Polypeptide for promoting synthesis of collagen, and preparation method, preparation and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide for promoting collagen synthesis as well as a preparation method, a preparation and application of the polypeptide. The polypeptide is nonapeptide of which the tail end is coupled with queen bee acid, is prepared by adopting a polypeptide solid-phase synthesis method, can improve the content of cell collagen, has weak effect on capsaicin receptor and mediated calcium influx, and has lower pain stimulation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide for promoting collagen synthesis, a preparation method thereof, a preparation and an application thereof. Background Art

[0002] The purpose of the background art information disclosed below is only to increase the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art known to those of ordinary skill in the art.

[0003] Collagen is an important component of human tissues such as skin and cartilage, and is crucial for maintaining the elasticity of skin and cartilage. Especially as a fibrous protein in the dermis of the skin, collagen is the key protein for maintaining skin elasticity and firmness, and is also a key target for the development of anti-wrinkle drugs and cosmetics. Fibroblasts are the main cells for synthesizing and secreting collagen. Therefore, enhancing the viability of fibroblasts is one of the strategies for promoting skin collagen synthesis. In addition, matrix metalloproteinase (MMP) is a class of zinc ion-dependent extracellular endopeptidases and is identified as a collagen-hydrolyzing enzyme. The hydrolysis of collagen by MMP is one of the direct causes of wrinkle formation. Therefore, MMP inhibitors are also one of the strategies for promoting skin collagen synthesis.

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

[0005] However, 10-hydroxy-2-decenoic acid still has many defects such as poor transdermal permeability and poor water solubility, and cosmetic peptides also have deficiencies such as poor transdermal permeability and low efficacy. So far, a complex emulsifier system or special preparations such as liposome preparations are usually required to improve the transdermal absorption effect of cosmetics containing such substances. However, the complex emulsifier system often has excipients suitable as microbial carbon sources, resulting in frequent problems of unqualified colony counts in cosmetics. And compared with other small molecule substances, cosmetic peptides are expensive. Increasing their activity as much as possible to reduce the dosage is beneficial to overcoming their high cost disadvantages. Summary of the Invention

[0006] In view of the above problems, the inventors investigated the combination strategy of 10-HDA and cosmetic peptides and found that it is difficult to obtain additional efficacy benefits when 10-HDA is used transdermally in combination with cosmetic peptides due to its poor skin penetration and absorption characteristics; when 10-HDA is covalently coupled with most cosmetic peptides, it is also difficult to obtain additional efficacy benefits, but covalent coupling with nonapeptide-3 has additional efficacy benefits, which is beneficial to reducing the dosage of the peptide (better efficacy under the same dosage). Therefore, the present invention provides a polypeptide for promoting collagen synthesis, its preparation method, preparation and application. The present invention provides the following technical solutions:

[0007] A polypeptide for promoting collagen synthesis, named "royal acid nonapeptide", and its structural formula is shown in Formula I:

[0008]

[0009] Specifically, the royal acid nonapeptide is a covalent conjugate of nonapeptide and royal acid (10-HDA) (equivalent to palmitoyl nonapeptide-3 modified by palmitic acid acylation to royal acid acylation), and can also be expressed as: 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH, and its molecular formula is C 58 H 100 N 12 O 12 , and the relative molecular weight is about 1157.51. Research shows that the activity of the royal acid nonapeptide in inhibiting MMP and promoting collagen synthesis under the same dosage is higher than that of royal acid, palmitoyl nonapeptide-3 and their mixture, with significant advantages.

[0010] The term "structural formula" can also be understood as "simplified structural formula", which is a chemical formula using element symbols and short lines to represent the arrangement and bonding mode of atoms in a compound (or simple substance) molecule, and is a method to simply describe the molecular structure. The structural formula can completely draw the chemical bonds between each atom in the molecule. When representing organic compounds, the simplified structural formula is generally used, usually omitting carbon-hydrogen bonds, and sometimes also omitting carbon-carbon single bonds, or using a bond-line formula to represent. Obviously, "structural formula" and "simplified structural formula" are only used to show the connection mode and bonding situation of atoms in organic compounds, and do not represent the true spatial configuration / conformation and true bond angle of the molecule.

[0011] As a display of the relationship between atoms and chemical bonds, the structural formula of Formula I first satisfies the basic valence bond rules of atoms: C, N, and O are tetravalent, trivalent, and divalent respectively. Therefore, the understanding of Formula I should be based on this valence bond rule as the basic premise. In any case, when the symbols, numbers, and bond lines of Formula I are unclear or there are mistakes in writing the structural formula, the understanding of Formula I should be based on C, N, and O being tetravalent, trivalent, and divalent respectively. When only a bond line is used at the end in the structural formula of Formula I, it represents that the end is -CH3 (methyl).

[0012] A preparation method of the aforementioned "nonapeptide of royal jelly acid" includes the following steps:

[0013] S1 Synthesis of linear peptide: Using solid-phase synthesis method, gradually couple and prepare

[0014] Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O-resin, where M1, M2, and M3 are amino protecting groups;

[0015] S2 Synthesis of nonapeptide of royal jelly acid covalently coupled with linear peptide: 10-HDA reacts with

[0016] 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;

[0017] S3 Cleavage of nonapeptide of royal jelly acid: Cleave 10-HDA-Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O-resin to obtain the crude product of 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH.

[0018] Preferably, the resin is selected from any one of Wang resin and CTC resin;

[0019] Preferably, M1, M2, and M3 are independently selected from any one of BOC protecting group (tert-butoxycarbonyl), Fmoc protecting group (9-fluorenylmethoxycarbonyl), Trt protecting group (triphenylmethyl), Dde protecting group, and Allyl (allyl) protecting group. Resins commonly used in solid-phase peptide synthesis, amino protecting groups, and coupling, deprotection, and cleavage conditions, etc. belong to the prior art in this field. For details, please refer to "Research and Development of Peptide Drugs" edited by Li Baoqiu (published by People's Medical Publishing House in 2011); "Peptide Synthesis" edited by Huang Weide and Chen Changqing (published by Science Press in 1985).

[0020] Preferably, the preparation method further includes the following steps:

[0021] S4 Product purification: The crude product of 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH is dissolved in an aqueous solution of acetonitrile with a volume percentage of 30%, and passed through a 0.22 μm organic filter membrane; the filtrate is purified and separated by a C8 reverse-phase column; the detection wavelength is 210 nm, and the sample is loaded at a flow rate of 20 ml / min. Gradient elution of the mobile phase: Mobile phase A is a 0.1% acetic acid solution, and mobile phase B is methanol or acetonitrile.

[0022] More preferably, mobile phase B is acetonitrile, and the mobile phase elution program is as follows:

[0023] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 90 10 30 65 35 33 20 80 38 20 80 40 90 10 45 90 10

[0024] A skin care product containing the aforementioned "nonapeptide of royal jelly acid" or the "nonapeptide of royal jelly acid" prepared by the aforementioned preparation method.

[0025] Preferably, the skin care product is a transdermal drug delivery preparation.

[0026] Preferably, the excipient of the transdermal drug delivery preparation is an aqueous solution of 5 wt% 1,2-pentanediol.

[0027] Use of the aforementioned "nonapeptide of royal jelly acid" or the "nonapeptide of royal jelly acid" prepared by the aforementioned preparation method or the aforementioned skin care product in the preparation of a preparation for inhibiting MMP-1 and increasing the collagen content of skin fibroblasts.

[0028] Beneficial effects:

[0029] (1) The nonapeptide of royal jelly acid of the present invention has good effects of inhibiting MMP and increasing the collagen content of the skin.

[0030] (2) The effect of the nonapeptide of royal jelly acid of the present invention in inhibiting MMP and increasing the content of skin collagen is superior to that of royal jelly acid, palmitoyl nonapeptide-3 and the physical mixture with the same dosage, overcoming the deficiencies of poor transdermal effect, large dosage or the need for special liposome preparations of royal jelly acid itself, and also overcoming the deficiency of large dosage and high cost of palmitoyl nonapeptide-3.

[0031] (3) The nonapeptide of royal jelly acid of the present invention has high safety, and the safe concentration for skin cells is much higher than that of palmitoyl nonapeptide-3, retinol, etc.; and it has little effect on the calcium ion flux related to pain.

[0032] Description of the drawings

[0033] Figure 1 It is the HPLC chromatogram for the preparation and purification of the nonapeptide of royal jelly acid in Example 1;

[0034] Figure 2 It is the mass spectrum of the nonapeptide of royal jelly acid;

[0035] Figure 3 It is the result graph of the influence of different test substances on cell viability in Example 3;

[0036] Figure 4 It is the fluorescence graph of the influence of different test substances on cell calcium ion flux in Example 3;

[0037] Figure 5 It is the result graph of the influence of different test substances on the content of MMP-1 in cells in Example 3. Detailed implementation manners

[0038] The following further illustrates the solutions and effects of the present invention in combination with the drawings, tables and examples to meet the requirements of clear, complete and implementable technical solutions. The reagents, instruments, reagent kits, cells, etc. used in the following examples are all materials and equipment that can be obtained in the market.

[0039] Example 1 Preparation and purification of the nonapeptide of royal jelly acid

[0040] Example 1 provides a method for the preparation and purification of the nonapeptide of royal jelly acid, and Table 1 is the feeding table for the preparation process of the nonapeptide of royal jelly acid.

[0041] Table 1 Feeding table for the preparation process of the nonapeptide of royal jelly acid

[0042]

[0043] Remarks: The use of each raw material is calculated by equivalent based on the initial resin substitution degree. The detailed feeding amount can be seen in the description of the preparation and purification steps of this example.

[0044] Preparation and purification steps:

[0045] Preparation of Fmoc-Phe-O-Wang Resin

[0046] Weigh Wang resin (0.5 mmol / g) and swell it in dichloromethane (DCM) for 30 min, then wash it twice with DMF (N,N-dimethylformamide). Weigh Fmoc-Phe-OH (3.0 eq) and HOBt (1-hydroxybenzotriazole; 3.0 eq), 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 it to the resin. Stir and react under nitrogen protection for 3 h. After the reaction is completed, drain. Wash three times with DMF. Add an acetic anhydride solution (volume ratio composition: acetic anhydride: pyridine: DMF = 1:1:3) that is 3 times the amount of the charged Wang resin (V / W, ml / g; for example, if 10 g of resin is charged, use 30 ml of acetic anhydride solution) to the obtained peptide resin, react under nitrogen protection for 30 min, drain after the reaction is completed, wash with DMF until neutral, add a 20% piperidine / DMF mixed solution for deprotection, drain, and wash with DMF until pH = 7 (the washing solvent can also be washed 3 times with the solvent used in the previous step first, and then washed with fresh DMF to save DMF solution).

[0047] Preparation of Fmoc-Ala-Phe-O-Wang Resin

[0048] Weigh Fmoc-Ala-OH and HOBt (2.0 eq), dissolve them in DMF, add DIC (2.0 eq), activate at 0 °C for 5 min, and add it to the product of step (1). Stir and react under nitrogen protection, and the resin is negative by ninhydrin detection. After the reaction is completed, drain. Wash three times with DMF, add a 20% piperidine / DMF mixed solution for deprotection, drain, and wash with DMF until pH = 7.

[0049] Preparation of Fmoc-Lys(BOC)-Ala-Phe-O-Wang Resin

[0050] Weigh Fmoc-Lys(BOC)-OH and HOBt (2.0 eq), dissolve them in DMF, add DIC (2.0 eq), activate at 0 °C for 5 min, and add it to the peptide resin. Stir and react under nitrogen protection, and the resin is negative by ninhydrin detection. After the reaction is completed, drain. Wash three times with DMF, then add a 20% piperidine / DMF mixed solution for deprotection, drain. Wash with DMF until pH = 7 (the washing solvent can be washed 3 times with the solvent used in the previous step first, and then washed with fresh DMF to save DMF solution).

[0051] Preparation of Fmoc-Ala-Lys(BOC)-Ala-Phe-O-Wang Resin

[0052] Weigh Fmoc-Ala-OH and HOBt (2.0 eq), dissolve them in DMF, add DIC (2.0 eq), activate at 0 °C for 5 min, and add to the peptide resin. Stir the reaction under nitrogen protection. The ninhydrin test shows that the resin is negative. After the reaction is completed, drain. Wash three times with DMF, add a 20% piperidine / DMF mixed solution for deprotection, drain, and wash with DMF until the pH = 7.

[0053] Step (5) Preparation of Fmoc-Leu-Ala-Lys(BOC)-Ala-Phe-O-Wang resin

[0054] Weigh Fmoc-Leu-OH and HOBt (2.0 eq), dissolve them in DMF, add DIC (2.0 eq), activate at 0 °C for 5 min, and add to the peptide resin. Stir the reaction under nitrogen protection. The ninhydrin test shows that the resin is negative. After the reaction is completed, drain. Wash three times with DMF, add a 20% piperidine / DMF mixed solution for deprotection, drain, and wash with DMF until the pH = 7 (the washing solvent can also be the solvent used in the previous step to wash 3 times first, and then wash with fresh DMF to save DMF solution).

[0055] Step (6) Preparation of Fmoc-Lys(BOC)-Leu-Ala-Lys(BOC)-Ala-Phe-O-Wang resin

[0056] Weigh Fmoc-Lys(BOC)-OH and HOBt (2.0 eq), dissolve them in DMF, add DIC (2.0 eq), activate at 0 °C for 5 min, and add to the peptide resin. Stir the reaction under nitrogen protection. The ninhydrin test shows that the resin is negative. After the reaction is completed, drain. Wash three times with DMF, add a 20% piperidine / DMF mixed solution for deprotection, drain, and wash with DMF until the pH = 7.

[0057] Step (7) Preparation of Fmoc-Lys(BOC)-Lys(BOC)-Leu-Ala-Lys-Ala-Phe-O-Wang resin

[0058] Weigh Fmoc-Lys(BOC)-OH and HOBt (2.0 eq), dissolve them in DMF, add DIC (2.0 eq), activate at 0 °C for 5 min, and add to the peptide resin. Stir the reaction under nitrogen protection. The ninhydrin test shows that the resin is negative. After the reaction is completed, drain. Wash three times with DMF, then add a 20% piperidine / DMF mixed solution for deprotection, drain. Wash with DMF until the pH = 7 (the washing solvent can also be the solvent used in the previous step to wash 3 times first, and then wash with fresh DMF to save DMF solution).

[0059] Preparation of Fmoc-Ala-Lys(BOC)-Lys(BOC)-Leu-Ala-Lys(BOC)-Ala-Phe-O-Wang resin in step (8)

[0060] Weigh Fmoc-Ala-OH and HOBt (2.0 eq), dissolve them in DMF, add DIC (2.0 eq), activate at 0 °C for 5 min, and add to the peptide resin. Stir the reaction under nitrogen protection, and detect that the resin is negative by ninhydrin. After the reaction is completed, drain. Wash three times with DMF, add 20% piperidine / DMF mixed solution for deprotection, drain, and wash with DMF until pH = 7.

[0061] Preparation of Fmoc-Leu-Ala-Lys(BOC)-Lys(BOC)-Leu-Ala-Lys(BOC)-Ala-Phe-O-Wang resin in step (9)

[0062] Weigh Fmoc-Leu-OH and HOBt (2.0 eq), dissolve them in DMF, add DIC (2.0 eq), activate at 0 °C for 5 min, and add to the peptide resin. Stir the reaction under nitrogen protection, and detect that the resin is negative by ninhydrin. After the reaction is completed, drain. Wash three times with DMF, add 20% piperidine / DMF mixed solution for deprotection, drain, and wash with DMF until pH = 7.

[0063] Preparation of 10-HDA-Leu-Ala-Lys(BOC)-Lys(BOC)-Leu-Ala-Lys(BOC)-Ala-Phe-O-Wang resin in step (10)

[0064] Weigh a certain amount of 10-hydroxy-2-decenoic acid (10-HDA), dissolve it in DMF, add the mixed solution to the peptide resin of the previous step, and react under nitrogen protection. Detect that the resin is negative by ninhydrin. After the reaction is completed. Wash three times with DMF. Swell and contract alternately with dichloromethane and methanol three times. Finally, contract with methanol. Dry the resin to constant weight.

[0065] Preparation of crude 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH (nonapeptide of royal jelly acid) in step (11)

[0066] Lysis solution 1 (composed by volume ratio: trifluoroacetic acid:phenol:1,2-ethanedithiol, anisole:water = 82.5:5:2.5:5:5), lysis solution 2 (composed by volume ratio: trifluoroacetic acid:1,2-ethanedithiol:water = 95:2.5:2.5), lysis solution 3 (composed by volume ratio: trifluoroacetic acid:water = 95:5), with a volume 10 times the weight of the peptide resin in the previous step (V / W, ml / g; for example, if the peptide resin is 10 g, 100 ml of lysis solution is used). After the lysis solution is prepared, it is cooled to 0 °C.

[0067] Add the resin to 10 times the amount of lysis solution, and stir and react for 2 hours. Filter off the resin with a sintered glass funnel. Concentrate the filtrate until a slight solid precipitation occurs, and add it to cold methyl tert-butyl ether with stirring (the volume of methyl tert-butyl ether is 10 times the volume of the concentrated solution). White solid precipitates. Continue to stir and precipitate for 1 hour. Centrifuge the suspension and pour out the supernatant. Wash the solid with methyl tert-butyl ether and then centrifuge again, repeating the washing and centrifugation 3 times. Dry to constant weight under vacuum to obtain crude nonapeptide of roylic acid.

[0068] Step (12) Purification and preparation of nonapeptide of roylic acid

[0069] After the crude nonapeptide of roylic acid is dissolved, it is prepared and purified using a reversed-phase C8 column (10 μm, 100 Å, 250*20 mm), and then freeze-dried. The purified product of nonapeptide of roylic acid is obtained.

[0070] The crude nonapeptide of roylic acid is dissolved in an aqueous acetonitrile solution with a volume percentage of 30% (the amount used is 20 times the mass, that is, 20 times the mass of the crude nonapeptide of roylic acid), and passed through a 0.22 μm organic filter membrane. The filtrate is purified and separated using a C8 reversed-phase column. The detection wavelength is 210 nm, and the sample is loaded at a flow rate of 20 ml / min. Gradient elution of the mobile phase: mobile phase A is 0.1% acetic acid solution, and mobile phase B is acetonitrile, by volume percentage.

[0071] Table 2 Gradient elution program

[0072] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 90 10 30 65 35 33 20 80 38 20 80 40 90 10 45 90 10

[0073] The HPLC chromatogram of chromatographic elution purification is as Figure 1 shown. The retention time of nonapeptide of roylic acid is 16.328 min (peak area 7180437), and there are two minor impurities at 15.955 min (peak area 39399) and 16.989 min (peak area 26358). The purity of nonapeptide of roylic acid in the product eluted in the 15 - 17 min section is 99.093% calculated by peak area.

[0074] The mass spectrum of nonapeptide of roylic acid is as Figure 2As shown, there are mass spectrometry peaks such as 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), 106.0 (m / z); the mass spectrometry peak with the largest mass-to-charge ratio m / z is 1158.1 (m / z). NMR data of nonacosapentanoic acid 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).

[0075] Preparation and purification of nonacosapentanoic acid nonapeptide in Example 2

[0076] In Example 2, referring to the preparation and purification method of Example 1, an additional preparation and purification method of nonacosapentanoic acid nonapeptide is provided. The difference from the example lies in the different substitution degrees of Wang resin.

[0077] Step (1) Preparation of Fmoc-Phe-O-Wang resin

[0078] Weigh 50 g of Wang resin (1.09 mmol / g), swell it in DCM for 30 min, and 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, 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 it to the resin. Stir and react for 3 hours under nitrogen protection. After the reaction is completed, drain it. Wash it three times with DMF. Add 150 ml of acetic anhydride solution (the volume ratio of each reagent is acetic anhydride:pyridine:DMF = 1:1:3) to the obtained resin, react for 30 min under N2 protection, drain it after the reaction is completed, wash it with DMF until neutral, add 20% piperidine / DMF mixed solution for deprotection, drain it, and wash it with DMF until pH = 7 (the washing solvent can be washed with the washing solvent in the previous step three times first, and then washed with fresh DMF to save DMF solution).

[0079] Step (2) Preparation of Fmoc-Ala-Phe-O-Wang resin

[0080] Weigh 33.9 g (2.0 eq) of Fmoc-Ala-OH and 14.7 g (2.0 eq) of HOBt, dissolve them in DMF, add 16.8 ml (2.0 eq) of DIC after dissolving clearly, activate at 0 °C for 5 min, and add it to the product of step (1). Stir and react under nitrogen protection, and the ninhydrin test shows that the resin is negative. After the reaction is completed, drain it. Wash it three times with DMF, add 20% piperidine / DMF mixed solution for deprotection, drain it, and wash it with DMF until pH = 7.

[0081] Steps (3)-(12) Preparation of crude 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH (nonapeptide of royal jelly acid)

[0082] Refer to Example 1 and step (2) of this example for conventional amino acid coupling and 10-HDA covalent coupling, and sequentially connect Fmoc-Lys(BOC)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Lys(BOC)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, 10-HDA to obtain 126 g of crude protected nonapeptide of royal jelly acid. Add 1250 ml of TFA solution (lysis solution, volume ratio of trifluoroacetic acid:water = 95:5) and lyse for about 2 hours. Centrifuge, wash, and dry in vacuum. Obtain 68.4 g of crude nonapeptide of royal jelly acid, purify it by preparation, and freeze-dry to obtain 24 g of freeze-dried powder.

[0083] The mass spectrum of the nonapeptide of royal jelly acid is as Figure 2 shown. The NMR data of the nonapeptide of royal jelly acid:1 1H 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).

[0084] Example 3: Preliminary Investigation of the Inhibitory Effect of Royalactin Nonapeptide on Matrix Metalloproteinase (MMP) in vitro

[0085] The royalactin nonapeptide used in this example was the royalactin nonapeptide prepared and purified according to the method of Example 1. The remaining materials, reagents, and reagent kits, such as palmitoyl nonapeptide - 3, were obtained by purchasing from the market. Unless otherwise specified, cell culture was carried out under conventional culture conditions.

[0086] 1 Materials

[0087] 1.1 Cells

[0088] The cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd.

[0089] 1.2 Reagents Related to Cell Culture

[0090] DMEM medium (Gibco, 11966025); fetal bovine serum (FBS) (gibco, 16140071); penicillin-streptomycin (P-S) (gibco, 15140122); phosphate buffer solution (PBS) (gibco, 10010023); trypsin-EDTA (0.05%) (gibco, 25300062); SteadyPure Quick RNA Extraction Kit (Accuri, AG21023); reverse transcription kit (Accuri, AG11728); SYBR GREEN (Accuri, AG11740); AR analytical pure alcohol (Greagent, G73537B); CCK8 (APExBIO, 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).

[0091] 1.3 Experimental drugs

[0092] Lipopolysaccharide (LPS), royalactin nonapeptide, palmitoyl nonapeptide-3, capsaicin.

[0093] 1.4 Instruments

[0094] Q-PCR instrument, microplate reader, fluorescence microscope.

[0095] 2 Experimental methods

[0096] 2.1 CCK-8 test for cytotoxicity

[0097] CCK8, full name Cell Counting Kit-8 reagent, can be used for simple and accurate cell proliferation and cytotoxicity analysis. HaCaT (human immortalized epidermal cells) cells were placed in DMEM medium (Gibco, 11966025) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and evenly plated into 96-well plates (5×10 4Cells / well) were incubated at 37 °C for 24 h. The cells were washed twice with PBS buffer and then incubated with lipopolysaccharide (LPS), palmitoyl nonapeptide-3, queen bee acid nonapeptide, and medium (negative control) for 24 h, respectively. Concentrations of the test substances: the concentrations of palmitoyl nonapeptide and queen bee acid nonapeptide were 0.001 wt%, 0.0025 wt%, 0.005 wt%, 0.01 wt%, 0.025 wt%, and 0.05 wt%, respectively; the concentration of LPS was 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. The absorbance was measured at OD 450 nm, and the cell viability (%) relative to the untreated control culture (negative control, representing 100% viability) was calculated. The data were the mean ± standard deviation (SD) of three independent experiments with three replicate samples.

[0098] 2.2 Effect of capsaicin on intracellular Ca 2+ flux

[0099] HaCaT cells were cultured in a 6-well culture plate for 24 h, and the test samples were added to the medium for treatment: 0.001 wt% retinol, 2 μM capsaicin, 3 μM Fluo-4AM (fluorescent probe), 0.05 wt% palmitoyl nonapeptide-3, 0.05 wt% queen bee acid nonapeptide, and then stained with Fluo-4AM at 37 °C for 1 h. After incubation, the cells were washed twice with PBS and observed under a fluorescence microscope.

[0100] 2.3 Analysis of the effect of test samples on the expression of related genes by real-time quantitative Q-PCR

[0101] The real-time fluorescence quantitative PCR (Bio-Rad, California, USA) was performed using the Sybr-Green-based method. The relative expression of mRNA was measured after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). After HaCaT cells were cultured for 24 h, the test samples were added for treatment, the concentration of LPS was 20 μg / ml, the concentrations of queen bee acid nonapeptide and palmitoyl nonapeptide-3 were both 0.05 wt%, and the concentration of retinol was 0.001 wt%; incubated at 37 °C for 24 h. Total RNA was isolated from the cells using TRIzol TM reagent (Invitrogen, USA).

[0102] 2.4 Analysis of the effect of test samples on the expression of related proteins by ELISA

[0103] Human collagen type I (COL-1) ELISA kit and MMP-1 ELISA kit were used to measure the expression levels of the two proteins in the test samples. COL-1 is a structural protein secreted by human skin fibroblasts, and MMP-1 is a senescence-secreted phenotype factor that can degrade collagen. Therefore, the up-regulation or down-regulation of these two types of proteins is closely related to the senescent phenotype of the skin. After culturing HaCaT cells with the test substances for 24 h, the expression level of MMP-1 protein was measured; after culturing human skin fibroblasts (HFF) with the test substances for 24 h, the expression level of COL-1 protein was measured. Concentrations of the test substances: the concentration of LPS was 20 μg / ml, the concentrations of nonapeptide queen bee acid and palmitoyl nonapeptide-3 were both 0.05 wt%, and the concentration of retinol was 0.001 wt%; incubation was carried out at 37 °C for 24 h.

[0104] 3 Results

[0105] The following are the research results of four experiments.

[0106] 3.1 CCK8

[0107] As Figure 3 shown, LPS is safe for HaCaT cells at concentrations within 50 μg / ml, and 20 μg / ml was selected for subsequent experiments to induce inflammation. The half-lethal concentration of retinol was 0.0025 wt%, and it was safe for cells below this concentration. 0.001 wt% was selected for subsequent experiments; the concentration of nonapeptide queen bee acid was below 1 wt% and did not reach the half-lethal level, and the concentration of palmitoyl nonapeptide-3 was below 0.05 wt% and did not reach the half-lethal level. 0.05 wt% was selected for subsequent experiments.

[0108] 3.2 Effect of capsaicin on intracellular Ca 2+ flux

[0109] Capsaicin stimulates the accumulation of [Ca 2+ by activating the capsaicin receptor TRPV1 and exerts a pain-stimulating effect. As Figure 4 shown, the accumulation of [Ca 2+ in HaCaT cells was measured using Fluo-4AM ester as a fluorescent indicator for Ca 2+ . The results showed that capsaicin significantly increased the accumulation of [Ca 2+ . The intensity of stimulating the accumulation of [Ca 2+ was in the order of: capsaicin > retinol > palmitoyl nonapeptide-3 > nonapeptide queen bee acid > NC (NC is the normal control, without adding any test substances or drugs). The pain-stimulating effect of nonapeptide queen bee acid was significantly lower than that of retinol and palmitoyl nonapeptide-3. 3.3 Q-PCR and ELISA experimental results

[0110] As Figure 5As shown, in the inflammatory cell model induced by LPS, palmitoyl nonapeptide-3, royal jelly acid nonapeptide, and retinol can all reduce the expression of MMP1 protein. Among them, the best effect is achieved by 0.05% royal jelly acid nonapeptide, and the MMP1 protein in the royal jelly acid nonapeptide group is significantly lower than that in other groups (p<0.05 or p<0.01). Three inflammatory factors, IL-1β, IL-8, and IL-6, were tested in the inflammatory cell model. Palmitoyl nonapeptide-3 and royal jelly acid nonapeptide both inhibited the expression level of IL-6, and there was no significant difference between the two groups.

[0111] As Figure 5 shown, ELISA detection shows that palmitoyl nonapeptide-3, royal jelly acid nonapeptide, and retinol can all increase the level of type I collagen (COL-1) in human skin fibroblasts (HFF). Among them. Among them, the best effect is achieved by 0.05% royal jelly acid nonapeptide, and the COL-1 level in the royal jelly acid nonapeptide group is significantly higher than that in other groups (p<0.05 or p<0.01).

[0112] Example 4 Investigation of the regulatory effect of royal jelly acid nonapeptide on total collagen

[0113] The royal jelly acid nonapeptide used in this example was prepared and purified according to the method of Example 1. The remaining materials, reagents, and kits were all obtained by purchasing from the market. Unless otherwise specified, cell culture was carried out under conventional culture conditions.

[0114] Human skin fibroblasts (HFF) were placed in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and evenly spread into 96-well plates (1.5x10 5 cells / well) and incubated at 37°C for 24 h. The cells were washed twice with PBS buffer, and different test substances were added and incubated at 37°C for 24 h. The Sircol soluble collagen detection kit was used to determine the total content of soluble collagen 1-IV in the culture. Test substance 1 was royal jelly acid (concentration 0.05 wt%); test substance 2 was palmitoyl nonapeptide-3 (concentration 0.05 wt%); test substance 3 was a mixture of royal jelly acid and palmitoyl nonapeptide-3, and the concentrations of both were 0.05 wt%; test substance 4 was royal jelly acid nonapeptide (concentration 0.05 wt%). The data were the mean ± standard deviation of three independent experiments with three replicate samples.

[0115] Compared with the negative control group (without adding test substances), all three substances and the mixture could increase the total content of soluble collagen 1-IV in the cells. The soluble collagen content in the test substance 3 group was 5.06±0.25 μg / 1x10 5 cells, and the soluble collagen content in the test substance 4 group was 6.47±0.21 μg / 1x10 5cells. The soluble collagen contents of both groups were significantly higher than those of the royal jelly acid group (p < 0.01) and the palmitoyl nonapeptide-3 group (p < 0.05). Among them, the soluble collagen content of the test substance 4 group was significantly higher than that of the test substance 3 group (p < 0.05).

[0116] The above are the preferred embodiments of the present invention. It should be noted that the embodiments are only used for the explanation and illustration of the present invention and are not regarded as a limitation of the protection scope. Under the main scheme, spirit and principle of the present invention, there can be more transformations / replacements / variations of the technical scheme of the present invention, such as the replacement of amino acid protecting groups, the replacement of resins, the replacement of skin care excipients, etc. Any transformations / replacements / variations made by those of ordinary skill in the art without creative work under the spirit and principle of the technical scheme in the specification shall fall within the protection scope of the present invention.

Claims

1. A polypeptide for promoting collagen synthesis, characterized in that: The structural formula of the polypeptide is shown in Formula I:

2. The method for preparing a polypeptide for promoting collagen synthesis according to claim 1, characterized in that: The steps include: Synthesis of S1 linear peptide: Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O-resin was prepared by step-wise coupling using solid phase synthesis, where M1, M2, and M3 are amino protecting groups; Synthesis of S2 Melissa officinalis covalently coupled linear peptide: 10-HDA and Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O-resin was reacted to prepare 10-HDA-Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O-resin; Cleavage of S3 beeswax acid nonapeptide: Cleavage of 10-HDA-Leu-Ala-Lys(M1)-Lys(M2)-Leu-Ala-Lys(M3)-Ala-Phe-O-resin gave the crude product 10-HDA-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Ala-Phe-OH.

3. The method for preparing a polypeptide for promoting collagen synthesis according to claim 2, characterized in that: The resin is selected from any one of Wang resin and CTC resin.

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

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

6. The method for preparing a polypeptide for promoting collagen synthesis according to claim 5, characterized in that: The mobile phase B is acetonitrile, and the mobile phase elution procedure is as follows:

7. A skin care product, characterized in that: Contains the polypeptide for promoting collagen synthesis according to claim 1 or the polypeptide prepared by the preparation method according to any one of claims 2 to 6.

8. A skin care product according to claim 7, characterized in that: The skin care product is a transdermal administration preparation.

9. A skin care product according to claim 8, characterized in that: The auxiliary material of the transdermal preparation is 5wt% 1,2-pentanediol aqueous solution.

10. Use of the polypeptide for promoting collagen synthesis according to claim 1 or the polypeptide prepared by the preparation method according to any one of claims 2 to 6 in preparing a preparation for increasing the collagen content of skin fibroblasts.

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