Cyclic pentapeptide-4, preparation method thereof and polypeptide cosmetic

Abstract

The invention relates to the technical field of biological polypeptides, in particular to cyclic pentapeptide-4, a preparation method thereof and a polypeptide cosmetic. The cyclic pentapeptide-4 has a chemical structural formula as shown in a formula I in the specification. Compared with the existing palmitoyl pentapeptide-4, the cyclopentapeptide-4 provided by the invention has good ability to penetrate through an epidermal barrier, so that the cyclopentapeptide-4 can penetrate into skin tissues more deeply, thereby significantly improving the anti-wrinkle and anti-aging ability of the skin. Meanwhile, the expression level of anti-aging genes in human skin fibroblasts can be remarkably improved. Therefore, the cyclic pentapeptide-4 disclosed by the invention can be used for preparing an anti-aging preparation so as to improve the expression level of an anti-aging gene in human skin fibroblasts and further relieve skin aging. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of biological polypeptides, and particularly relates to a cyclopentapeptide-4, a preparation method thereof, and a polypeptide cosmetic. Background Art

[0002] Type I and type III collagens in the skin are key proteins for maintaining skin health and youth. They are synthesized and secreted by fibroblasts in human skin. These two collagens work together to make the skin smooth, delicate and moist, thus effectively slowing down the pace of skin aging and keeping the skin youthful. Hyaluronic acid is one of the components of the extracellular matrix, which is composed of repeating glucuronic acid and N-acetylglucosamine. It is embedded between collagen fiber bundles and can bind to water, contributing to skin hydration. CD44 is a glycoprotein and a receptor for hyaluronic acid. It is widely distributed on the cell membranes of the dermis, epidermis, vascular endothelial cells and white blood cells, and is involved in regulating the movement of circulating white blood cells through the blood vessel wall to the inflammatory site. The TIMP metallopeptidase inhibitor 2 gene is a member of the TIMP gene family. The proteins encoded by this gene family are natural inhibitors of matrix metalloproteinases, which are extracellular matrix degrading enzymes.

[0003] Currently, palmitoyl pentapeptide-4 is widely used in various anti-aging and anti-wrinkle cosmetics. However, during its application, it is easily enzymatically hydrolyzed by trypsin in human skin, resulting in a short residence time in the skin, thus affecting the persistence of its anti-aging effect. In addition, although palmitoyl pentapeptide-4 can promote collagen synthesis, in actual application, the efficiency of promoting collagen regeneration and the effect of alleviating skin aging still need to be improved. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a cyclopentapeptide-4, a preparation method thereof, and a polypeptide cosmetic. By adopting the cyclopentapeptide-4 with a cyclic structure, the present invention effectively improves the anti-enzymatic hydrolysis ability of pentapeptide-4, enables it to penetrate deeper into the skin, improves the bioavailability, and significantly enhances its anti-wrinkle and anti-aging effects, overcoming the problems of easy enzymatic hydrolysis, low bioavailability and limited anti-aging effect of palmitoyl pentapeptide-4 in existing products. At the cellular level, the cyclopentapeptide-4 of the present invention can significantly increase the expression levels of anti-aging genes in human skin fibroblasts, including hyaluronic acid synthase 2 gene, hyaluronic acid receptor gene, metalloproteinase inhibitor 2 gene, type I collagen gene and type III collagen gene, thus showing broad application prospects in promoting collagen regeneration, maintaining skin moisture and alleviating skin aging.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The first object of the present invention is to provide a cyclopentapeptide-4, whose structural formula is: .

[0006] The second object of the present invention is to provide a preparation method of the above-mentioned cyclopentapeptide-4, which comprises the following steps: Synthesize by the solid-phase method, using dichloride resin as the solid-phase carrier, N,N-diisopropylcarbodiimide and 1-hydroxybenzotriazole as condensing agents. Under alkaline conditions, starting from the C-terminus of cyclopentapeptide-4, in the sequence order of Lys-Thr-Thr-Lys-Ser, Fmoc-protected amino acids are added in turn and condensation reactions are carried out. After deprotecting the terminal amino protecting group, full-protection cleavage treatment is carried out to obtain a linear pentapeptide with fully protected side chains; the linear pentapeptide with fully protected side chains is placed in a solvent, and N,N-diisopropylethylamine and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate are used as condensing agents for condensation reactions. Through liquid-phase cyclization synthesis, the linear pentapeptide is converted into a cyclopentapeptide-4 with fully protected side chains; the cyclopentapeptide-4 with fully protected side chains is deprotected using trifluoroacetic acid to obtain cyclopentapeptide-4.

[0007] Preferably, in the sequence order of Lys-Thr-Thr-Lys-Ser, the Fmoc-protected amino acids are N-fluorenylmethoxycarbonyl-N'-tert-butoxycarbonyl-L-lysine, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, N-fluorenylmethoxycarbonyl-N'-tert-butoxycarbonyl-L-lysine, and fluorenylmethoxycarbonyl-O-tert-butyl-L-serine in turn.

[0008] The third object of the present invention is to provide a polypeptide cosmetic, which includes the above-mentioned cyclopentapeptide-4.

[0009] Preferably, in the polypeptide cosmetic, the cyclopentapeptide-4 exists in the form of cyclopentapeptide-4 acetate.

[0010] Preferably, the cyclopentapeptide-4 acetate is prepared according to the following steps: After mixing ammonium acetate with cyclopentapeptide-4, salt conversion treatment and solvent replacement are carried out in turn, and after freeze-drying, cyclopentapeptide-4 acetate is obtained.

[0011] Preferably, the cyclopentapeptide-4 delays skin aging by enhancing the expression level of anti-aging genes.

[0012] Preferably, the anti-aging genes are selected from at least one of hyaluronic acid synthase 2 gene, hyaluronic acid receptor gene, tissue inhibitor of metalloproteinase 2 gene, type I collagen gene, and type III collagen gene.

[0013] Preferably, the polypeptide cosmetic further includes pharmaceutically acceptable excipients.

[0014] Preferably, the pharmaceutically acceptable excipients are one or more of diluents, thickeners, antioxidants, preservatives, humectants, pH regulators, dispersants and stabilizers; among them, antioxidants are preferably vitamin E or vitamin C; thickeners are preferably xanthan gum or carbomer; humectants are preferably hyaluronic acid, trehalose or glycerol; preservatives are preferably phenoxyethanol or potassium sorbate; dispersants are preferably water, glycerol or propylene glycol; pH regulators are preferably citric acid or sodium hydroxide.

[0015] Preferably, the polypeptide cosmetics include essence, toner, emulsion or cream.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention provides a cyclopentapeptide-4, which forms a rigid cyclic structure by cyclization of a linear pentapeptide. This closed structure endows cyclopentapeptide-4 with excellent anti-enzymatic hydrolysis ability. At the same time, the high permeability of the cyclic peptide further enhances the ability to penetrate the epidermal barrier, enabling it to penetrate deeper into the skin, improve bioavailability, and exert anti-wrinkle and anti-aging effects.

[0017] 2. Compared with palmitoyl pentapeptide in the prior art, the cyclopentapeptide-4 of the present invention has a stronger anti-aging effect. At the cellular level, the cyclopentapeptide-4 of the present invention can significantly increase the expression levels of anti-aging genes in human skin fibroblasts, including type I collagen gene, type III collagen gene, hyaluronan synthase 2 gene, hyaluronic acid receptor gene and tissue inhibitor of metalloproteinase 2 gene. Therefore, the cyclopentapeptide-4 of the present invention can be used to prepare anti-aging preparations to increase the expression levels of anti-aging genes in human skin fibroblasts, thereby alleviating skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the MS chromatogram of cyclopentapeptide-4 of the present invention.

[0019] Figure 2 It is the HPLC chromatogram of cyclopentapeptide-4 of the present invention.

[0020] Figure 3 It is the relative expression quantity diagram of cyclopentapeptide-4 of the present invention on COL1A1 gene.

[0021] Figure 4 It is the relative expression quantity diagram of cyclopentapeptide-4 of the present invention on COL3A1 gene.

[0022] Figure 5 It is the relative expression quantity diagram of cyclopentapeptide-4 of the present invention on HAS2 gene.

[0023] Figure 6 It is the relative expression quantity diagram of cyclopentapeptide-4 of the present invention on CD44 gene.

[0024] Figure 7 This is the relative expression level diagram of TIMP2 gene by the cyclopentapeptide-4 of the present invention. Detailed implementation manners

[0025] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the data in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be obtained through market purchase or prepared by existing methods.

[0027] Among them, human skin fibroblasts were purchased from Wuhan Pusaisai Biotechnology Co., Ltd. and denoted as HFB cells; type I collagen was denoted as COL1A1; type III collagen was denoted as COL3A1; hyaluronan synthase 2 was denoted as HAS2; hyaluronan receptor was denoted as CD44; tissue inhibitor of metalloproteinase 2 was denoted as TIMP2; fetal bovine serum was denoted as FBS and was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; DMEM and trypsin were both purchased from Thermo Fisher, and PBS, phosphate buffer solution, was purchased from Merck; RNA EASY FAST total RNA extraction kit, Tiangen, DP452; Prime Script TM RT Master Mix, TAKARA, RR06A; TB Green ® premixExTaq TM II, TAKARA, RR820A.

[0028] Cyclopentapeptide-4, the single-character corresponding to Lys-Thr-Thr-Lys-Ser is KTTKS, and the sequence information is SEQ ID NO.13.

[0029] The raw material information used in the present invention is shown in Table 1, wherein the substitution degree of the dichloro resin is 0.8 nmol / g.

[0030] Table 1 Raw material information table used in the present invention In the prior art, palmitoyl pentapeptide is often applied in cosmetics to achieve the effect of anti-aging. However, palmitoyl pentapeptide-4 is easily enzymatically hydrolyzed by trypsin in human skin during application, resulting in a short residence time in the skin, thus affecting the persistence of its anti-aging effect. In addition, although palmitoyl pentapeptide-4 can promote the synthesis of collagen, its efficiency in promoting collagen regeneration and the effect of alleviating skin aging are limited and still need to be improved.

[0031] The present invention provides a cyclopentapeptide-4, and its structural formula is: 。

[0032] Compared with the existing palmitoyl pentapeptide-4, its linear structure has a high sensitivity to enzymatic degradation, resulting in limited bioavailability and insufficient anti-aging efficacy. The cyclopentapeptide-4 provided by the present invention forms a rigid cyclic structure through intramolecular cyclization. This closed structure endows cyclopentapeptide-4 with significant anti-enzymatic hydrolysis ability. At the same time, the high permeability of the cyclic peptide further enhances the ability to penetrate the epidermal barrier. This improvement enables cyclopentapeptide-4 to penetrate more deeply into the skin tissue, thus significantly enhancing the anti-wrinkle and anti-aging effects.

[0033] The cyclopentapeptide-4 of the present invention plays a significant anti-aging role by enhancing the expression levels of a series of anti-aging genes in human skin fibroblasts. It effectively promotes the regeneration of collagen, enhances the elasticity and firmness of the skin, and thus significantly alleviates the signs of skin aging. These anti-aging genes include: hyaluronic acid synthase 2 gene, hyaluronic acid receptor gene, tissue inhibitor of metalloproteinase 2 gene, type I collagen gene, and type III collagen gene. They play a crucial role in maintaining the healthy state of the skin, promoting the process of collagen synthesis, and alleviating the phenomenon of skin aging. Through this mechanism, cyclopentapeptide-4 can effectively promote the regeneration of collagen, thereby enhancing the elasticity and firmness of the skin, and thus significantly alleviating the signs of skin aging.

[0034] The preparation method of the cyclopentapeptide-4 of the present invention includes the following steps: S1. Preparation of Fmoc-Ser(tbu)-O-2-CTC Resin: Dissolve 2.4 g of Fmoc-Ser(tbu)-OH in 50 mL of DCM, then add 2 g of DIEA and add it to 5 g of dichloride resin swollen with DCM. Stir and react at 25 °C under nitrogen protection for 2 h, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of N,N-dimethylformamide to obtain Fmoc-Ser(tbu)-O-2-CTC Resin.

[0035] Preparation of Fmoc-Lys(boc)-Ser(tbu)-O-2-CTC Resin: Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20%, the Fmoc protecting group on Fmoc-Ser(tbu)-O-2-CTC Resin in step S1 was removed at 25 °C for 40 min, followed by suction filtration. Then, it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Ser(tbu)-O-2-CTC Resin. 3.7 g of Fmoc-lys(boc)-OH, 1.6 g of 1-hydroxybenzotriazole, and 1.5 g of N,N-diisopropylcarbodiimide were dissolved in 50 mL of DMF to obtain a mixed solution. Subsequently, the mixed solution was added to H-Ser(tbu)-O-2-CTC Resin, and the reaction was stirred at 25 °C for 3 h under nitrogen protection, followed by suction filtration. Then, it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Lys(boc)-Ser(tbu)-O-2-CTC Resin.

[0036] Preparation of Fmoc-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin: Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20%, the Fmoc protecting group on Fmoc-Lys(boc)-Ser(tbu)-O-2-CTC Resin in step S2 was removed at 25 °C for 40 min, followed by suction filtration. Then, it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Lys(boc)-Ser(tbu)-O-2-CTC Resin. 3.2 g of Fmoc-Thr(tbu)-OH, 1.6 g of 1-hydroxybenzotriazole, and 1.5 g of N,N-diisopropylcarbodiimide were dissolved in 50 mL of DMF to obtain a mixed solution. Subsequently, the mixed solution was added to H-Lys(boc)-Ser(tbu)-O-2-CTC Resin, and the reaction was stirred at 25 °C for 3 h under nitrogen protection, followed by suction filtration. Then, it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin.

[0037] Preparation of Fmoc-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin: Deprotect the Fmoc protecting group on Fmoc-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin in step S3 with a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20% at 25 °C for 40 min, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin; dissolve 3.2 g of Fmoc-Thr(tbu)-OH, 1.6 g of 1-hydroxybenzotriazole and 1.5 g of N,N-diisopropylcarbodiimide in 50 mL of DMF to obtain a mixed solution; then add the mixed solution to H-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin, stir and react at 25 °C for 3 h under nitrogen protection, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin.

[0038] S5. Preparation of Fmoc-Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin: Deprotect the Fmoc protecting group on Fmoc-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin in step S4 with a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20% at 25 °C for 40 min, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain H--Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin; dissolve 3.7 g of Fmoc-Lys(boc)-OH, 2 g of 1-hydroxybenzotriazole and 1.9 g of N,N-diisopropylcarbodiimide in 50 mL of DMF to obtain a mixed solution; then add the mixed solution to Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin, stir and react at 25 °C for 3 h under nitrogen protection, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin.

[0039] Preparation of S6, H-Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-OH: Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20%, the Fmoc protecting group on Fmoc-Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin in step S5 was removed at 25 °C for 40 min, then filtered by suction. It was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin. 10 mL of acetic acid, 20 mL of trifluoroethanol, and 70 mL of DCM were mixed and added to H-Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-O-2-CTC Resin and reacted for 2 h. After filtration, the reaction solution was taken. The reaction solution was concentrated to dryness at 40 °C, 100 mL of water was added, and sodium bicarbonate was added to adjust the pH to 8. After stirring, filtration, and drying, 4 g of white solid of H-Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-OH was obtained.

[0040] Preparation of S7, Cyclo(Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)): 4 g of H-Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)-OH was dissolved in 20 mL of DMF. Subsequently, 2 g of DIEA was added, diluted to 600 mL with tetrahydrofuran, and 1.5 g of HBTU was added thereto. The reaction was carried out at room temperature for 3 h. Subsequently, the tetrahydrofuran was evaporated to dryness at 40 °C. Then, water and ethyl acetate were continuously added, stirred until layered, and the ethyl acetate layer was taken and evaporated to dryness to obtain 3.5 g of white Cyclo(Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)).

[0041] Preparation of S8, Cyclo(Lys-Thr-Thr-Lys-Ser): Prepare a 100 mL mixed solution by mixing trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:4:1. Add 3.5 g of Cyclo(Lys(boc)-Thr(tbu)-Thr(tbu)-Lys(boc)-Ser(tbu)) to the mixed solution and stir at room temperature for 2 h. Then, evaporate the solvent at 40 °C, add tert-butyl ether for extraction, stir for 30 min, and filter to obtain 2 g of white crude Cyclo(Lys-Thr-Thr-Lys-Ser).

[0042] Dissolve the crude Cyclo(Lys-Thr-Thr-Lys-Ser) and purify it multiple times by reverse high-performance liquid chromatography. Use a chromatographic column packed with C18, 10 μm, 100 Å packing material and adopt the method of gradient elution and fractionated sampling. Through high-performance liquid chromatography interval detection and impurity analysis, obtain cyclopentapeptide-4 with a purity of over 90%.

[0043] Perform salt conversion treatment on cyclopentapeptide-4 with ammonium acetate at a mass concentration of 32 g / L and use a nanofiltration device to replace organic solvents to reduce the residual amount of organic solvents and achieve enrichment of substances. Subsequently, freeze-dry the solution to obtain freeze-dried cyclopentapeptide-4 powder, denoted as freeze-dried Cyclo(Lys-Thr-Thr-Lys-Ser) powder; the mass is 0.2 g.

[0044] I. Testing of cyclopentapeptide-4 sample: Observe Figure 1 It is concluded that the chromatographic peak at 8.598 min is the peak of cyclopentapeptide-4 of the present invention, and its purity exceeds 90%.

[0045] Observe Figure 2 It is concluded that 546 is the mass spectrometry signal of cyclopentapeptide-4, showing a molecular weight +1, and the molecular weight of the product is 547.

[0046] Combine Figure 1 and Figure 2 It is concluded that the molecular weight of cyclopentapeptide-4 of the present invention is highly consistent with the theoretical molecular weight, further indicating that the synthesized cyclopentapeptide-4 of the present invention has high purity and accuracy and conforms to the expected molecular structural characteristics.

[0047] II. Application method: a. Cell resuscitation and culture: Take out HFB cells from liquid nitrogen for resuscitation. When the HFB cells proliferate to occupy 80% of the surface area of the culture flask, perform digestion treatment on the HFB cells, and then perform cell counting on the HFB cells to obtain a cell suspension.

[0048] b. Cell seeding: When performing HFB cell seeding, use 6×10 4Inoculation density per well, add the HFB cell suspension dropwise into a 12-well plate at a volume of 1 mL per well; after inoculation, place the 12-well plate into a cell culture incubator containing 5 vt% CO 2 and continue to culture at 37 °C for 24 h.

[0049] c. Grouping: Set up a control group, a palmitoyl pentapeptide-4 group, and a cyclopentapeptide-4 group, with 3 replicate wells in each group.

[0050] d. Solution preparation: Prepare the working solution, as shown in Table 2 specifically.

[0051] Table 2 Working solution information table e. RNA extraction of HFB cells: ① After ensuring that the number of HFB cells is correct, thoroughly aspirate the culture medium supernatant in each well and immediately perform cell lysis operation.

[0052] ② Add 350 μL of lysis buffer RIPA to the well plate and let it stand for 2 min. Then, gently pipette and aspirate the HFB cells on the surface of the well plate, and then transfer the lysis buffer containing the HFB cell lysate to a centrifuge tube and perform vortex oscillation treatment to ensure thorough mixing.

[0053] ③ After centrifuging at 12000 rpm for 5 min, take the supernatant and add it to the genomic DNA removal column, and centrifuge again at 12000 rpm for 30 s. After centrifugation, retain the filtrate flowing through the column.

[0054] ④ Slowly add 70% ethanol solution with a volume equivalent to twice the volume of the supernatant to the filtrate and mix well. Then, transfer all the mixed solution to the RNase-free adsorption column CR4 placed in the collection tube. Then, centrifuge the RNase-free adsorption column CR4 at a speed of 12000 rpm for 30 s. After centrifugation, pour out the waste liquid and place the adsorption column back into the collection tube.

[0055] ⑤ Add 700 μL of protein removal solution RW3 to the RNase-free adsorption column CR4 processed in step ④ and centrifuge at a speed of 12000 rpm for 30 s. After centrifugation, pour out the waste liquid and place the adsorption column back into the collection tube.

[0056] ⑥ Add 500 μL of washing solution RW3 to the RNase-free adsorption column CR4 processed in step ⑤, let it stand at room temperature for 2 min, and then centrifuge at a speed of 12000 rpm for 30 s. After centrifugation, pour out the waste liquid and place the adsorption column back into the collection tube; repeat this step and then place the RNase-free adsorption column CR4 at room temperature for 2 min to thoroughly dry the residual washing solution.

[0057] ⑦ Transfer the RNase-free adsorption column CR4 into a new RNase-free centrifuge tube. Drop 35 μL of diethyl pyrocarbonate solution onto the middle part of the adsorption membrane in a suspended manner. After standing at room temperature for 2 min, centrifuge at a speed of 12,000 rpm for 2 min to obtain an RNA solution; repeat this step, concentrate the RNA solution, measure the concentration of RNA with a ultra-micro nucleic acid and protein analyzer, and store the extracted RNA in a -80 °C refrigerator.

[0058] f. cDNA cloning: Calculate the total input amount according to the concentrations of RNA in the control group, palmitoyl pentapeptide-4 group, and cyclopentapeptide-4 group, prepare a cDNA template, and store it in a -20 °C refrigerator. The specific operations are shown in Table 3.

[0059] Table 3 Reverse transcription system table g. Real-time fluorescence quantitative polymerase chain reaction: Using the obtained cDNA as a template, perform fluorescence quantitative polymerase chain reaction amplification. GAPDH is used as an internal reference gene, and the rest are target anti-aging genes. Among them, the primer sequences corresponding to the COL1A1 gene, COL3A1 gene, HAS2 gene, CD44 gene, and TIMP2 gene are shown in Table 4.

[0060] Table 4 Primer sequence table Prepare a mixed solution according to the number of reactions, dispense it into 8-well tubes, add the cDNA of the control group, palmitoyl pentapeptide-4 group, and cyclopentapeptide-4 group. The control group, palmitoyl pentapeptide-4 group, and cyclopentapeptide-4 group are each repeated three times. The reaction conditions are shown in Table 5, and the results are expressed as 2 --Δ Δct for presentation.

[0061] Table 5 PCR system table The reaction conditions of the above PCR system are: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, for 30 cycles; extension at 72 °C for 10 min; preservation at 4 °C.

[0062] III. Results: Summary table of the detection results of the COL1A1 gene level, as shown in Table 6 specifically.

[0063] Table 6 Summary table of the detection results of the COL1A1 gene level Note: * represents the significance analysis between "Palmitoyl Pentapeptide-4", "Cyclic Pentapeptide-4" and the "Control Group". * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and / indicates none.

[0064] Combined with Figure 3 and Table 6, it can be concluded that compared with the control group and palmitoyl pentapeptide-4, the relative expression level of COL1A1 gene in cyclic pentapeptide-4 increased extremely significantly, with an increase rate of 38.23%.

[0065] Summary table of the detection results of COL3A1 gene level, as shown in Table 7 specifically.

[0066] Table 7 Summary table of the detection results of COL3A1 gene level Note: * represents the significance analysis between "Palmitoyl Pentapeptide-4", "Cyclic Pentapeptide-4" and the "Control Group". * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and / indicates none.

[0067] Combined with Figure 4 and Table 7, it can be concluded that compared with the control group and palmitoyl pentapeptide-4, the relative expression level of COL1A1 gene in cyclic pentapeptide-4 increased extremely significantly, with an increase rate of 61.51%.

[0068] Summary table of the detection results of HAS2 gene level, as shown in Table 8 specifically.

[0069] Table 8 Summary table of the detection results of HAS2 gene level Note: * represents the significance analysis between "Palmitoyl Pentapeptide-4", "Cyclic Pentapeptide-4" and the "Control Group". * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and / indicates none.

[0070] Combined with Figure 5 and Table 8, it can be concluded that compared with the control group, the relative expression levels of HAS2 gene in cyclic pentapeptide-4 and palmitoyl pentapeptide-4 increased extremely significantly, with increase rates of 24.00% and 51.22% respectively.

[0071] Summary table of the detection results of CD44 gene level, as shown in Table 9 specifically.

[0072] Table 9 Summary table of the detection results of CD44 gene level Note: * represents the significance analysis between "Palmitoyl Pentapeptide-4", "Cyclic Pentapeptide-4" and the "Control Group". * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and / indicates none.

[0073] Combined with Figure 6 Table 9, it can be obtained that compared with the control group and palmitoyl pentapeptide-4, the relative expression level of CD44 gene in cyclic pentapeptide-4 increased extremely significantly, with a promotion rate of 31.62%.

[0074] Summary table of TIMP2 gene level detection results, as shown in Table 10 specifically.

[0075] Table 10 Summary table of TIMP2 gene level detection results Note: * represents the significance analysis between "Palmitoyl Pentapeptide-4", "Cyclic Pentapeptide-4" and the "Control Group". * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and / indicates none.

[0076] Combined with Figure 5 Table 10, it can be obtained that compared with the control group and palmitoyl pentapeptide-4, the relative expression level of TIMP2 gene in cyclic pentapeptide-4 increased extremely significantly, with a promotion rate of 21.01%.

[0077] In summary, cyclic pentapeptide-4 can significantly increase the relative expression levels of COL1A1, COL3A1, CD44 and TIMP2 genes. Among them, the promotion rate of COL3A1 gene is the highest, reaching 61.51%. Compared with the control group and palmitoyl pentapeptide-4, cyclic pentapeptide-4 shows a significant promoting effect on the expression of multiple genes, further indicating that the cyclic pentapeptide-4 of the present invention can significantly improve the expression level of anti-aging genes in human skin fibroblasts, thereby alleviating skin aging.

[0078] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the adopted step methods are the same as those in the embodiments, in order to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

Claims

1. A cyclopentapeptide-4, characterized in that: The structural formula of cyclopentapeptide-4 is: 。 2. A method for preparing cyclopentapeptide-4 according to claim 1, characterized in that: The steps include: The solid phase method is adopted for synthesis, with dichlororesin as a solid phase carrier, N,N-diisopropylcarbodiimide and 1-hydroxybenzotriazole as condensation agents, and under alkaline conditions, starting from the C-terminus of cyclopentapeptide-4, Fmoc amino acids are added in sequence in the order of Lys-Thr-Thr-Lys-Ser to carry out condensation reaction, deprotect the terminal amino protecting group, and cut to obtain a straight-chain pentapeptide with fully protected side chains; The side chain fully protected straight-chain pentapeptide is placed in a solvent, and a condensation reaction is carried out using N,N-diisopropylethylamine and O-benzotriazole-tetramethyluronium hexafluorophosphate as condensation agents, and the straight-chain pentapeptide is converted into the side chain fully protected cyclopentapeptide-4 through a liquid phase cyclosynthesis method; The side chain fully protected cyclopentapeptide-4 is deprotected by using trifluoroacetic acid to obtain cyclopentapeptide-4.

3. The method for preparing cyclopentapeptide-4 according to claim 2, characterized in that: According to the sequence order of Lys-Thr-Thr-Lys-Ser, the Fmoc-protected amino acids are N-fluorenylmethoxycarbonyl-N'-tert-butyloxycarbonyl-L-lysine, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, N-fluorenylmethoxycarbonyl-N'-tert-butyloxycarbonyl-L-lysine, and fluorenylmethoxycarbonyl-O-tert-butyl-L-serine.

4. A polypeptide cosmetic, characterized in that: The polypeptide cosmetics include the cyclopentapeptide-4 described in claim 1.

5. The polypeptide cosmetic according to claim 4, characterized in that: In the polypeptide cosmetics, the cyclopentapeptide-4 exists in the form of cyclopentapeptide-4 acetate.

6. The polypeptide cosmetic according to claim 5, characterized in that: Cyclopentapeptide-4-acetate was prepared according to the following steps: After ammonium acetate and cyclopentapeptide-4 are mixed, salt conversion treatment and solvent replacement are carried out in sequence, and cyclopentapeptide-4 acetate is obtained by freeze drying.

7. The polypeptide cosmetic according to claim 3, characterized in that: The cyclic pentapeptide-4 delays skin aging by increasing the expression level of anti-aging genes.

8. The polypeptide cosmetic according to claim 7, characterized in that: The anti-aging gene is selected from at least one of hyaluronan synthase 2 gene, hyaluronan receptor gene, metalloproteinase inhibitor 2 gene, type I collagen gene, and type III collagen gene.

9. The polypeptide cosmetic according to claim 3, characterized in that: The polypeptide cosmetics also include pharmaceutically acceptable excipients.

10. The polypeptide cosmetic according to claim 9, characterized in that: The pharmaceutically acceptable excipient is one or more of a diluent, a thickener, an antioxidant, a preservative, a humectant, a dispersant and a stabilizer.

Images