.93% and a DPPH free radical scavenging rate of 84.72% at a concentration of 500 ppm, indicating that the short peptide compound prepared in the embodiment of the present application has a significant antioxidant effect compared with vitamin C.

[0104] It should be understood that the application is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. A short peptide compound, characterized in that The structural formula of the short peptide compound is shown below:

2. The method for preparing a short peptide compound according to claim 1, characterized in that: Including steps: (1) Preparation of Lys: Fmoc-L-Lys(Boc)-Wang Resin was added to a reactor, and a dimethylformamide solution of piperidine was added to remove Fmoc, and solid-liquid separation was performed to obtain L-Lys(Boc)-Wang Resin; (2) Coupling of Pro: Fmoc-L-Pro-OH, 1-hydroxybenzotriazole and a dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to a reactor containing the L-Lys(Boc)-Wang Resin, and the mixture was reacted. Then, Fmoc removal was performed using a dimethylformamide solution of piperidine, and solid-liquid separation was performed to obtain L-Pro-L-Lys(Boc)-Wang Resin. (3) Coupling of Cys: Fmoc-L-Cys(Dpm)-OH, 1-hydroxybenzotriazole and a dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to a reactor containing the L-Pro-L-Lys(Boc)-Wang Resin, and the mixture was reacted. Then, Fmoc removal was performed using a dimethylformamide solution of piperidine, and solid-liquid separation was performed to obtain L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin. (4) Coupling of Arg: Fmoc-L-Arg(Pbf)-OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide in dimethylformamide were added to the reactor containing the L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and the reaction was carried out. Then, Fmoc removal was carried out using a dimethylformamide solution of piperidine, and solid-liquid separation was performed to obtain L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin; (5) Coupling of Ile: Fmoc-L-Ile-OH, 1-hydroxybenzotriazole and a dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to a reactor containing the L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and the reaction was carried out. Then, Fmoc removal was carried out using a dimethylformamide solution of piperidine, and solid-liquid separation was performed to obtain L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin; (6) Thr coupling: Fmoc-L-Thr(tBu)-OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide in dimethylformamide were added to the reactor containing the L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and the mixture was reacted. Then, Fmoc removal was performed using a dimethylformamide solution of piperidine, and solid-liquid separation was performed to obtain L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin. (7) Coupling of Lys: Fmoc-L-Lys(Boc)-OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide in dimethylformamide were added to the reactor containing the L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and the mixture was reacted. Then, Fmoc removal was performed using a dimethylformamide solution of piperidine, and solid-liquid separation was performed to obtain L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin. (8) Coupling of Phe: Fmoc-Phe-OH, 1-hydroxybenzotriazole and a dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to a reactor containing the L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and the mixture was reacted. Then, Fmoc removal was performed using a dimethylformamide solution of piperidine, and solid-liquid separation was performed to obtain L-Phe-L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin. (9) Coupling of Fmoc-β-alanine: Fmoc-β-alanine, 1-hydroxybenzotriazole and a dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to a reactor containing the L-Phe-L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and the mixture was reacted. Then, Fmoc removal was performed using a dimethylformamide solution of piperidine, and solid-liquid separation was performed to obtain β-Ala-L-Phe-L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin. (10) Coupling of nicotinic acid: add a dimethylformamide solution of nicotinic acid, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide to a reactor containing the β-Ala-L-Phe-L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and react for 2 to 3 hours. Wash and dry the reaction product to obtain a solid phase resin; (11) The solid phase resin in step (10) is cracked using a cracking solution, and a reaction residue is obtained after rotary evaporation. The reaction residue is precipitated with isopropyl ether, and the short peptide compound is obtained after separation and purification.

3. The method for preparing a short peptide compound according to claim 2, characterized in that: In steps (1) to (9), the mass concentration of the piperidine dimethylformamide solution is 10 to 20 wt%.

4. The method for preparing a short peptide compound according to claim 2, wherein: In steps (1) to (9), the solid-liquid separation step includes: separating the solid phase material by suction filtration, and repeatedly washing the solid phase material with dimethylformamide, methanol and dichloromethane in sequence.

5. The method for preparing a short peptide compound according to claim 2, characterized in that: In step (10), the molar ratio of the nicotinic acid, 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide and the Fmoc-L-Lys(Boc)-Wang Resin is (1.8-2.5):(1.8-2.5):(1.8-2.5):

1.

6. The method for preparing a short peptide compound according to claim 2, characterized in that: In step (11), the lysis solution is trifluoroacetic acid lysis solution, and the lysis time is 2 to 3 hours.

7. A cosmetic composition, characterized in that The invention comprises the short peptide compound according to claim 1 and a cosmetically acceptable carrier, wherein the cosmetically acceptable carrier is one or more of water, alcohols, vegetable oils, and vaseline.

8. The cosmetic composition according to claim 7, wherein The antioxidant effective concentration of the short peptide compound in the cosmetic composition is 100 to 4000 ppm.

9. The cosmetic composition according to claim 7, wherein The repair-effective concentration of the short peptide compound in the cosmetic composition is 100 to 1000 ppm.

10. Use of the cosmetic composition according to any one of claims 7 to 9 in the preparation of skin care products and cosmetics.

Images