Polypeptide for promoting collagen synthesis, preparation method therefor, formulation and use thereof

Abstract

A polypeptide for promoting collagen synthesis, a preparation method therefor, a formulation and use thereof. The polypeptide is a nonapeptide having 10-hydroxy-2-decenoic acid coupled at the terminus, and is prepared by means of a solid-phase peptide synthesis method. The polypeptide can increase the content of cellular collagen, has a weak effect on capsaicin receptors and mediated calcium influx, and causes relatively low pain stimulation.

Description

A polypeptide that promotes collagen synthesis, its preparation method, formulation, and application. Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide that promotes collagen synthesis, its preparation method, formulation, and application. Background Technology

[0002] The background information disclosed below is intended only to enhance understanding of the overall background of the present invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Collagen is an essential component of human skin, cartilage, and other tissues, playing a crucial role in maintaining their elasticity. As a fibrous protein in the dermis, collagen is key to maintaining skin elasticity and firmness, and is a key target for the development of anti-wrinkle drugs and cosmetics. Fibroblasts are the main cells that synthesize and secrete collagen; therefore, enhancing fibroblast activity is one strategy to promote collagen synthesis in the skin. Furthermore, matrix metalloproteinases (MMPs), zinc-dependent extracellular endopeptidases, have been identified as collagen-hydrolyzing enzymes. MMP hydrolysis of collagen is one of the direct causes of wrinkles. Therefore, MMP inhibitors are also a strategy to promote collagen synthesis in the skin.

[0004] 10-Hydroxy-2-decenoic acid (10-HDA), also known as royal jelly acid, is a natural substance found only in honey. Applications CN116790685A (application number CN202311048043.2), CN117159530A (application number CN202311332540.5), and CN117338613A (application number CN202311413437.3) disclose the preparation method, activity, and formulation of 10-hydroxy-2-decenoic acid. 10-Hydroxy-2-decenoic acid has been found to inhibit MMPs and promote collagen synthesis. Cosmetic peptides, such as palmitoyl tripeptide-1, acetyl tetrapeptide-22, acetyl hexapeptide-30, heptapeptide-7, nonapeptide-1, and palmitoyl nonapeptide-3, also exhibit certain anti-aging effects. These effects are partly related to inhibiting MMPs, thereby inhibiting collagen breakdown, as described in CN117642415A (application number CN202280044238.7).

[0005] However, 10-hydroxy-2-decenoic acid still suffers from many drawbacks, such as poor transdermal absorption and poor water solubility, while cosmetic peptides also have shortcomings such as poor transdermal absorption and low efficacy. To date, complex emulsifier systems or special formulations such as liposomes are typically required to improve the transdermal absorption of cosmetics containing these substances. However, complex emulsifier systems often contain excipients suitable as microbial carbon sources, leading to frequent issues with substandard bacterial counts in cosmetics. Furthermore, cosmetic peptides are relatively expensive compared to other small molecule substances; maximizing their activity to reduce dosage helps overcome this high cost. Summary of the Invention

[0006] To address the aforementioned issues, the inventors investigated the combination strategy of 10-HDA with cosmetic peptides. They found that due to the poor skin penetration and absorption characteristics of 10-HDA, it was difficult to obtain additional efficacy benefits when used transdermally in combination with cosmetic peptides. Furthermore, covalently coupling 10-HDA with most cosmetic peptides also yielded limited benefits, but covalently coupling with nonapeptide-3 provided additional efficacy benefits, which was beneficial for reducing the amount of peptide used (resulting in better efficacy at the same dosage). Therefore, this invention provides a polypeptide that promotes collagen synthesis, its preparation method, formulation, and application. The invention provides the following technical solution:

[0007] A polypeptide that promotes collagen synthesis, named "royal acid nonapeptide," has the structural formula shown in Formula I:

[0008] .

[0009] Specifically, this royal acetic acid nonapeptide is a nonapeptide covalently coupled with royal acetic acid (10-HDA) (equivalent to palmitoyl nonapeptide-3 being modified by royal acetic acid acylation instead of palmitic acid acylation), and can also be represented as: 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH, with the molecular formula C 58 H 100 N 12 O 12 The relative molecular weight is approximately 1157.51. Studies have shown that this royal acetic acid nonapeptide, at the same dosage, has a higher activity in inhibiting MMP and promoting collagen synthesis than royal acetic acid, palmitoyl nonapeptide-3, and their mixtures, demonstrating a significant advantage.

[0010] The term "structural formula" can also be understood as "reduced structural formula," which is a chemical formula using element symbols and short lines to represent the arrangement and bonding of atoms in a compound (or element) molecule. It is a simple method for describing molecular structure. A structural formula can completely depict the chemical bonds between every atom in the molecule. Organic compounds are generally represented using reduced structural formulas, often omitting carbon-hydrogen bonds, sometimes carbon-carbon single bonds, or using bond-line representation. It is obvious that "structural formula" and "reduced structural formula" are only used to show the way atoms are connected and bonded in organic compounds; they do not represent the actual spatial configuration / conformation or actual bond angles of the molecule.

[0011] Formula I, as a representation of the relationships between atoms and chemical bonds, first and foremost satisfies the basic valence rules of atoms: C, N, and O are 4, 3, and 2 valences, respectively. Therefore, understanding Formula I should be based on this valence rule. In any case, if the symbols, numbers, bond lines, or structural formula of Formula I are unclear, or if there are errors in its writing, Formula I should still be understood as C, N, and O being 4, 3, and 2 valences, respectively. In the structural formula of Formula I, when only a bond line is used at the end, it represents a -CH3 (methyl) terminal.

[0012] A method for preparing the aforementioned "royal acid nonapeptide" includes the following steps:

[0013] Synthesis of S1 linear peptide: Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O- resin was prepared by stepwise coupling using a solid-phase synthesis method, wherein M1, M2, and M3 are amino protecting groups;

[0014] Synthesis of S2 royal ic acid covalently coupled linear peptide: 10-HDA was reacted with Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O- resin to prepare 10-HDA-Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O- resin;

[0015] Cleavage of S3 royal jelly acid nonapeptide: Cleavage of 10-HDA-Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O- resin yields crude 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH.

[0016] Preferably, the resin is selected from either royal jelly resin or CTC resin;

[0017] Preferably, M1, M2, and M3 are independently selected from any one of the following protecting groups: BOC (tert-butyloxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Trt (triphenylmethyl), Dde (allyl), and Allyl (allyl). The resins, amino protecting groups, and coupling, decoupling, and cleavage conditions commonly used in polypeptide solid-phase synthesis are existing technologies in this field. For details, please refer to *Polypeptide Drug Research and Development* (edited by Li Baoqiu, People's Medical Publishing House, 2011) and *Polypeptide Synthesis* (edited by Huang Weide and Chen Changqing, Science Press, 1985).

[0018] Preferably, the preparation method further includes the following steps:

[0019] S4 Product Purification: The crude 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH was dissolved in 30% (v / v) acetonitrile aqueous solution and filtered through a 0.22 μm organic filter membrane. The filtrate was purified and separated using a C8 reversed-phase column. The sample was loaded at a flow rate of 20 mL / min and a detection wavelength of 210 nm. Gradient elution was performed using mobile phases: mobile phase A was 0.1% acetic acid solution, and mobile phase B was methanol or acetonitrile.

[0020] More preferably, the mobile phase B is acetonitrile, and the mobile phase elution procedure is as follows:

[0021] Time (min) Mobile phase A (%) Mobile phase B (%) 0 9 0 10 30 65 35 33 20 80 38 20 80 40 90 10 45 90 10

[0022] A skincare product containing the aforementioned "royal acid nonapeptide" or "royal acid nonapeptide" prepared by the aforementioned preparation method.

[0023] Preferably, the skin care product is a transdermal drug delivery formulation.

[0024] Preferably, the excipient of the transdermal drug delivery formulation is a 5 wt% aqueous solution of 1,2-pentanediol.

[0025] The aforementioned "royal acid nonapeptide" or the "royal acid nonapeptide" prepared by the aforementioned preparation method or the aforementioned skin care products are used in the preparation of formulations that inhibit MMP-1 and increase the collagen content of skin fibroblasts. Beneficial effects

[0026] (1) The royal jelly nonapeptide of the present invention has good effects of inhibiting MMP and increasing the collagen content of the skin.

[0027] (2) The effects of the royal jelly nonapeptide of the present invention in inhibiting MMP and increasing skin collagen content are better than those of royal jelly, palmitoyl nonapeptide-3 and physical mixtures in the same amount. It overcomes the shortcomings of royal jelly itself having poor transdermal effect, requiring large amount or special liposome preparations, and also overcomes the shortcomings of palmitoyl nonapeptide-3 requiring large amount and high cost.

[0028] (3) The royal jelly nonapeptide of the present invention has high safety, and its safe concentration for skin cells is much higher than that of palmitoyl nonapeptide-3, retinol, etc.; and it has little effect on pain-related calcium ion flux. Instruction manual illustrations

[0029] Figure 1 is an HPLC chromatogram of the separation and purification of royal jelly nonapeptide prepared in Example 1;

[0030] Figure 2 shows the mass spectrum of royal jelly acid nonapeptide;

[0031] Figure 3 shows the effect of different test substances on cell viability in Example 3;

[0032] Figure 4 shows the fluorescence spectrum of the effect of different test substances on cellular calcium ion flux in Example 3.

[0033] Figure 5 shows the effect of different test substances on the MMP-1 content of cells in Example 3. Detailed Implementation

[0034] The following description, in conjunction with the accompanying drawings, tables, and embodiments, further illustrates the solution and effects of the present invention to ensure that the technical solution is clear, complete, and feasible. The reagents, instruments, kits, cells, etc., used in the following embodiments are all commercially available materials and equipment.

[0035] Example 1: Preparation and purification of royal jelly nonapeptide

[0036] Example 1 provides a method for preparing and purifying royal jelly nonapeptide, wherein Table 1 is the material feeding table for the preparation process of royal jelly nonapeptide.

[0037] Table 1. Raw material feeding table for the preparation process of royal jelly nonapeptide

[0038] Serial Number Resin / Amino Acid Weight / Equivalent HOBt Weight / Equivalent DIC Volume / Equivalent 1 King Resin 1.0eq-- 2 Fmoc-Phe-OH 3.0eq 3.0eq 3.0eq 3 Fmoc-Ala-OH 2.0eq 2.0eq 2.0eq 4 Fmoc-Lys(BOC)-OH 2.0eq 2.0eq 2.0eq 5 Fmoc-Ala-OH 2.0eq 2.0eq 2.0eq 6 Fmoc-Leu-OH 2.0eq 2.0eq 7 Fmoc-Lys(BOC)-OH 2.0eq 2.0eq 8 Fmoc-Lys(BOC)-OH 2.0eq 2.0eq 2.0eq 9 Fmoc-Ala-OH 2.0eq 2.0eq 10 Fmoc-Leu-OH 2.0eq 2.0eq 2.0eq 11 10-Hydroxy-2-decenoic acid 5eq 5eq 5eq

[0039] Note: The equivalent amounts of each raw material are calculated based on the initial resin substitution degree. Detailed feed amounts are described in the preparation and purification steps of this embodiment.

[0040] Preparation and purification steps:

[0041] Step (1) Preparation of Fmoc-Phe-O-King Resin

[0042] Weigh out 0.5 mmol / g of the resin and swell it in dichloromethane (DCM) for 30 min, then wash it twice with DMF (N,N-dimethylformamide). Weigh out 3.0 eq of Fmoc-Phe-OH and 3.0 eq of HOBt (1-hydroxybenzotriazole); dissolve them in DMF, add DIC (N,N'-diisopropylcarbodiimide; 3.0 eq) and DMAP (4-dimethylaminopyridine; 0.10 eq), activate at 0 °C for 5 min, and add to the resin. Stir the reaction under nitrogen protection for 3 h. After the reaction is complete, dry the mixture. Wash three times with DMF. Add 3 times the volume (V / W, ml / g; for example, 10g of resin feed with 30ml of acetic anhydride solution) of the obtained peptide resin to an acetic anhydride solution (volume ratio: acetic anhydride:pyridine:DMF = 1:1:3). React under nitrogen protection for 30 minutes. After the reaction is complete, dry the solution and wash with DMF until neutral. Add a 20% piperidine / DMF mixed solution to deprotect the solution, dry the solution, and wash with DMF until pH=7 (the washing solvent can also be the same solvent used in the previous step, used to wash 3 times first, and then washed with fresh DMF to save DMF solution).

[0043] Step (2) Preparation of Fmoc-Ala-Phe-O-King Resin

[0044] Weigh out Fmoc-Ala-OH and HOBt (2.0 eq) and dissolve them in DMF. Add DIC (2.0 eq), activate at 0℃ for 5 min, and add to the product from step (1). Stir the reaction under nitrogen protection. The ninhydrin test on the resin is negative. After the reaction is complete, dry the solution. Wash three times with DMF, add 20% piperidine / DMF mixed solution to deprotect, dry the solution, and wash with DMF until pH=7.

[0045] Step (3) Preparation of Fmoc-Lys(BOC)-Ala-Phe-O-King Resin

[0046] Weigh out Fmoc-Lys(BOC)-OH and HOBt (2.0 eq) and dissolve them in DMF. Add DIC (2.0 eq) and activate at 0°C for 5 min. Add the solution to the peptide resin. Stir the reaction under nitrogen protection. The resin showed a negative result for ninhydrin detection. After the reaction was complete, dry the solution under vacuum. Wash three times with DMF, then add a 20% piperidine / DMF mixed solution to deprotect the solution and dry under vacuum. Wash with DMF until pH=7 (the washing solvent can be reused from the previous step for three washes, followed by washing with fresh DMF to save DMF solution).

[0047] Step (4) Preparation of Fmoc-Ala-Lys(BOC)-Ala-Phe-O-King Resin

[0048] Weigh out Fmoc-Ala-OH and HOBt (2.0 eq) and dissolve them in DMF. Add DIC (2.0 eq) and activate at 0°C for 5 min. Add the solution to the peptide resin. Stir the reaction under nitrogen protection. The resin is negative for ninhydrin. After the reaction is complete, dry the solution. Wash three times with DMF, add 20% piperidine / DMF mixed solution to deprotect, dry the solution, and wash with DMF until pH=7.

[0049] Step (5) Preparation of Fmoc-Leu-Ala-Lys(BOC)-Ala-Phe-O-King Resin

[0050] Dissolve Fmoc-Leu-OH and HOBt (2.0 eq) in DMF, add DIC (2.0 eq), activate at 0℃ for 5 min, and add to peptide resin. Stir the reaction under nitrogen protection; the resin showed a negative result for ninhydrin detection. After the reaction was complete, dry under vacuum. Wash three times with DMF, add 20% piperidine / DMF mixed solution for deprotection, dry under vacuum, and wash with DMF until pH=7 (the washing solvent can also be the same solvent used in the previous step for three washes, followed by washing with fresh DMF to save DMF solution).

[0051] Step (6) Preparation of Fmoc-Lys(BOC)-Leu-Ala-Lys(BOC)-Ala-Phe-O-King Resin

[0052] Weigh out Fmoc-Lys(BOC)-OH and HOBt (2.0 eq) and dissolve them in DMF. Add DIC (2.0 eq) and activate at 0°C for 5 min. Add the solution to the peptide resin. Stir the reaction under nitrogen protection. The resin showed a negative result for ninhydrin detection. After the reaction was complete, dry the solution under vacuum. Wash three times with DMF, add 20% piperidine / DMF mixed solution to deprotect, dry the solution under vacuum, and wash with DMF until pH=7.

[0053] Step (7) Preparation of Fmoc-Lys(BOC)-Lys(BOC)-Leu-Ala-Lys-Ala-Phe-O-King Resin

[0054] Weigh out Fmoc-Lys(BOC)-OH and HOBt (2.0 eq) and dissolve them in DMF. Add DIC (2.0 eq) and activate at 0°C for 5 min. Add the solution to the peptide resin. Stir the reaction under nitrogen protection. The resin showed a negative result for ninhydrin detection. After the reaction was complete, dry the solution under vacuum. Wash three times with DMF, then add a 20% piperidine / DMF mixed solution to deprotect the solution and dry under vacuum. Wash with DMF until pH=7 (the washing solvent can also be the same as the solvent used in the previous step, washing three times first, then washing with fresh DMF to save DMF solution).

[0055] Step (8) Preparation of Fmoc-Ala-Lys(BOC)-Lys(BOC)-Leu-Ala-Lys(BOC)-Ala-Phe-O-King Resin

[0056] Weigh out Fmoc-Ala-OH and HOBt (2.0 eq) and dissolve them in DMF. Add DIC (2.0 eq) and activate at 0°C for 5 min. Add the solution to the peptide resin. Stir the reaction under nitrogen protection. The resin is negative for ninhydrin. After the reaction is complete, dry the solution. Wash three times with DMF, add 20% piperidine / DMF mixed solution to deprotect, dry the solution, and wash with DMF until pH=7.

[0057] Step (9) Preparation of Fmoc-Leu-Ala-Lys(BOC)-Lys(BOC)-Leu-Ala-Lys(BOC)-Ala-Phe-O-King Resin

[0058] Weigh out Fmoc-Leu-OH and HOBt (2.0 eq) and dissolve them in DMF. Add DIC (2.0 eq) and activate at 0°C for 5 min. Add the solution to the peptide resin. Stir the reaction under nitrogen protection. The resin is negative for ninhydrin. After the reaction is complete, dry the solution. Wash three times with DMF, add 20% piperidine / DMF mixed solution to deprotect, dry the solution, and wash with DMF until pH=7.

[0059] Step (10) Preparation of 10-HDA-Leu-Ala-Lys(BOC)-Lys(BOC)-Leu-Ala-Lys(BOC)-Ala-Phe-O-King Resin

[0060] Weigh a measured amount of 10-hydroxy-2-decenoic acid (10-HDA), dissolve it in DMF, and add the mixed solution to the peptide resin from the previous step. React under nitrogen protection. The resin showed a negative result for ninhydrin. The reaction was complete. Wash three times with DMF. Swell and shrink three times alternately with dichloromethane and methanol. Finally, shrink with methanol. Dry the resin to constant weight.

[0061] Step (11) Preparation of crude 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH (royal acid nonapeptide)

[0062] Lysis buffer 1 (composition by volume: trifluoroacetic acid: phenol: 1,2-ethanedithiol, anisole sulfide: water = 82.5:5:2.5:5:5), lysis buffer 2 (composition by volume: trifluoroacetic acid: 1,2-ethanedithiol: water = 95:2.5:2.5), and lysis buffer 3 (composition by volume: trifluoroacetic acid: water = 95:5) should each have a volume 10 times the weight of the peptide resin from the previous step (V / W, ml / g; for example, 10g of peptide resin requires 100ml of lysis buffer). After preparing the lysis buffers, cool them to 0°C.

[0063] The resin was added to 10 times its volume of lysis buffer, and the mixture was stirred for 2 hours. The resin was filtered off using a sintered glass funnel. The filtrate was concentrated until a slight solid precipitate appeared, and then added to cold methyl tert-butyl ether (methyl tert-butyl ether volume was 10 times the volume of the concentrated solution) with stirring. A white solid precipitated. Stirring was continued for 1 hour to allow precipitation. The suspension was centrifuged, and the supernatant was discarded. The solid was washed with methyl tert-butyl ether and centrifuged again, repeating the washing and centrifugation three times. The solid was dried under vacuum to constant weight to obtain crude royal jelly nonapeptide.

[0064] Step (12) Purification and preparation of royal jelly acid nonapeptide

[0065] The crude royal jelly nonapeptide was dissolved and purified using a reverse-phase C8 column (10 μm, 100 Å, 250*20 mm), followed by lyophilization. The purified royal jelly nonapeptide was obtained.

[0066] Crude royal jelly acid nonapeptide was dissolved in 30% (v / v) acetonitrile aqueous solution (20 times its mass), and filtered through a 0.22 μm organic filter membrane. The filtrate was purified and separated using a C8 reversed-phase column. The detection wavelength was 210 nm, and the sample was loaded at a flow rate of 20 mL / min. Gradient elution was used: mobile phase A was 0.1% acetic acid solution, and mobile phase B was acetonitrile, by volume percentage.

[0067] Table 2 Gradient elution program

[0068] Time (min) Mobile phase A (%) Mobile phase B (%) 0 9 0 10 30 65 35 33 20 80 38 20 80 40 90 10 45 90 10

[0069] The HPLC chromatogram of the purified product after elution is shown in Figure 1. The retention time of royal jelly acid nonapeptide was 16.328 min (peak area 7180437), with two very small impurities present at 15.955 min (peak area 39399) and 16.989 min (peak area 26358). The purity of royal jelly acid nonapeptide in the product obtained from the elution range of 15–17 min was 99.093% based on peak area.

[0070] The mass spectrum of royal jelly nonapeptide is shown in Figure 2, exhibiting mass peaks at 1158.1 (m / z), 772.6 (m / z), 718.8 (m / z), 660.9 (m / z), 601.6 (m / z), 590.7 (m / z), 579.8 (m / z), 386.9 (m / z), 348.5 (m / z), 296.6 (m / z), 222.3 (m / z), 180.3 (m / z), 163.6 (m / z), 111.1 (m / z), and 106.0 (m / z). The mass-to-charge ratio (m / z) peak with the highest value is at 1158.1 (m / z). NMR data for royal jelly nonapeptide are also provided. 1 H NMR (400 MHz, dmso) δ 12.77 (s, 3H), 8.27 – 7.96 (m, 3H), 7.95 – 7.79 (m, 2H), 7.69 (s, 5H), 7.36–7.08 (m, 3H), 6.71 – 6.48 (m, 2H), 5.98 (d,J= 15.5 Hz, 1H), 4.47 – 4.04 (m, 5H), 3.47 – 3.10 (m, 45H), 3.04 (dd,J= 13.8, 5.1 Hz, 1H), 2.89 (dd,J= 14.0, 8.7 Hz, 1H), 2.70 (d,J= 26.4 Hz, 3H), 2.12 (dd,J= 14.0, 6.9 Hz, 2H), 1.40 (dddd,J= 13.0, 10.5, 10.0, 4.9 Hz, 16H), 0.85 (dt,J= 13.8, 6.5 Hz, 6H).

[0071] Example 2: Preparation and purification of royal jelly acid nonapeptide

[0072] Example 2 provides an additional method for preparing and purifying royal jelly nonapeptide, following the preparation and purification methods of Example 1. The difference from the example lies in the degree of royal jelly resin substitution.

[0073] Step (1) Preparation of Fmoc-Phe-O-King Resin

[0074] Weigh 50 g (1.09 mmol / g) of the resin and swell it in DCM for 30 min, then wash it twice with DMF. Weigh 63.3 g (3.0 eq) of Fmoc-Phe-OH and 22.1 g (3.0 eq) of HOBt and dissolve them in DMF. Add 25.3 ml (3.0 eq) of DIC and 0.67 g (0.10 eq) of DMAP, activate at 0 °C for 5 min, and add to the resin. Stir and react for 3 hours under nitrogen protection. After the reaction is complete, dry the mixture. Wash three times with DMF. Add 150 ml of acetic anhydride solution to the obtained resin (the volume ratio of each reagent is acetic anhydride:pyridine:DMF = 1:1:3), react under N2 protection for 30 min, dry under vacuum after the reaction is complete, wash with DMF until neutral, add 20% piperidine / DMF mixed solution to deprotect, dry under vacuum, wash with DMF until pH=7 (the washing solvent can be the same solvent used in the previous step to wash 3 times first, then wash with fresh DMF to save DMF solution).

[0075] Step (2) Preparation of Fmoc-Ala-Phe-O-King Resin

[0076] Weigh 33.9 g (2.0 eq) of Fmoc-Ala-OH and 14.7 g (2.0 eq) of HOBt and dissolve them in DMF. After dissolving, add 16.8 ml (2.0 eq) of DIC and activate at 0 °C for 5 min. Add the activated DIC to the product from step (1). Stir the reaction under nitrogen protection. The ninhydrin test on the resin was negative. After the reaction was complete, dry the solution. Wash the product three times with DMF, add 20% piperidine / DMF mixed solution to deprotect it, dry the solution, and wash the product with DMF until pH=7.

[0077] Steps (3)-(12) Preparation of crude 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH (royal acid nonapeptide)

[0078] Following the routine steps (2) of Example 1 and this Example, amino acid coupling and 10-HDA covalent coupling were performed sequentially, connecting Fmoc-Lys(BOC)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Lys(BOC)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, and 10-HDA to obtain 126g of crude royal acetic acid nonapeptide with a protecting group. 1250ml of TFA solution (lysis buffer, volume ratio of trifluoroacetic acid:water = 95:5) was added for lysis for approximately 2 hours. After centrifugation, washing, and vacuum drying, 68.4g of crude royal acetic acid nonapeptide was obtained. After purification, 24g of lyophilized powder was obtained.

[0079] The mass spectrum of royal jelly nonapeptide is shown in Figure 2. NMR data of royal jelly nonapeptide: 1 H NMR (400 MHz, dmso) δ 12.77 (s, 3H), 8.27 – 7.96 (m, 3H), 7.95 – 7.79 (m, 2H), 7.69 (s, 5H), 7.36 – 7.08 (m, 3H), 6.71 – 6.48 (m, 2H), 5.98 (d,J= 15.5 Hz, 1H), 4.47 – 4.04 (m, 5H), 3.47 – 3.10 (m, 45H), 3.04 (dd,J= 13.8, 5.1 Hz, 1H), 2.89 (dd,J= 14.0, 8.7 Hz, 1H), 2.70 (d,J= 26.4 Hz, 3H), 2.12 (dd,J= 14.0, 6.9 Hz, 2H), 1.40 (dddd,J= 13.0, 10.5, 10.0, 4.9 Hz, 16H), 0.85 (dt,J= 13.8, 6.5 Hz, 6H).

[0080] Example 3: Preliminary investigation of the in vitro matrix metalloproteinase (MMP) inhibitory effect of royal jelly nonapeptide.

[0081] The royal jelly nonapeptide used in this embodiment was prepared and purified according to the method in Example 1. All other materials, reagents, and kits, such as palmitoyl nonapeptide-3, were commercially available. Unless otherwise specified, cell culture was performed under standard conditions.

[0082] 1. Materials

[0083] 1.1 Cells

[0084] The cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd.

[0085] 1.2 Cell Culture Related Reagents

[0086] DMEM medium (Gibco, 11966025); Fetal bovine serum (FBS) (gibco, 16140071); Penicillin-streptomycin (PS) (gibco, 15140122); Phosphate-buffered saline (PBS) (gibco, 10010023); Trypsin-EDTA (0.05%) (gibco, 25300062); SteadyPure Rapid RNA Extraction Kit (Akerui, AG21023); Reverse Transcription Kit (Akerui, AG11728); SYBR Green (Akerui, AG11740); AR analytical grade ethanol (Greagent, G73537B); CCK8 (APE×BIO, K1018); ELISA Kit (YEASEN, 97028ES96 / 97035ES96 / 97068ES96); CUSABIO (CSB-E08082h / CSB-E04672h); Hank's balanced salt solution (HBSS) (Adamas life, C8025); Fluo-4, AM fluorescent probe (Yeasen, HB220823).

[0087] 1.3 Experimental Drugs

[0088] Lipopolysaccharide (LPS), royal jelly nonapeptide, palmitoyl nonapeptide-3, capsaicin.

[0089] 1.4 Instruments

[0090] Q-PCR instrument, ELISA reader, fluorescence microscope.

[0091] 2 Experimental Methods

[0092] 2.1 Cytotoxicity CCK-8 assay

[0093] CCK8, short for Cell Counting Kit-8, is a simple and accurate assay for cell proliferation and toxicity. HaCaT (human immortalized epidermal cells) cells are placed in DMEM medium (Gibco, 11966025) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and evenly seeded into 96-well plates (5 × 10⁻⁶). 4Cells were incubated at 37°C for 24 h (cells / well). Cells were washed twice with PBS buffer and incubated for 24 h with lipopolysaccharide (LPS), palmitoyl nonapeptide-3, royal jelly nonapeptide, and culture medium (negative control), respectively. Assay concentrations: palmitoyl nonapeptide and royal jelly nonapeptide concentrations were 0.001 wt%, 0.0025 wt%, 0.005 wt%, 0.01 wt%, 0.025 wt%, and 0.05 wt%, respectively; LPS concentrations were 1 μg / ml, 2 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, and 50 μg / ml. Subsequently, CCK8 solution was prepared and incubated for 30–60 min. Absorbance was measured at OD 450 nm, and cell viability (%) relative to untreated control culture (negative control, representing 100% viability) was calculated. Data are the mean ± standard deviation (SD) of three replicates from three independent experiments.

[0094] 2.2 Capsaicin's effect on intracellular calcium 2+ Effects of flux

[0095] HaCaT cells were cultured in 6-well plates for 24 h. The culture medium was then treated with the following solutions: 0.001 wt% retinol, 2 μM capsaicin, 3 μM Fluo-4 AM (fluorescent probe), 0.05 wt% palmitoyl nonapeptide-3, and 0.05 wt% royal jelly nonapeptide. Cells were then stained with Fluo-4 AM at 37 °C for 1 h. After incubation, the cells were washed twice with PBS and observed under a fluorescence microscope.

[0096] 2.3 Analysis of the expression of relevant genes in the test samples using real-time quantitative Q-PCR

[0097] Real-time quantitative PCR was performed using the Sybr-Green method (Bio-Rad, California, USA). The relative expression level of mRNA was determined after normalization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). HaCaT cells were cultured for 24 h, and then treated with the following samples: LPS at 20 μg / ml, royal jelly nonapeptide and palmitoyl nonapeptide-3 at 0.05 wt%, and retinol at 0.001 wt%; incubation was performed at 37°C for 24 h. Total RNA was isolated from the cells using TRIzol™ reagent (Invitrogen, USA).

[0098] 2.4 Analysis of the expression of relevant proteins in the test samples using ELISA method

[0099] The expression levels of two proteins in the test samples were determined using a human collagen type I (COL-1) ELISA kit and an MMP-1 ELISA kit, respectively. COL-1 is a structural protein secreted by human skin fibroblasts, while MMP-1 is an aging secretion phenotype factor that degrades collagen. Therefore, the upregulation or downregulation of these two proteins is closely related to the aging phenotype of the skin. The expression level of MMP-1 was tested after HaCaT cells were cultured with the test samples for 24 hours; the expression level of COL-1 was tested after human skin fibroblasts (HFF) were cultured with the test samples for 24 hours. Test sample concentrations: LPS 20 μg / ml, royal jelly nonapeptide and palmitoyl nonapeptide-3 0.05 wt%, retinol 0.001 wt%; incubation at 37℃ for 24 hours.

[0100] 3 Results

[0101] The following are the results of four experiments.

[0102] 3.1 CCK8

[0103] As shown in Figure 3, LPS is safe for HaCaT cells at concentrations below 50 μg / ml, and 20 μg / ml was used for subsequent experiments to establish an inflammation model. The median lethal concentration (LD50) of retinol is 0.0025 wt%, and it is safe for cells below this concentration; 0.001 wt% was used in subsequent experiments. Royal venom nonapeptide concentrations below 1 wt% did not reach the LD50, and palmitoyl nonapeptide-3 concentrations below 0.05 wt% did not reach the LD50; 0.05 wt% was used in subsequent experiments.

[0104] 3.2 Capsaicin's effect on intracellular calcium 2+ Effects of flux

[0105] Capsaicin stimulates [Ca] by activating the capsaicin receptor TRPV1. 2+ Accumulation, exerting a pain-stimulating effect. As shown in Figure 4, Fluo-4 AM ester is used as Ca... 2+ The fluorescent indicator was used to determine [Ca] in HaCaT. 2+ Cellular accumulation. Results showed that capsaicin significantly increased [Ca]. 2+ The accumulation of ] . Stimulus [Ca 2+ The intensity of accumulation was as follows: capsaicin > retinol > palmitoyl nonapeptide-3 > royal jelly nonapeptide > NC (NC was the normal control, without any test substance or drug added). The analgesic effect of royal jelly nonapeptide was significantly lower than that of retinol and palmitoyl nonapeptide-3.

[0106] 3.3 Results of Q-PCR and ELISA experiments

[0107] As shown in Figure 5, in the LPS-induced inflammatory cell model, palmitoyl nonapeptide-3, royal jelly nonapeptide, and retinol all reduced MMP1 protein expression. The 0.05% royal jelly nonapeptide group showed the best effect, with significantly lower MMP1 protein levels compared to the other groups (p<0.05 or p<0.01). In the inflammatory cell model, three inflammatory factors, IL-1β, IL-8, and IL-6, were tested. Both palmitoyl nonapeptide-3 and royal jelly nonapeptide inhibited IL-6 expression levels, with no significant difference between the two groups.

[0108] As shown in Figure 5, ELISA analysis revealed that palmitoyl nonapeptide-3, royal jelly nonapeptide, and retinol all increased the level of type I collagen (COL-1) in human skin fibroblasts (HFF). Among these, the 0.05% royal jelly nonapeptide group showed the best effect, with significantly higher COL-1 levels compared to the other groups (p<0.05 or p<0.01).

[0109] Example 4: Investigation on the regulatory effect of royal jelly nonapeptide on total collagen

[0110] The royal jelly nonapeptide used in this embodiment was prepared and purified according to the method in Example 1. All other materials, reagents, and kits were commercially available. Unless otherwise specified, cell culture was performed under standard conditions.

[0111] Human skin fibroblasts (HFF) were placed in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and evenly seeded into 96-well plates (1.5 x 10⁻⁶). 5 Cells were incubated at 37°C for 24 h (cells / well). Cells were washed twice with PBS buffer and incubated at 37°C for 24 h with different analytes. The total content of soluble collagen 1-IV in the culture was determined using the Sircol Soluble Collagen Assay Kit. Assay 1 was royal jelly acid (0.05 wt%); Assay 2 was palmitoyl nonapeptide-3 (0.05 wt%); Assay 3 was a mixture of royal jelly acid and palmitoyl nonapeptide-3, both at 0.05 wt%; Assay 4 was royal jelly nonapeptide (0.05 wt%). Data are the mean ± standard deviation of three replicates from three independent experiments.

[0112] Compared with the negative control group (without test substances), all three substances and the mixture increased the total content of soluble collagen 1–IV in cells. The soluble collagen content in the three test substance groups was 5.06 ± 0.25 μg / 1x10⁻⁶. 5 The soluble collagen content in the four test groups was 6.47 ± 0.21 μg / 1 x 10⁻⁶ cells. 5Cells. The soluble collagen content in both groups was significantly higher than that in royal jelly (p<0.01) and palmitoyl nonapeptide-3 (p<0.05), with the soluble collagen content in test substance 4 being significantly higher than that in test substance 3 (p<0.05).

[0113] The above are preferred embodiments of the present invention. It should be noted that these embodiments are for explanation and illustration only and are not intended to limit the scope of protection. Under the main scheme, spirit, and principles of the present invention, the technical solution of the present invention can have many variations / substitutions / changes, such as replacement of the amino acid protecting group, replacement of the resin, replacement of skin care excipients, etc. Any variations / substitutions / changes made by those skilled in the art without creative effort under the spirit and principles of the technical solution in this specification should fall within the scope of protection of the present invention.

Claims

1. A polypeptide that promotes collagen synthesis, characterized in that, The structural formula of the polypeptide is shown in Formula I: 。 2. The method for preparing a polypeptide that promotes collagen synthesis according to claim 1, characterized in that, Includes the following steps: Synthesis of S1 linear peptide: Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O- resin was prepared by stepwise coupling using a solid-phase synthesis method, wherein M1, M2, and M3 are amino protecting groups; Synthesis of S2 royal ic acid covalently coupled linear peptide: 10-HDA was reacted with Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O- resin to prepare 10-HDA-Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O- resin; Cleavage of S3 royal jelly acid nonapeptide: Cleavage of 10-HDA-Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O- resin yields crude 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH.

3. The method for preparing a polypeptide that promotes collagen synthesis according to claim 2, characterized in that, The resin is selected from either royal jelly resin or CTC resin.

4. The method for preparing a polypeptide that promotes collagen synthesis according to claim 2, characterized in that, M1, M2, and M3 are independently selected from any one of the following protecting groups: BOC, Fmoc, Trt, Dde, and Allyl.

5. The method for preparing a polypeptide that promotes collagen synthesis according to claim 2, characterized in that, The preparation method further includes the following steps: S4 Product Purification: The crude 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH was dissolved in 30% (v / v) acetonitrile aqueous solution and filtered through a 0.22 μm organic filter membrane; the filtrate was purified and separated using a C8 reversed-phase column; the sample was loaded at a flow rate of 20 ml / min with a detection wavelength of 210 nm; gradient elution was performed using mobile phases: mobile phase A was 0.1% acetic acid solution, and mobile phase B was methanol or acetonitrile.

6. The method for preparing a polypeptide that promotes collagen synthesis according to claim 5, characterized in that, The mobile phase B is acetonitrile, and the elution procedure for the mobile phase is as follows: Time (min) Mobile phase A (%) Mobile phase B (%) 0 9 0 10 30 65 35 33 20 80 38 20 80 40 90 10 45 90 10.

7. A skincare product, characterized in that, The polypeptide containing the collagen-promoting polypeptide of claim 1 or the polypeptide prepared by any one of claims 2 to 6.

8. A skincare product according to claim 7, characterized in that, The skin care product is a transdermal drug delivery formulation.

9. A skincare product according to claim 8, characterized in that, The excipient of the transdermal drug delivery formulation is a 5 wt% aqueous solution of 1,2-pentanediol.

10. The use of the polypeptide that promotes collagen synthesis according to claim 1 or the polypeptide prepared by any one of claims 2 to 6 in the preparation of a formulation that increases the collagen content of skin fibroblasts.

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