Collagen peptide, collagen peptide salt, polypeptide composition and application thereof

Abstract

The application discloses a collagen peptide, a collagen peptide salt, a polypeptide composition and application thereof, and belongs to the technical field of active peptide materials. The collagen peptide is a short peptide with an amino acid sequence as shown in SEQ ID NO:1 or a cyclic peptide containing the amino acid sequence as shown in SEQ ID NO:1. The collagen peptide can significantly inhibit tyrosinase activity and significantly inhibit melanin content in cells, thereby achieving a whitening effect.

Description

Technical Field

[0001] This invention relates to the field of active peptide materials technology, and in particular to a collagen peptide, collagen peptide salt, polypeptide composition and its application. Background Technology

[0002] Skin whitening is one of the core demands in the skincare industry. There is an increasing need for safe and effective whitening products to address issues such as dark spots, dull skin tone, and pigmentation caused by UV exposure. Excessive melanin production is the main cause of skin darkening and the formation of dark spots. Tyrosinase is a key rate-limiting enzyme in the melanin synthesis pathway, and its activity directly determines the efficiency of melanin production. Therefore, inhibiting tyrosinase activity has become one of the core research directions in whitening technology.

[0003] Currently, existing skin whitening technologies mainly include chemical whitening agents, natural extracts, and physical whitening methods. Among them, chemical whitening agents, such as hydroquinone, kojic acid, and arbutin, can inhibit tyrosinase activity to a certain extent, but they have problems such as strong irritation, poor stability, and the potential for skin sensitivity or cytotoxicity with long-term use. Natural extracts, such as licorice flavonoids and mulberry leaf extract, have mild ingredients but low levels of active ingredients, resulting in weak whitening effects. They also require high concentrations, which can lead to complex product formulations and increased costs. Physical whitening methods, such as sun umbrellas, can only reduce pigmentation by blocking ultraviolet rays and cannot inhibit melanin production at the root.

[0004] Therefore, there is an urgent need to develop new whitening active ingredients. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a collagen peptide, collagen peptide salt, polypeptide composition and its application, to overcome the problem of poor whitening effect of whitening active substances in the prior art.

[0006] In a first aspect, the present invention provides a collagen peptide, which is a short peptide with an amino acid sequence as shown in SEQ ID NO:1 or a cyclic peptide containing an amino acid sequence as shown in SEQ ID NO:1.

[0007] Compared with the prior art, the present invention degrades type 21 collagen (disclosed in patent CN 114195884 A) in the prior art. After degradation, multiple short peptides are screened, and a collagen short peptide with the amino acid sequence APGMPGLMG (SEQ ID NO:1) is obtained and named AG-9. The collagen short peptide AG-9 or its cyclic peptide can significantly inhibit tyrosinase activity and significantly inhibit melanin content and formation in cells, thereby achieving a whitening effect.

[0008] Secondly, the present invention provides a collagen peptide salt, which is a pharmaceutically or cosmetically acceptable salt formed by the above-mentioned collagen peptide with organic acid, inorganic acid, organic base or inorganic base.

[0009] Compared with existing technologies, the collagen peptides of this invention, when reacted with acids or alkalis to form collagen peptide salts, also significantly inhibit tyrosinase activity and significantly inhibit melanin content in cells, thereby achieving the effect of skin whitening.

[0010] Furthermore, the organic acids include acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pyric acid, or gluconic acid; and / or, Inorganic acids include hydrochloric acid, sulfuric acid, boric acid, or carbonic acid; and / or, Organic bases include triethylamine or diisopropylethylamine; and / or, Inorganic bases include sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, or ammonia.

[0011] Thirdly, the present invention provides a method for preparing collagen peptides, comprising the following steps: The Fmoc solid-phase peptide synthesis method was used to sequentially link glycine, methionine, leucine, glycine, proline, methionine, glycine, proline, and alanine on a solid resin to form a peptide chain. The peptide chain is cleaved from the solid resin using a lysis buffer, and the protecting groups of all amino acid side chains are removed simultaneously to obtain the short peptide.

[0012] It should be understood that the preparation method of the present invention is a conventional peptide solid-phase synthesis technique, without any special conditions or limitations.

[0013] Furthermore, by performing head-to-tail condensation on the aforementioned short peptides, cyclic peptides are prepared.

[0014] In the preparation of cyclic peptides, the above-mentioned technical solution can first dissolve the linear short peptide in a dilute solution, such as DMF or DMSO, with a solution concentration of 0.01M to 0.05M, and then carry out an intramolecular cyclization reaction under highly diluted conditions to avoid intermolecular coupling side reactions.

[0015] When linear short peptides undergo intramolecular cyclization reactions, highly efficient condensation reagents commonly used in the field can be used, such as HATU / HOAt, PyBOP, EDC·HCl / HOBt, etc.; and the reaction pH can be adjusted by adding alkaline auxiliaries (such as DIPEA or NMM); the reaction temperature can be controlled as needed at room temperature or under heating conditions.

[0016] Compared with existing technologies, the present invention synthesizes collagen peptides through the Fmoc solid-phase polypeptide synthesis method. This method has mild reaction conditions, pure reaction products, and easy-to-monitor reaction process.

[0017] Fourthly, the present invention provides a polypeptide composition comprising: component A, myophoton peptide C, and nonapeptide-1, wherein component A comprises the aforementioned collagen peptide and / or collagen peptide salt.

[0018] It should be understood that a polypeptide composition may contain only one of collagen peptides or collagen peptide salts, or it may contain both collagen peptides and collagen peptide salts.

[0019] Compared with the prior art, the collagen peptides and / or collagen peptide salts of the present invention, when combined with myophotopeptide C and nonapeptide-1, have a synergistic effect, further enhancing the inhibitory effect on melanin production and effectively improving the whitening effect.

[0020] Furthermore, the mass ratio of myophotopeptide C, component A, and nonapeptide-1 is (1~6):(5~15):(5~15).

[0021] More preferably, the mass ratio of myophotopeptide C, component A, and nonapeptide-1 is 5:10:10.

[0022] When a polypeptide composition contains both collagen peptides and collagen peptide salts, the collagen peptides and collagen salts can be mixed in any proportion, for example, the mass ratio of the two can be (1~5):(1~5).

[0023] The above technical solution combines myophotopeptide C, collagen peptide and / or collagen peptide salt, and nonapeptide-1 in the above mass ratio, further improving the whitening effect of the polypeptide composition.

[0024] Fifthly, the present invention provides the application of the above-mentioned collagen peptides, collagen peptide salts, or polypeptide compositions in the preparation of whitening products.

[0025] Furthermore, skin whitening products achieve their whitening effect by inhibiting tyrosinase activity or reducing melanin production.

[0026] Furthermore, whitening products include whitening serums, whitening lotions, whitening creams, whitening masks, whitening toners, whitening gels, or whitening freeze-dried powders. Attached Figure Description

[0027] Figure 1 This refers to the cellular tyrosinase activity in Example 2.

[0028] Figure 2 The cell melanin formation rate in Example 2. Detailed Implementation

[0029] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0030] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0031] Example 1 Preparation of collagen short peptide AG-9 1. Select 2-Cl resin as the solid-phase synthesis resin and couple it sequentially from the C-terminus to the N-terminus in the solid-phase synthesis.

[0032] 2. Resin modification: Weigh an appropriate amount of resin and put it into the reactor, add DCM and soak for 20-30 minutes, then dry it and wash it with DMF 3 times.

[0033] 3. Coupling the first amino acid: Weigh the calculated first amino acid, glycine (Fmoc-Gly-OH), activating reagent (such as DIC / HOBt or HBTU), DIEA, and DMF. After activation, add them to the reaction vessel (containing the resin after Fmoc deprotection) and react for 2 hours. Then add an appropriate amount of DIEA and methanol, and continue the reaction for 0.5 hours. Dry the reaction solution under vacuum, wash it three times with DMF, and dry it under vacuum again.

[0034] 4. Removal of Fmoc protecting group: Add 20% piperidine solution to the reaction vessel and react for 20 minutes. After removal, wash with DMF 5 times. Detect the resin with ninhydrin; a blue color indicates that Fmoc has been successfully removed.

[0035] 5. Condensation reaction (coupling the next amino acid): Weigh the calculated methionine Fmoc-Met-OH and HOBT, dissolve them in DMF, then add an appropriate amount of alkali (such as DIPEA) and DIC, activate them, and then add them to the reaction vessel to react for 2 hours. After that, wash the resin 5 times with DMF. The resin is colorless when tested by the ninhydrin method.

[0036] 6. Repeat steps 4 and 5, sequentially coupling leucine (Leu), glycine (Gly), proline (Pro), methionine (Met), glycine (Gly), proline (Pro), and alanine (Ala). After the last amino acid, alanine, is coupled, remove its Fmoc protecting group. Analyze the resin using the ninhydrin method; it should show a colorless result. Finally, dry the resin.

[0037] 7. Use a lysis buffer to cleave the peptide chain from the solid-phase resin and simultaneously remove the protecting groups of all amino acid side chains to obtain the short peptide AG-9.

[0038] It should be understood that the preparation method in this embodiment is a conventional peptide solid-phase synthesis technique without any special conditions. All reagents used are conventional reagents in peptide solid-phase synthesis techniques. When coupling each amino acid, the amino acid is activated first and then coupled according to methods known in the art, and appropriate amounts of coupling reagents, bases and other corresponding reagents are added as needed.

[0039] Example 2 Whitening efficacy test of collagen short peptides The cell line used in this embodiment is mouse skin melanoma cell line B16F10 (provided by Qisai Biotechnology), with cell passage number 5-6. The cell culture medium used is DMEM (containing 4.5g / L LD-Glucose) + 10% fetal bovine serum + 1% mixed antibiotics (100×).

[0040] The B16F10 cell density was adjusted to 2.5 × 10⁶ cells using cell culture medium. 3 Cells / mL were seeded into five 6-well plates, 2 mL per well. The five 6-well plates were divided into five groups: blank control group, negative control group, positive control group, and two experimental groups, with three replicates in each group.

[0041] 24 hours after B16F10 cells were plated, the negative control group, positive control group, and two experimental groups were induced with α-MSH (0.1 μM) for 48 hours (the blank control group was not induced with α-MSH).

[0042] After induction, the cell culture medium was changed: the positive control group was replaced with cell culture medium containing 30 nM Tyrosinase-IN-22 (5-chloro-2-mercaptobenzimidazole); experimental group 1 was replaced with cell culture medium containing 10 ppm (i.e. 0.001 wt%) collagen peptide AG-9; experimental group 2 was replaced with cell culture medium containing 25 ppm (i.e. 0.0025 wt%) collagen peptide AG-9; the blank control group and negative control group were replaced with the original cell culture medium.

[0043] Continue incubation for 24 hours, then freeze and thaw the cells three times, centrifuge at 10,000 rpm for 5 minutes, collect the supernatant and detect tyrosinase activity and melanin formation rate (refer to T-SHRH027-2019 "In vitro test of B16 cell melanin synthesis inhibition experiment").

[0044] Experimental results are as follows Figure 1 , Figure 2 As shown: Control is the blank control group, and Model is the negative control group. # indicates that, relative to the blank control group, P < 0.05; Representative: Compared with the negative control group, P < 0.05; Representative: Compared with the negative control group, P < 0.01; Mean: P < 0.001 compared to the negative control group; Mean ± SD, n = 3.

[0045] Depend on Figure 1 It can be seen that: compared with the blank control group (Control), the tyrosinase activity of the negative control group (Model) was significantly increased; compared with the negative control group (Model), the tyrosinase activity of the positive control group containing 30 nM Tyrosinase-IN-22 was significantly decreased, indicating that Tyrosinase-IN-22 significantly inhibited tyrosinase activity, proving that the cell model was successfully constructed.

[0046] Compared with the negative control group (Model), the tyrosinase activity in the experimental groups containing collagen short peptide AG-9 at concentrations of 10 ppm and 25 ppm was significantly reduced, indicating that collagen short peptide AG-9 at concentrations of 10 ppm and 25 ppm could significantly inhibit tyrosinase activity, with inhibition rates of 18.48% and 22.42%, respectively.

[0047] Depend on Figure 2 It can be seen that: compared with the blank control group (Control), the melanin formation rate of the negative control group (Model) was significantly increased; compared with the negative control group (Model), the melanin formation rate of the positive control group containing 30nM Tyrosinase-IN-22 cell culture medium was significantly decreased, indicating that Tyrosinase-IN-22 significantly inhibited melanin formation, and the cell model was successfully constructed.

[0048] Compared with the negative control group (Model), the melanin formation rate in experimental groups 1 and 2, which contained collagen peptide AG-9 cell culture medium at concentrations of 10 ppm and 25 ppm, was significantly reduced, indicating that collagen peptide AG-9 at concentrations of 10 ppm and 25 ppm could significantly inhibit melanin formation, with inhibition rates of 27.95% and 36.24%, respectively.

[0049] Example 3 Whitening efficacy test of peptide composition The cell line used in this embodiment is mouse skin melanoma cell line B16F10 (provided by Qisai Biotechnology), with cell passage number 5-6. The cell culture medium used is DMEM (containing 4.5g / L LD-Glucose) + 10% fetal bovine serum + 1% mixed antibiotics (100×).

[0050] The B16F10 cell density was adjusted to 2.5 × 10⁶ cells using cell culture medium. 3Cells / mL were seeded into five 6-well plates, 2 mL per well. The five 6-well plates were divided into five groups: blank control group, negative control group, positive control group, and two experimental groups, with three replicates in each group.

[0051] 24 hours after B16F10 cells were plated, the negative control group, positive control group, and two experimental groups were induced with α-MSH (0.1 μM) for 48 hours (the blank control group was not induced with α-MSH).

[0052] After induction, the cell culture medium was changed: the positive control group was replaced with cell culture medium containing 4 μM 377 (phenylethyl resorcinol); experimental group 1 was replaced with cell culture medium containing 5 ppm (i.e., 0.0005 wt%) myoglobin C + 10 ppm (i.e., 0.001 wt%) AG-9 + 10 ppm (i.e., 0.001 wt%) nonapeptide-1; experimental group 2 was replaced with cell culture medium containing 5 ppm (i.e., 0.0005 wt%) myoglobin C + 20 ppm (i.e., 0.002 wt%) nonapeptide-1; the blank control group and negative control group were replaced with the original cell culture medium.

[0053] Continue incubation for 24 hours, then freeze and thaw the cells three times, centrifuge at 10,000 rpm for 5 minutes, collect the supernatant and detect the melanin formation rate (refer to T-SHRH027-2019 "In vitro test of B16 cell melanin synthesis inhibition experiment").

[0054] The test results showed that the melanin formation rate of the negative control group was significantly increased compared with the blank control group; the melanin formation rate of the positive control group containing 4 μM 377 was significantly decreased compared with the negative control group, indicating that 377 significantly inhibited melanin formation and the cell model was successfully constructed. The melanin inhibition rate (%) of each experimental group, positive control group and negative control group is shown in Table 1.

[0055] Table 1 The results above show that, compared with Group 1, the inhibitory effect on melanin formation was significantly reduced in Group 2 after replacing 10 ppm AG-9 with nonapeptide-1. This indicates that there is a synergistic effect among myophotopeptide C, AG-9, and nonapeptide-1 in Group 1, which further improved the inhibitory effect on melanin formation.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A collagen peptide, characterized in that, The collagen peptide is a short peptide with an amino acid sequence as shown in SEQ ID NO:1 or a cyclic peptide containing an amino acid sequence as shown in SEQ ID NO:

1.

2. A collagen peptide salt, characterized in that, The collagen peptide salt is a pharmaceutically or cosmetically acceptable salt formed by the collagen peptide of claim 1 and an organic acid, inorganic acid, organic base, or inorganic base.

3. The collagen peptide salt according to claim 2, characterized in that, The organic acids include acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, primordial acid, or gluconic acid; and / or, The inorganic acid includes hydrochloric acid, sulfuric acid, boric acid, or carbonic acid; and / or, The organic base includes triethylamine or diisopropylethylamine; and / or, The inorganic base includes sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, or ammonia.

4. A method for preparing collagen peptides, used to prepare the collagen peptides of claim 1, characterized in that, Includes the following steps: The Fmoc solid-phase peptide synthesis method was used to sequentially link glycine, methionine, leucine, glycine, proline, methionine, glycine, proline, and alanine on a solid resin to form a peptide chain. The peptide chain is cleaved from the solid-phase resin using a lysis buffer, and all protecting groups on the amino acid side chains are removed simultaneously to obtain the short peptide; and / or, The cyclic peptide was prepared by first-to-last condensation using the short peptide as a raw material.

5. A polypeptide composition, characterized in that, The composition comprises: component A, myophotopeptide C, and nonapeptide-1, wherein component A comprises the collagen peptide of claim 1 and / or the collagen peptide salt of claim 2.

6. The polypeptide composition according to claim 5, characterized in that, The mass ratio of myophoton peptide C, component A, and nonapeptide-1 is (1~6):(5~15):(5~15).

7. The polypeptide composition according to claim 6, characterized in that, The mass ratio of myophotopeptide C, component A, and nonapeptide-1 is 5:10:

10.

8. The use of the collagen peptide of claim 1, the collagen peptide salt of claim 2, or the polypeptide composition of any one of claims 5 to 7 in the preparation of whitening products.

9. The application according to claim 8, characterized in that, The whitening product works by inhibiting tyrosinase activity or reducing melanin production.

10. The application according to claim 8, characterized in that, The whitening products include whitening serums, whitening lotions, whitening creams, whitening masks, whitening toners, whitening gels, or whitening freeze-dried powders.

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