Oligopeptide compound, preparation method thereof, beautifying composition and application

Abstract

The invention belongs to the technical field of skin care product raw materials, and discloses an oligopeptide compound, a preparation method thereof, a beautifying composition and application. The oligopeptide compound has a structural formula of # imgabs0. The oligopeptide compound provided by the invention contains peptide fragments of antioxidant amino acids such as tyrosine (Tyr), beta-alanine (beta-Ala), cysteine (Cys) and proline (Pro), and the amino acids can directly participate in removal of free radicals or form a stable peptide structure to enhance the antioxidant effect; therefore, the oligopeptide compound with anti-oxidation and physiological repair effects is obtained. Meanwhile, a mild Fmoc route is adopted in the preparation method, the condensation reaction time of each step is short, the production period is greatly shortened, raw materials are convenient to obtain, the process is stable, the quality is controllable, the product yield and purity are high, and large-scale production can be achieved.

Description

Technical Field

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

[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 moisturizing, whitening, anti-wrinkle and anti-aging. Beauty peptides can usually be divided into several types according to their mechanism of action, such as signal peptides, neurotransmitter inhibitory peptides, carrier peptides, enzyme inhibitory peptides, etc. In addition, some specific types of peptides are also used in cosmetics, such as antibacterial peptides used in acne, dandruff and oral products, and composite bioactive peptide cosmetics in improving skin barrier function and increasing the moisture content of the stratum corneum.

[0003] Peptides are compounds formed by the condensation reaction of two or more amino acids. The active groups and sequences on the amino acid residues create its diversity in structure and efficacy, and show great prospects in the fields of biomedicine 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 enzymatic hydrolysis and simulated digestion conditions. Therefore, it is necessary to discover ideal functional peptide raw materials through reasonable structural design and screening, so as to provide more options for the development and application of skin care products. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a short peptide compound and 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 solution of the present invention is as follows:

[0006] The first aspect of the present invention provides a short peptide compound having the structural formula shown below:

[0007]

[0008] The second aspect of the present invention provides a method for preparing the short peptide compound, comprising the steps of:

[0009] (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;

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

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

[0012] (4) Coupling of Arg: Fmoc-L-Arg(Pbf)-OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide in dimethylformamide were added to a reactor containing L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin for reaction, followed by removal of Fmoc using a dimethylformamide solution of piperidine, followed by solid-liquid separation to obtain L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin;

[0013] (5) Coupling of Ile: Fmoc-L-Ile-OH, 1-hydroxybenzotriazole and dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to a reactor containing L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin, and after reaction, Fmoc removal was performed with 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;

[0014] (6) Coupling of Thr: Fmoc-L-Thr(tBu)-OH, 1-hydroxybenzotriazole and dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to the reactor containing L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin to react, and then Fmoc removal was carried out with 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;

[0015] (7) Coupling of Lys: Fmoc-L-Lys(Boc)-OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide in dimethylformamide were added to a reactor containing L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin to react, and then Fmoc removal was performed with 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;

[0016] (8) Coupling of Phe: Fmoc-L-Phe-OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide in dimethylformamide were added to a reactor containing L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin to react, and 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;

[0017] (9) Coupling of Fmoc-β-Ala: Fmoc-β-Ala, 1-hydroxybenzotriazole and a dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to a 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 to react, and 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;

[0018] (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, react for 2 to 3 hours, wash and dry the reaction product to obtain a solid phase resin;

[0019] (11) using a cleavage solution to cleave the solid phase resin in step (10), obtaining a reaction residue after rotary evaporation, precipitating the reaction residue with isopropyl ether, and obtaining the short peptide compound after separation and purification.

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

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

[0022] Optionally, 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.

[0023] Optionally, in step (11), the lysis solution is a trifluoroacetic acid lysis solution, and the lysis time is 2 to 3 hours.

[0024] The third aspect of the present invention provides a cosmetic composition, characterized in that it comprises the short peptide compound according to claim 1 and a cosmetically acceptable carrier, wherein, optionally, the cosmetically acceptable carrier comprises one or more of water, glycerin, alcohols, vegetable oils, and vaseline.

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

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

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

[0028] Beneficial effects:

[0029] The short peptide compound provided by the present invention has significant antioxidant and repair physiological effects by using peptide segments containing antioxidant amino acids such as tyrosine (Tyr), β-alanine (β-Ala), cysteine ​​(Cys) and proline (Pro). These amino acids can directly participate in the scavenging 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. At the same time, the preparation method adopts a mild Fmoc route, and the condensation reaction time of each step is short, which greatly shortens the production cycle, and the raw material source is convenient, the process is stable, the quality is controllable, the product yield and purity are high, and large-scale production can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the mass spectrometer of the short peptide compound K9 prepared in Example 1 of the present invention.

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

[0032] Figure 3 This is a graph showing the effect of the short peptide compound K9 prepared in Example 1 of the present invention on cell proliferation in a cytotoxicity test.

[0033] Figure 4 This is a graph showing the effect of the short peptide compound K9 prepared in Example 1 of the present invention on cell apoptosis in a cytotoxicity test.

[0034] Figure 5 This is the gene expression of keratin 1 (KRT-1) in the cell repair test of the short peptide compound K9 prepared in Example 1 of the present invention.

[0035] Figure 6 This is the gene expression of keratin 5 (KRT-5) in the cell repair test of the short peptide compound K9 prepared in Example 1 of the present invention.

[0036] Figure 7 This is the gene expression of keratin 10 (KRT-10) in the cell repair test of the short peptide compound K9 prepared in Example 1 of the present invention.

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

[0038] Fig. 9 This is the gene expression of hyaluronan synthase 3 (HAS3) in the cell repair experiment of the short peptide compound K9 prepared in Example 1 of the present invention.

[0039] Fig.10 The gene expression of aquaporin 3 (AQP3) in a cell repair test of the short peptide compound K9 prepared in Example 1 of the present invention.

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

[0041] Fig.12 This is a graph showing the ABTS free radical scavenging rate results of the short peptide compound K9 prepared in Example 1 of the present invention in the antioxidant efficacy test.

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

[0043] Fig.14 This is a graph showing the DPPH free radical scavenging rate results of the short peptide compound K9 prepared in Example 1 of the present invention in the antioxidant efficacy test. DETAILED DESCRIPTION

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

[0045] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0046] The following describes it in detail through specific embodiments.

[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)), Fmoc-L-Arg(Pbf)-OH(9-Fluorenylmethoxycarbonyl-L-arginine(2,2,4,6,7-pentamethyldihydrobenzofuran-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)), 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), glacial isopropyl ether.

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

[0053] (1) Preparation of the first Lys: Weigh 0.67mmol / g of Fmoc-L-Lys(Boc)-Wang Resin (12g, 8mmol, 1.0 equivalent), remove the Fmoc protecting group with 20% piperidine in DMF solution (150mL), and react for about 30min. Take a small amount of resin for ninhydrin test, the result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, wash the obtained resin with 200mL DMF twice, each time for 2 minutes; wash once with dichloromethane, each time for 2 minutes.

[0054] (2) Second coupling of Pro: Add raw materials Fmoc-L-Pro-OH (5.4 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), and DMF (100 mL) to the reactor, react for 2-3 hours, take a small amount of resin for ninhydrin detection, the result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Use 20% piperidine DMF solution (150 mL) to remove the Fmoc protecting group, and react for about 30 minutes. Take a small amount of resin for ninhydrin detection, the result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes; wash once with 100 mL dichloromethane, each time for 2 minutes.

[0055] (3) Coupling of the third Cys: Add raw materials Fmoc-L-Cys(Dpm)-OH (8.15 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), and DMF (100 mL) into the reactor and react for 2-3 hours. Take a small amount of resin for ninhydrin detection. The result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Use 20% piperidine in DMF solution (150 mL) to remove the Fmoc protecting group and react for about 30 minutes. Take a small amount of resin for ninhydrin detection. The result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes; wash once with 100 mL dichloromethane, each time for 2 minutes.

[0056] (4) Coupling of the fourth Arg: Add the raw materials Fmoc-L-Arg(Pbf)-OH (10.38 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), and DMF (100 mL) to the reactor, and react for 2-3 hours. Take a small amount of resin for ninhydrin detection, and the result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Use 20% piperidine DMF solution (150 mL) to remove the Fmoc protecting group, and react for about 30 minutes. Take a small amount of resin for ninhydrin detection, and the result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes; wash once with 100 mL dichloromethane, each time for 2 minutes.

[0057] (5) Coupling of the fifth Ile: Add raw materials 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), and DMF (100 mL) to the reactor, react for 2-3 hours, take a small amount of resin for ninhydrin detection, the result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Use 20% piperidine DMF solution (150 mL) to remove the Fmoc protecting group, and react for about 30 minutes. Take a small amount of resin for ninhydrin detection, the result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes; wash once with 100 mL dichloromethane, each time for 2 minutes.

[0058] (6) Coupling of the sixth Thr: Add raw materials 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), and DMF (100 mL) to the reactor and react for 2-3 hours. Take a small amount of resin for ninhydrin detection. The result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Use 20% piperidine DMF solution (150 mL) to remove the Fmoc protecting group and react for about 30 minutes. Take a small amount of resin for ninhydrin detection. The result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes; wash once with 100 mL dichloromethane, each time for 2 minutes.

[0059] (7) Coupling of the seventh Lys: Add the raw materials 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), and DMF (100 mL) to the reactor and react for 2-3 hours. Take a small amount of resin for ninhydrin detection. The result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Use 20% piperidine in DMF solution (150 mL) to remove the Fmoc protecting group and react for about 30 minutes. Take a small amount of resin for ninhydrin detection. The result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes; wash once with 100 mL dichloromethane, each time for 2 minutes.

[0060] (8) The eighth coupling of Phe: Add raw materials Fmoc-Phe-OH (6.20 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), and DMF (100 mL) to the reactor, and react for 2-3 hours. Take a small amount of resin for ninhydrin detection. The result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Use 20% piperidine DMF solution (150 mL) to remove the Fmoc protecting group, and react for about 30 minutes. Take a small amount of resin for ninhydrin detection. The result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes; wash once with 100 mL dichloromethane, each time for 2 minutes.

[0061] (9) Ninth coupling of Fmoc-β-Ala-OH: Add raw materials Fmoc-β-alanine (5.0 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), and DMF (100 mL) to the reactor, and react for 2-3 hours. Take a small amount of resin for ninhydrin detection. The result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Use 20% piperidine DMF solution (150 mL) to remove the Fmoc protecting group, and react for about 30 minutes. Take a small amount of resin for ninhydrin detection. The result is positive, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes; wash once with 100 mL dichloromethane, each time for 2 minutes.

[0062] (10) Coupling of the 10th nicotinic acid: Add raw nicotinic acid (2.0 g, 16 mmol, 2.0 equivalents), HOBT (2.7 g, 20 mmol, 2.5 equivalents), DIC (2.52 g, 20 mmol, 2.5 equivalents), and DMF (100 mL) to the reactor and react for 2-3 hours. Take a small amount of resin for ninhydrin detection. The result is negative, indicating that the reaction is complete. After stopping the reaction, drain the reaction liquid, and wash the obtained resin with 200 mL DMF twice, each time for 2 minutes. Wash once with 100 mL dichloromethane, each time for 2 minutes. Wash once with 100 mL methanol, each time for 2 minutes. Wash once with 100 mL dichloromethane, each time for 2 minutes. Wash once with 100 mL methanol, each time for 2 minutes. Transfer to a 40°C vacuum drying oven and dry overnight.

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

[0064] 3. Purification steps

[0065] (1) First, the crude peptide obtained above is subjected to sample purity analysis. After the retention time of the main peak is determined, the sample is 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 with a high performance liquid chromatograph, collect the corresponding mobile phase at the retention time of the target absorption peak, and freeze-dry to obtain the target peptide, that is, obtain 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: 1mL / 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 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 the 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 taken. This step was used to mass produce the short peptide compound K9;

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

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

[0081] (6) Concentrate the collected qualified solution to 40-50 mL at 40°C;

[0082] (7) Place the concentrated solution in a 100 mL beaker and freeze it in a freezer;

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

[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 , proving that the short peptide compound K9 was successfully synthesized in the embodiment of the present invention.

[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 were as follows: Figure 2 As shown, according to the peak area in the figure, the purity was determined to be 96.11%.

[0090] Test Example 3

[0091] Cytotoxicity test of short peptide compound K9

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

[0093] HaCaT cells and RAW264.7 cells were plated in 96-well plates at 10,000 cells per well for 24 hours, and 100 μl of the complete medium containing the short peptide compound K9 (0-2000 ppm) was added. Each group of 6 replicates was incubated for 24 hours, and the old medium was removed. 110 μl of medium containing CCK8 solution (complete medium: CCK8 = 10:1) was added. After incubation for 2 hours in a cell culture incubator at 37°C and 5% CO2, the absorbance of each well was measured at 450 nm by a microplate reader to calculate the cell survival rate.

[0094] The experimental results are as follows Figure 3As shown, the results showed that within the range of 0-1000 ppm, the short peptide compound K9 had no effect on the proliferation of HaCaT cells and RAW264.7 cells.

[0095] Experiment 2: Flow cytometry was used to detect the effect on cell apoptosis. The specific operation was as follows: Human immortalized epidermal cells (HaCaT cells) were cultured in a complete medium of DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin & streptomycin (hereinafter referred to as complete medium) at 37°C in a cell culture incubator with 5% CO2. HaCaT cells were plated in 6-well plates with 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 replicates in each group. After incubation for 48 hours, the cells were collected and flow cytometry was used to detect cell apoptosis.

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

[0097] Test Example 5

[0098] Test of the effect of short peptide compound K9 on cell repair

[0099] Human immortalized epidermal cells (HaCaT cells) were cultured in a complete medium (hereinafter referred to as complete medium) of DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin & streptomycin at 37°C in a cell culture incubator with 5% CO2. HaCaT cells were plated in 6-well plates with 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 replicates in each group. After incubation for 48 hours, the cells were collected and the expression of keratin 1 (KRT-1), keratin 5 (KRT-5), keratin 10 (KRT-10), keratin 14 (KRT-14), hyaluronan synthase 3 (HAS3), and aquaporin 3 (AQP3) genes was detected by fluorescent quantitative PCR. The results are as follows Figure 5-10 As shown, 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 of the present invention has a good repair effect.

[0100] Test Example 6

[0101] Antioxidant efficacy test of short peptide compound K9

[0102] Referring to the standard GB / T 39100-2020, the antioxidant activity of peptides was determined by DPPH and ABTS methods.

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

[0104] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

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

2. The method for preparing a short peptide compound according to claim 1, characterized in that: Includes 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 are added to a reactor containing the L-Lys(Boc)-Wang Resin to react, and then Fmoc removal is performed using a dimethylformamide solution of piperidine, and solid-liquid separation is 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 are added to a reactor containing the L-Pro-L-Lys(Boc)-Wang Resin to react, and then Fmoc removal is performed using a dimethylformamide solution of piperidine, and solid-liquid separation is 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 dimethylformamide solution of N,N'-diisopropylcarbodiimide are added to the reactor containing the L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin to react, and then Fmoc removal is carried out with a dimethylformamide solution of piperidine, and solid-liquid separation is 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 to react, and then Fmoc removal was performed 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) Coupling of Thr: Fmoc-L-Thr(tBu)-OH, 1-hydroxybenzotriazole and dimethylformamide solution of N,N'-diisopropylcarbodiimide were added to the reactor containing the L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin to react, and then Fmoc removal was performed with 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 dimethylformamide solution of N,N'-diisopropylcarbodiimide 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 to react, and then Fmoc removal was performed with 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 dimethylformamide solution of N,N'-diisopropylcarbodiimide are added to the reactor containing the L-Lys(Boc)-L-Thr(tBu)-L-Ile-L-Arg(Pbf)-L-Cys(Dpm)-L-Pro-L-Lys(Boc)-Wang Resin to react, and then Fmoc removal is carried out with a dimethylformamide solution of piperidine, and solid-liquid separation is carried out 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 are 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 to react, and then Fmoc removal is carried out using a dimethylformamide solution of piperidine, and solid-liquid separation is 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, react for 2 to 3 hours, wash and dry the reaction product to obtain a solid phase resin; (11) using a cleavage solution to cleave the solid phase resin in step (10), obtaining a reaction residue after rotary evaporation, precipitating the reaction residue with isopropyl ether, and obtaining the short peptide compound 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, characterized in that: In steps (1) to (9), the solid-liquid separation step comprises: separating a solid phase substance by suction filtration, and repeatedly washing the solid phase substance 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: It comprises the short peptide compound according to claim 1 and a cosmetically acceptable carrier, wherein the cosmetically acceptable carrier comprises one or more of water, glycerin, alcohols, vegetable oils, and vaseline.

8. The cosmetic composition according to claim 7, characterized in that 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, characterized in that 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 skin care products and cosmetics.

Images