Polypeptide, polypeptide derivative and composition and application thereof

Abstract

The invention discloses a polypeptide (NP1706, SEQ ID NO: 6) derived from the skin of nanorana parkeri, a polypeptide derivative as well as a composition and application of the polypeptide derivative. A natural parent peptide (NP1711, SEQ ID NO: 11) is optimized by combining a polypeptide activity prediction system with a molecular design technology, systematic mutation screening finds that the 5th site, the 9th site and the 14th site are active non-essential residues, and variant derivatives obtained by performing site-directed mutagenesis on the 5th site, the 9th site and the 14th site do not significantly change the original biological activity. The pharmacological activity and safety parameters of the NP1706 show optimal balance, so that the NP1706 is determined as the optimal candidate polypeptide. Based on the polypeptide and the polypeptide derivative, the activities of promoting cell proliferation and migration, resisting oxidation, resisting inflammation and the like are provided, and the application of the polypeptide and the polypeptide derivative in cosmetics and medicines is provided. The invention has the advantages of simple synthesis, good safety and excellent biological activity, and can be widely applied to the fields of skin repair, aging resistance, wound healing, inflammation resistance and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and biomedicine / cosmetic technology, in particular to a novel anti-inflammatory repair polypeptide isolated and identified from the skin secretions of Nanorana parkeri, a polypeptide obtained by mutating the parent polypeptide, a polypeptide derivative and a composition and application thereof. BACKGROUND

[0002] Frog skin secretions have long been considered a rich source of natural drugs and bioactive molecules. Numerous studies have found that frogs, especially those of the family Ranidae, which adapt to diverse habitats, can secrete a series of small molecule polypeptides. These polypeptides were initially reported to have antibacterial and antifungal activities, and play an important role in defending against pathogenic microorganisms and maintaining skin ecological homeostasis.

[0003] Plateau frog polypeptide (NP1711: amino acid sequence: SFMPLSIPIMCKMLSKC, SEQ ID NO: 11) is a novel bioactive polypeptide derived from the skin of Nanorana parkeri. Previous studies have shown that this polypeptide plays an important role in helping the body adapt to the extreme environment of the Qinghai-Tibet Plateau, such as high ultraviolet radiation and low microbial diversity. Previous studies (Adaptation of Plateau Frog Peptide: From Antimicrobial to Angiogenic and Proliferative Functions) have confirmed that NP1711 exhibits significant anti-inflammatory, angiogenic promoting activity, cell migration ability, and antioxidant properties, and can activate downstream signaling pathways by interacting with the epidermal growth factor receptor (EGFR). However, this study has not involved the structural optimization of the polypeptide and the discovery of the activity of specific mutation sites, resulting in the structure-function relationship of the polypeptide not being fully elucidated, which to some extent limits its further optimization and application. SUMMARY

[0004] The technical problem to be solved by the present application is to provide new optimized polypeptides, polypeptide derivatives and compositions thereof and to develop their applications.

[0005] To solve the above technical problems, the present application adopts the following technical solutions:

[0006] In a first aspect, the present application provides a polypeptide, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 6.

[0007] In a second aspect, the present application further provides a polypeptide derivative, which comprises a sequence having at least 70% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 11, and the sequence is a polypeptide derivative with original biological activity obtained by site-directed amino acid mutation at the site of active non-essential residues at positions 5, 9 and / or 14 based on the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 11; the original biological activity is having the activity of promoting cell proliferation, migration, antioxidant activity and / or anti-inflammatory activity.

[0008] Further, the polypeptide derivative is a polypeptide derivative with original biological activity obtained by modification based on the amino acid sequence shown in SEQ ID NO: 6 or based on the mutated polypeptide derivative.

[0009] Further, the modification is acylation, PEGylation or fatty acid coupling modification at the N-terminus or C-terminus.

[0010] In a third aspect, the present application further provides a preparation method of an optimized polypeptide derivative, comprising:

[0011] site-directed amino acid mutation at the site of active non-essential residues at positions 5, 9 and / or 14 based on the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 11; safety evaluation and biological activity detection of the mutated polypeptide derivative, thereby obtaining an optimized polypeptide derivative with original biological activity; the original biological activity is having the activity of promoting cell proliferation, migration, antioxidant activity and / or anti-inflammatory activity.

[0012] In a fourth aspect, the present application further provides a composition, which is a cosmetic composition or a pharmaceutical composition; the active ingredient of the composition comprises the above-mentioned polypeptide or polypeptide derivative or the optimized polypeptide derivative prepared by the above-mentioned preparation method; the composition further comprises a cosmetically or pharmaceutically acceptable base, carrier and / or adjuvant.

[0013] Further, the cosmetic composition is an external emulsion, serum or mask; the pharmaceutical composition is an external gel, dressing, topical drug delivery system or injection; the composition has the activity of promoting cell proliferation, migration, antioxidant activity and / or anti-inflammatory activity.

[0014] Further, the cosmetic composition has the effects of anti-inflammatory, soothing, promoting skin barrier repair and / or antioxidant aging; the pharmaceutical composition has the effects of anti-inflammatory, promoting wound repair, ulcer healing, burn repair, ischemic injury and / or other tissue regeneration.

[0015] In a fifth aspect, the present application further provides a use of the polypeptide, the polypeptide derivative, the optimized polypeptide derivative prepared by the preparation method, or the composition, in the preparation of a cosmetic or a medicine for treating a cell proliferation-promoting, migration-activity-promoting, antioxidant-activity-promoting, and / or anti-inflammatory activity-promoting.

[0016] In a sixth aspect, the present application further provides a use of the polypeptide, the polypeptide derivative, the optimized polypeptide derivative prepared by the preparation method, or the composition, in the preparation of a cosmetic or a medicine for treating a cell proliferation-promoting, migration-activity-promoting, antioxidant-activity-promoting, and / or anti-inflammatory activity-promoting.

[0017] The use is in the preparation of a cosmetic for anti-inflammatory, soothing, skin barrier repair-promoting, and / or antioxidant-aging; or the use is in the preparation of a medicine for anti-inflammatory, wound repair-promoting, ulcer healing, burn repair, ischemic injury, and / or other tissue regeneration.

[0018] The present application has at least the following beneficial effects:

[0019] 1. The present application is based on the high mountain Buergeria skin-derived polypeptide (NP1711) reported in the existing literature, taking NP1711 as the parent peptide, combining polypeptide library design, internal large model activity prediction and molecular docking screening, and then using modern pharmacological means to evaluate the biological activity, so as to obtain the optimal variant and identify the key sites. Through systematic research, it is found for the first time and successfully identified that the 5th, 9th and 14th positions of the original polypeptide are active non-essential residues (key sites that do not affect the biological activity of the polypeptide group), which breaks through to limit the mutation of derivatives only at the three positions, and the biological activity is retained, successfully elucidating the structure-function relationship, and providing a direction for further optimization and application of polypeptide sequences.

[0020] 2. The present application is based on the optimal variant (a plurality of polypeptide derivatives) obtained by screening, and comprehensively compares and evaluates the biological activity and safety characteristics, and finally finds that NP1706 (SEQ ID NO: 6) has a synergistic, optimal balance between efficacy and safety, taking an unexpected effect.

[0021] 3. The polypeptide, the polypeptide derivative, and the composition and application thereof of the present application also have the following outstanding advantages:

[0022] 1. Unique multiple effects: cell proliferation-promoting, migration-activity-promoting, antioxidant-activity-promoting, anti-inflammatory activity-promoting, etc., with anti-inflammatory, antioxidant, migration-promoting, angiogenesis-promoting, and wound repair functions;

[0023] 2. Excellent developability: simple structure, easy to synthesize and optimize;

[0024] 3. Wide application prospects: suitable for multiple fields of medicine and cosmetics; can be widely used in skin repair, anti-aging, wound healing, etc.

[0025] 4) Good safety: low immunogenicity, safe for use;

[0026] 5) Significant production advantage: low cost, easy to scale up production;

[0027] 6) Outstanding innovation: first time to reveal the relationship between the new function and structure of highland frog skin peptide, and to find the optimized sequence, with high application value. BRIEF DESCRIPTION OF DRAWINGS

[0028] The above is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, the following will further describe the present application in detail in combination with the drawings and specific embodiments.

[0029] Figure 1 , 2 are respectively the test results of the cytotoxicity of polypeptides NP1701-NP1717 on Raw264.7 cells and THP-1 cells in Example 2;

[0030] Figure 3 are respectively the test results of the sensitization of polypeptides NP1701-NP1717 in Example 3 (a) and the comparison results of the sensitization of NP1706 at different concentrations (b);

[0031] Figure 4 is the result graph of the influence of polypeptides NP1701-NP1717 on the proliferation activity of HaCaT cells in Example 4 of the present application;

[0032] Figures 5-7 are respectively the 8h and 12h result graphs of the scratch repair ability of polypeptides NP1701-NP1717 on HaCaT cells in Example 5 and the comparison photos;

[0033] Figure 8 is the test result graph of the DPPH scavenging ability of polypeptides NP1701-NP1717 in Example 6 of the present application;

[0034] Figures 9-11 are respectively the influence result graphs of polypeptides NP1701-NP1717 on the release of TNF-α, IL-6 and NO in the LPS-induced Raw 264.7 cell inflammation model in Example 7 of the present application;

[0035] Figure 12 is the test result graph of the skin wound healing of the animal model. DETAILED DESCRIPTION

[0036] The application will be further described below in conjunction with examples, but not used to limit the scope of the present application. In the examples, the experimental methods used are performed according to the conventional methods unless otherwise specified; the reagents, cell lines, instruments and equipment used are commercially available or prepared by the inventors and have passed the corresponding ethical approval.

[0037] The present application is based on the high mountain Rana skin-derived polypeptide (NP1711) reported in the prior art literature, and the following studies are carried out:

[0038] 1) Polypeptide sequence optimization method based on activity prediction and molecular docking

[0039] By combining polypeptide library design, molecular docking screening and pharmacological model activity verification, the anti-inflammatory repair polypeptide sequence is optimized.

[0040] 2) Polypeptide activity detection method

[0041] Safety evaluation: in vitro cytotoxicity (CCK-8), allergic test verification.

[0042] Promote migration / proliferation detection: wound healing assay: after scratching on HaCaT cells, add polypeptide treatment, observe the migration speed; CCK-8 method detects cell proliferation.

[0043] Antioxidant protection: DPPH free radical scavenging experiment detects the free radical scavenging ability of the sample.

[0044] Anti-inflammatory activity in vitro detection: for example, using LPS to induce RAW264.7 or THP-1 derived macrophage inflammation model, detecting the secretion level (ELISA) of inflammatory factors (TNF-α, IL-6, IL-1β, etc.) after treatment; or inducing inflammatory stimulation (such as UVB or chemical stimulation) in human keratinocytes (HaCaT) or skin fibroblasts (HSF), detecting the inhibitory effect of polypeptide on cell inflammatory factor expression.

[0045] Based on the above research, a plurality of candidate polypeptides (NP1701-NP1710, NP1712-NP1717, respectively corresponding to SEQ ID NO: 1-10, SEQ ID NO: 12-17) are screened out, and based on the sequence of the parent peptide (NP1711, corresponding to SEQ ID NO: 11), by comparing and analyzing the active key sites, the present application first discovers and successfully identifies that the 5th, 9th and 14th positions in the original polypeptide are active non-essential residues (key sites that do not affect the biological activity of the polypeptide group), which breaks through and limits the mutation of derivatives only at the three sites, which retains the biological activity, successfully elucidates the structure-function relationship, and provides a direction for further optimization and application of polypeptide sequences.

[0046] In addition, in the above process, the application aims to comprehensively compare and evaluate the biological activity and safety characteristics of the plurality of candidate polypeptides obtained by screening, and ultimately finds that NP1706 (SEQ ID NO: 6) has a synergistic and optimal balance between efficacy and safety. For example, NP1706 has a significantly better comprehensive performance than the other 16 comparison peptides in various important indicators (such as cytotoxicity, cell migration promotion, TNF-α inhibition rate, etc.), highlighting its superior activity and safety in anti-inflammatory and tissue repair.

[0047] Based on this, corresponding polypeptides, polypeptide derivatives, compositions and applications are developed.

[0048] For example, the application provides a polypeptide, the amino acid sequence of which is the amino acid sequence shown in SEQ ID NO: 6 (the sequence is SFMPWSIPWMCKMWSKC). NP1706 (SFMPWSIPWMCKMWSKC) exhibits multiple repair functions such as significant anti-inflammatory, promotion of cell migration and proliferation, antioxidant, promotion of collagen synthesis, and promotion of angiogenesis in various in vitro models. Compared with common growth factors (such as VEGF, EGF), the polypeptide has a smaller molecular weight (easy to synthesize, lower cost), optimized structural stability, high in vivo safety (no obvious toxicity), low immunogenicity, and can obtain better pharmacokinetic properties through optimization and modification. It can be prepared by solid-phase synthesis or recombinant expression method.

[0049] For example, the application provides a polypeptide derivative, which comprises a sequence having at least 70% sequence identity (such as 70%-99%, more than 70%, more than 80%, more than 85%, more than 90%, more than 95%, etc.) with SEQ ID NO: 6 or SEQ ID NO: 11, and the sequence is a polypeptide derivative with original biological activity obtained by site-directed amino acid mutation at the site of an active non-essential residue at position 5, 9 and / or 14 based on the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 11; the original biological activity is the activity of promoting cell proliferation, migration, antioxidant activity and / or anti-inflammatory activity. Those skilled in the art can understand that the mutation can be deletion, substitution, or addition, etc.

[0050] The skilled in the art can understand that the polypeptide derivative can also be a polypeptide derivative with original biological activity obtained by modification based on the amino acid sequence shown in SEQ ID NO: 6, or a polypeptide derivative with original biological activity obtained by modification based on the aforementioned mutated polypeptide derivative. Such conventional modification forms can include acylation at the N- or C-terminus, or PEGylation or fatty acid coupling modification, etc., so that the in vivo half-life or stability is improved. The skilled in the art can understand that the sequence after the aforementioned modification and modification should also retain the original biological activity, such as promoting cell proliferation, migration activity, antioxidant activity and / or anti-inflammatory activity, etc.

[0051] The application also provides a method for obtaining an optimized polypeptide derivative, comprising:

[0052] Based on the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 11, site-directed amino acid mutation is carried out at the position of active non-essential residues at positions 5, 9 and / or 14. The skilled in the art can understand that the mutation can be deletion, substitution, or addition, etc.

[0053] The mutated polypeptide derivative is subjected to safety evaluation and biological activity detection, so as to obtain an optimized polypeptide derivative with original biological activity. The original biological activity is promoting cell proliferation, migration activity, antioxidant activity and / or anti-inflammatory activity.

[0054] In addition, the application also provides a composition, which can be a cosmetic composition or a pharmaceutical composition; the active ingredient of the cosmetic composition or the pharmaceutical composition comprises the aforementioned polypeptide or polypeptide derivative or the optimized polypeptide derivative prepared by the aforementioned preparation method; the composition further comprises a cosmetically or pharmaceutically acceptable matrix, carrier and / or adjuvant. The composition has promoting cell proliferation, migration activity, antioxidant activity and / or anti-inflammatory activity.

[0055] For the cosmetic composition, the polypeptide or polypeptide derivative or optimized polypeptide derivative can be used as the active ingredient to formulate cosmetic preparations such as external emulsion, essence, mask, etc., which can be combined with commonly used matrix (such as glycerol, hyaluronic acid, small molecule peptide, collagen, etc.) and adjuvant-assisted ingredients (preservatives, emulsifiers, humectants, excipients) to prepare cosmetics with promoting cell proliferation and migration activity, so as to become functional cosmetics with anti-inflammatory, soothing, repair, anti-sensitivity, anti-aging, promoting skin barrier repair and microcirculation improvement, etc.

[0056] For pharmaceutical compositions, polypeptides or polypeptide derivatives or optimized polypeptide derivatives can be used as active ingredients to prepare external preparations (gels, dressings, etc.) for wound repair (burns, trauma, ulcers, etc.); external preparations for skin diseases (eczema, psoriasis, repair of photoaging damage, etc.); and injection preparations or local administration systems (such as microneedle patches, nanocarriers, sustained-release systems) for promoting tissue repair, vascular regeneration, and improving ischemic injury, etc.

[0057] Administration method: local administration to the skin can be used; systemic administration (such as injection) can also be studied for tissue repair and regenerative medicine; carriers (nanoparticles, microspheres, transdermal drug delivery systems, etc.) can be used to improve delivery efficiency and stability, such as nanocarrier-loaded polypeptide preparations, which can be used for targeted delivery and prolonged release.

[0058] In addition, other active ingredients can also be added to the above-mentioned compositions, such as combinations with other growth factors (such as low-dose EGF, FGF, etc.), antioxidants, antibacterial ingredients, or other active peptides, to synergistically enhance the repair effect.

[0059] In addition, the above-mentioned polypeptides or polypeptide derivatives can also be used for in vitro anti-inflammatory detection or cell migration promotion detection, which includes adding polypeptides or polypeptide derivatives to a LPS-induced macrophage inflammation model and detecting the level of inflammatory factors, or adding polypeptides or polypeptide derivatives to a scratch test and detecting the cell migration speed.

[0060] In addition, the polypeptides or polypeptide derivatives can also be locally administered in a mouse full-thickness skin wound model and the wound closure rate, histological indicators, etc. can be detected.

[0061] The following will be specifically illustrated by examples:

[0062] Example 1: Sequence optimization of NP1711 based on activity prediction and molecular docking

[0063] This example uses NP1711 (SFMPWSIPWMCKMWSKC) as the polypeptide mother nucleus, and details how to combine polypeptide library design, internal large model activity prediction and molecular docking screening to obtain the optimal variant and identify the key sites that do not affect the biological activity of the polypeptide.

[0064] 1) Polypeptide library design

[0065] A. Mother peptide and optimization target: mother peptide NP1711 (SFMPWSIPWMCKMWSKC) has known anti-inflammatory and repair activity, and is set to mutate the active site for further study of the structure optimization of the polypeptide and the activity rules of specific mutation sites.

[0066] B. Selected mutation sites and candidate residues

[0067] a) Based on multiple sequence alignment, receptor docking model, enzyme sensitive site analysis, etc., the following optimization sites are selected in advance: 3, 5, 7, 9, 12, 14, 16 sites.

[0068] b) In order to improve the scale and diversity of the library, the following candidate amino acids are set at the 3rd, 5th, 7th, 9th, 12th, 14th, and 16th positions in this embodiment:

[0069] 3rd position (original M): {M, L, F, I, V}

[0070] 5th position (original L): {W, Y, F, L, I}

[0071] 7th position (original I): {I, V, L, T, S}

[0072] 9th position (original I): {W, Y, F, L, I}

[0073] 12th position (original K): {C, S, T, A, D}

[0074] 14th position (original L): {W, Y, F, L, I}

[0075] 16th position (original K): {K, R, H, N, Q}

[0076] c) Thus, 5^7=78,125 full combination sequences can be generated, covering the possible variant space.

[0077] C. Library file generation and management: use python script to generate all variant sequences from the above combination, record the sequence and corresponding mutation site information according to the number (P00001-P78125), save as FASTA format file, for subsequent activity prediction and docking use.

[0078] 2) Polypeptide activity prediction

[0079] A. Introduction of prediction system: Nova System is a polypeptide activity prediction system independently developed by Novateinbio. Its design is based on a large model of neural network, which can predict the anti-inflammatory and repair activity scores of a given polypeptide sequence. This system has been verified on multiple known peptides and experimental data, and has high prediction accuracy and reliability.

[0080] B. Batch prediction process: For the above 78,125 candidate sequences, call the activity prediction system for batch prediction. Each sequence can return two dimensions of prediction scores: anti-inflammatory activity potential score and repair activity potential score. Record the prediction results of each sequence, and finally generate a ranked list of comprehensive scores of each polypeptide.

[0081] C. Candidate polypeptide screening: Sort the 250 sequences with the highest predicted scores from high to low, and select them as the next step of molecular docking screening objects. These sequences have better anti-inflammatory and repair potential than the parent polypeptide in model prediction. Record the number, sequence, and each predicted score of the 250 sequences for subsequent analysis and experimental comparison.

[0082] 3) Molecular docking screening

[0083] A. Receptor model preparation: Obtain the three-dimensional structure of the target receptor EGFR, and perform preprocessing and docking site definition before docking. The selected docking sites are selected based on the docking results of the parent polypeptide.

[0084] B. Polypeptide conformation generation: Based on the linear conformation of the 250 candidate sequences, generate an initial three-dimensional conformation model;

[0085] C. Docking process and scoring

[0086] a) Preliminary docking: Use the GPU fast mode of the open source framework uni-dock to dock the 250 candidate polypeptides with the receptor, and obtain the preliminary binding score and binding site interaction information.

[0087] b) Fine docking: For the top 50 polypeptides with good preliminary docking scores or reasonable binding modes, use HADDOCK for accurate molecular docking, and use PyMOL for visualization to evaluate binding energy, key residue interactions, and binding mode stability.

[0088] c) Record the docking score, key interaction residues, and complex stability indicators of each peptide.

[0089] 4) Candidate polypeptide screening

[0090] a) Based on the comprehensive docking score, binding mode, binding stability, and physicochemical properties (such as net charge, hydrophobic distribution, solubility prediction), select the 16 peptide sequences with the most outstanding performance and the best predicted binding as the final candidates.

[0091] b) Save the detailed docking result summary of the 17 sequences in the record, including docking score, key residue contact with receptor, binding conformation example, etc.

[0092] Among them: The 17 polypeptide sequences are as follows (where NP1711: sequence SFMPLSIPIMCKMLSKC, is the parent peptide, and the other 16 polypeptides from R. amolus are used for comparison and combination research)

[0093] NP1701: sequence SFMPISIPIMCKMYSKC (SEQ ID NO: 1)

[0094] NP1702: sequence SFMPISIPIMCKMLSKC (SEQ ID NO: 2)

[0095] NP1703: sequence SFMPISIPIMCKMWSKC (SEQ ID NO: 3)

[0096] NP1704: sequence SFMPWSIPWMCKMYSKC (SEQ ID NO: 4)

[0097] NP1705: sequence SFMPYSIPIMCKMLSKC (SEQ ID NO: 5)

[0098] NP1706: sequence SFMPWSIPWMCKMWSKC (SEQ ID NO: 6)

[0099] NP1707: sequence SFMPISIPWMCKMYSKC (SEQ ID NO: 7)

[0100] NP1708: sequence SFMPLSIPFMCKMYSKC (SEQ ID NO: 8)

[0101] NP1709: sequence SFMPISIPIMCKMISKC (SEQ ID NO: 9)

[0102] NP1710: sequence SFMPYSIPIMCKMWSKC (SEQ ID NO: 10)

[0103] NP1711: sequence SFMPLSIPIMCKMLSKC (SEQ ID NO: 11)

[0104] NP1712: sequence SFMPYSIPFMCKMLSKC (SEQ ID NO: 12)

[0105] NP1713: sequence SFMPLSIPFMCKMLSKC (SEQ ID NO: 13)

[0106] NP1714: sequence SFMPLSIPIMCKMYSKC (SEQ ID NO: 14)

[0107] NP1715: sequence SFMPISIPFMCKMLSKC (SEQ ID NO: 15)

[0108] NP1716: sequence SFMPWSIPIMCKMYSKC (SEQ ID NO: 16)

[0109] NP1717: sequence SFMPWSIPWMCKMLSKC (SEQ ID NO: 17)

[0110] 5) Non-active key site analysis

[0111] A. Site contribution evaluation

[0112] a) Align the sequences of the above 17 candidate peptides and observe the mutation distribution pattern.

[0113] b) Statistics of mutations at positions 3, 5, 7, 9, 12, 14, 16, etc. compared with the parent peptide, especially the mutation types with higher frequency or significant improvement in docking results.

[0114] B. Determine non-active key sites

[0115] The analysis results show that in all 17 optimal sequences, the mutation patterns at positions 5, 9, and 14 have no significant correlation with activity strength. Therefore, positions 5, 9, and 14 are determined as non-key active sites.

[0116] Example 2: Cytotoxicity test

[0117] 1) Reagents and materials

[0118] High-sugar medium (DMEM), 1640 medium, fetal bovine serum (FBS), DPBS, dimethyl sulfoxide (DMSO), CCK-8.

[0119] 2) Instruments

[0120] CO2 incubator, biological safety cabinet, inverted microscope, microplate reader.

[0121] 3) Cell lines

[0122] Mouse monocyte macrophage leukemia cells (Raw264.7), human acute mononuclear leukemia cells (THP-1).

[0123] 4) Samples to be tested

[0124] Sample group: NP1701-NP1717, test concentration 300 μg / mL, 100 μg / mL, 30 μg / mL, 10 μg / mL, 3 μg / mL, 1 μg / mL. Solvent control group: DMEM complete medium, or 1640 complete medium; positive control group: DMEM complete medium containing 10% DMSO, or 1640 complete medium containing 10% DMSO; zero adjustment group: DMEM complete medium (without cells), or 1640 complete medium (without cells).

[0125] 5) Experimental method

[0126] Raw264.7 cells or THP-1 cells were seeded in 96-well plates at a density of 2.5 x 10 5 cells / mL cell suspension per well 100 μL, and cultured until the cell fusion rate reached about 40%, the supernatant was removed, and different concentrations of samples were added in turn for biological activity treatment. After 24 h of treatment, CCK-8 reagent was added at a ratio of 1:10, and after 40 min, the absorbance of each well at 450 nm was detected using an enzyme-labeled instrument. The cell survival rate was calculated according to the following formula:

[0127]

[0128] 6) Results

[0129] CCK-8 method is a method for detecting cell survival and growth, and the OD value measured is proportional to cell activity. The results of the effect of the test sample on the toxicity of Raw264.7 cells and THP-1 cells are as follows Figures 1-2 , and the results show that the cell viability of NP1706 in RAW 264.7 and THP-1 cells is greater than 75% in the range of greater than 300 μg / mL, and no obvious cytotoxicity is shown.

[0130] Example 3: Sensitization performance test

[0131] 1) Reagents and materials

[0132] 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), 2-mercaptoethanol, DPBS, bovine serum albumin (BSA), dinitrochlorobenzene (DNCB), lactic acid (LA), 7-AAD, FITC anti-human CD86 Antibody, PE anti-human CD54 Antibody, FITC Mouse IgG1 (k Isotype Ctrl Antibody), PE Mouse IgG1 (k Isotype Ctrl Antibody).

[0133] 2) Instruments

[0134] CO2 incubator, biological safety cabinet, inverted microscope, flow cytometer.

[0135] 3) Cell strains

[0136] Human acute monocytic leukemia cells (THP-1).

[0137] 4) Samples to be tested

[0138] Dosing group: NP1701-NP1717, sample test concentration 40 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, positive control group: 3.125 μg / mL dinitrochlorobenzene (DNCB); negative control group: 2 mg / mL lactic acid (LA); blank control group: culture medium containing 1% FBS (1% FBS + 1% P / S + 1640 culture medium); solvent control group, culture medium containing 16% PBS (1% FBS + 1% P / S + 1640 culture medium + 16% PBS).

[0139] 5) Experimental method

[0140] The density of the suspended cells THP-1 was adjusted to 1.5 x 10 6 cells / mL of cell suspension, then 200 μL of the cell suspension and 200 μL of 2 times the concentration of the sample to be tested were mixed at 1:1 and inoculated in a 48-well plate for culture. After 24 h of culture, the cells in each well were mixed by blowing and divided into two 96-well plates, each 150 μL, one as the test group and the other as the isotype control group. Next, fluorescence staining was performed, the cells were washed with PBS, and dead cells were stained with 7-AAD staining solution; the cells were washed with PBS, the test group was stained with FITC-CD86 and PE-CD54, and the isotype control group was stained with FITC-IgG1 and PE-IgG1; finally, the cells were fixed with paraformaldehyde. The mean fluorescence intensity (MFI) of FITC, PE, and 7-AAD dyes of each group of cells was determined by flow cytometry. The relative fluorescence intensity value (RFI) of the test substance was calculated using the following formula.

[0141]

[0142] 6) Results

[0143] When the sensitizer contacts the skin, the dendritic cells differentiate and mature during the process of moving to the lymphatic organs and up-regulate the expression of a series of surface molecules, and staining the cell surface molecules CD86 and CD54 with fluorescent antibody dyes and determining them by flow cytometry can determine whether the test substance has sensitization.

[0144] The experimental results are as follows: Figure 3As shown, the CD86 and CD54 molecules of THP-1 cells produced positive expression to DNCB (RFICD86>150, RFICD54>200) and the cell survival rate was >50%; produced negative expression to LA (RFICD86<150, RFICD54<200) and the cell survival rate was >50%; produced negative expression to solvent (RFICD86<150, RFICD54<200) and the cell survival rate was >90%; indicating that the cells passed the stability test; the cell survival rate of NP1706 at the highest concentration (40 μg / mL) was higher than 90%, and the CD86 and CD54 molecules of THP-1 cells did not produce positive expression to NP1706, indicating that NP1706 did not have sensitization.

[0145] Example 4: Proliferation activity test

[0146] 1) Reagents and materials

[0147] High-sugar medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, EGF, CCK-8.

[0148] 2) Instruments

[0149] CO2 incubator, biological safety cabinet, inverted microscope, microplate reader.

[0150] 3) Cell strain

[0151] Human immortalized keratinocytes (HaCaT).

[0152] 4) Samples to be tested

[0153] Sample group: NP1701-NP1717, test concentration: 2.5, 5, 10, 20, 40, 80 μg / mL; positive control group: DMEM medium containing 10 ng / mL EGF; blank control group: DMEM medium containing 0.5% FBS.

[0154] 5) Experimental method

[0155] HaCaT cells were inoculated in a 96-well cell culture plate at a density of 3×10 4 cells / mL cell suspension per well 100 μL, and when the cell confluence rate reached 20%, the supernatant was removed. The test samples were sequentially added for biological activity substance treatment. After the administration was completed, the 96-well plate was placed in a CO2 incubator (37°C, 5% CO2) for culture for 72 h. After 72 h of treatment, CCK-8 reagent was added at a ratio of 1:10, and after 40 min, the absorbance of each well at 450 nm was detected using a microplate reader, and the cell survival rate was calculated according to the following formula:

[0156]

[0157] 6) Experimental results

[0158] The HaCat proliferation activity evaluation model was selected for the experiment. As shown in Table 1, the NP1701-NP1717 in the sample group can significantly enhance the relative activity of HaCat cells at a concentration of 20 pg / mL, and NP1706 shows strong proliferation activity, which can significantly enhance the relative activity of HaCat cells at 10 pg / mL, but does not show concentration dependence. Figure 4

[0159] Example 5: HaCat cell scratch repair activity

[0160] 1) Reagents and materials

[0161] High glucose medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, epidermal growth factor (EGF).

[0162] 2) Instruments

[0163] CO2 incubator, biological safety cabinet, inverted microscope, microplate reader, 2 Well Culture-lnserts for self-insertion (Ibidi, 80209).

[0164] 3) Cell lines

[0165] Human immortalized keratinocytes (HaCaT).

[0166] 4) Samples to be tested

[0167] Sample group: NP1701-NP1717, test concentration is 20 pg / mL; model group: 5 ng / mL EGF; blank control group: DMEM medium containing 0.1% FBS.

[0168] 5) Experimental method

[0169] HaCaT cells were inoculated in the scratch insert at a density of 6x10 5 cells / mL cell suspension per well 70 pL, and the supernatant was removed when the cell fusion rate reached 100%. The test sample was added in turn for biological activity treatment. After the administration was completed, the 6-well plate was placed in a CO2 incubator (37°C, 5% CO2) for culture. At 0h, 8h and 12h after administration, a 5x microscope was used to take photos in the same field of view to record the scratch gap at the corresponding time. The area of the scratch in the photo was calculated using Image J, and the scratch healing rate was calculated using the following formula:

[0170] ​

[0171] 6) Results

[0172] The differentiation ability of keratinocytes plays a crucial role in the process of skin wound healing, and the cell scratch experiment is a common method for detecting cell migration ability.

[0173] The experimental results are shown in Table 1. Figures 5-7 As shown in Table 1, compared with the blank control, the sample group NP1706 can significantly improve the healing rate of HaCaT cells at 8h and 12h, indicating that NP1706 has strong repair activity. In addition, sample groups NP1701-NP1705, NP1708, NP1710 and NP1711 can improve the healing rate of HaCaT cells at 8h and 12h, showing scratch repair activity.

[0174] Example 6: In vitro antioxidant protection

[0175] 1) Reagents and materials

[0176] DPPH, anhydrous ethanol, GSH, 96-well plate, collection tube, sample addition slot.

[0177] 2) Instruments

[0178] Multifunctional enzyme label instrument, one hundred thousandth micro- balance.

[0179] 3) Samples to be tested

[0180] Sample group: NP1701-NP1717, test concentration is 1000, 200, 40, 8, 1.6, 0.32, 0.064 μg / mL; positive control group: GSH, concentration is 1000, 200, 40, 8, 1.6, 0.32, 0.064 μg / mL;

[0181] 4) Experimental method

[0182] 1.5 mg of DPPH powder was weighed in a 50 mL centrifuge tube, wrapped with tin foil to avoid light, 30 mL of anhydrous ethanol was added, mixed well, ultrasonic assisted dissolution to completely dissolve the powder, and 50 μg / mL DPPH working solution was obtained. Gradient dilution method was used to prepare GSH working solution and test substance working solution with corresponding concentration, 50 μL of test substance working solution, or 50 μL of GSH working solution, or 50 μL of anhydrous ethanol was added to each well, and 150 μL of DPPH working solution, or 150 μL of anhydrous ethanol was added to each well; after the addition was completed, the 96-well microplate mixer was used to mix, and the absorbance at 517 nm was read using the enzyme label instrument after 30 min of reaction at room temperature in the dark;

[0183] The DPPH scavenging rate P of the test substance and GSH was calculated using the following formula:

[0184]

[0185] wherein As represents the absorbance of the test substance (or VE) and DPPH mixture, Ac represents the absorbance of the test substance (or GSH) and EtOH mixture, Ab represents the absorbance of EtOH and DPPH mixture, and A0 represents the absorbance of EtOH and EtOH mixture.

[0186] After calculating the scavenging rate, the GraphPad Prism software was used for plotting, with the final concentration of the sample in the reaction system as the abscissa and the scavenging rate as the ordinate, to establish a non-linear fitting equation of the concentration of the solution to be tested and the scavenging rate, and to calculate the half scavenging amount EC50. The antioxidant capacity AO value of the polypeptide or small molecule active substance sample was calculated according to the following formula:

[0187]

[0188] wherein EC50(S) represents the concentration (μg / mL) corresponding to the half scavenging rate of the test substance, and EC50(R) represents the concentration (μg / mL) corresponding to the half scavenging rate of GSH

[0189] 5) Results

[0190] When a free radical scavenger (reducing agent) is present, DPPH reacts with its single electron pairing, causing the characteristic purple color of the DPPH ethanol solution to gradually disappear, turning colorless or light yellow, thereby causing the light absorption of DPPH to weaken. Studies have shown that the degree of fading of DPPH ethanol solution is linearly related to the number of electrons it receives, and thus the ability of the sample to scavenge free radicals, i.e., the size of the antioxidant activity, can be evaluated by measuring the light absorption of the reaction solution of the sample and DPPH at 517 nm.

[0191] The test results are shown in Table 1. Figure 8 All 17 polypeptides have a certain ability to scavenge DPPH, and according to the AO value, it can be seen that the AO value of NP1706 is smaller, i.e., the DPPH scavenging ability of NP1706 is stronger, close to the positive control GSH, indicating that NP1706 has strong antioxidant activity.

[0192] And as can be seen from Table 2, Figure 8 the EC50 of NP1706 decreased from 80 to 21 compared to the parent peptide NP1711, significantly reducing the concentration required to achieve a half-maximal effect, thereby doubling its activity. 50

[0193] Example 7: In vitro anti-inflammatory activity detection

[0194] 1) Reagents and materials​

[0195] High glucose medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, lipopolysaccharide (LPS), IL-1a (Mouse) ELISAs kit, IL-6 (Mouse) ELISAs kit, NO detection kit.

[0196] 2) Instruments

[0197] CO2 incubator, biological safety cabinet, inverted microscope, microplate reader.

[0198] 3) Cell lines

[0199] Mouse monocyte macrophage leukemia cells (Raw264.7).

[0200] 4) Samples to be tested

[0201] Sample group: NP1701-NP1717, test concentration is 20 μg / mL; blank control group: DMEM medium containing 1% FBS and 1% P / S; model group: 25 ng / mL LPS; positive control group: 0.5 μg / mL dexamethasone.

[0202] 5) Experimental method

[0203] Raw264.7 cells were inoculated in a 48-well plate at a density of 1.5 x 10 5 cells / mL cell suspension per well 500 μL, and cultured until the cell fusion rate reached about 40%, and the supernatant was removed. The samples to be tested were added in turn for biological activity treatment. After 24 h of treatment, the supernatant was collected and centrifuged at 1000 rpm for 20 min; according to the steps of the ELISA kit instructions, the cell supernatant after centrifugation was detected for NO, IL-6, and TNF-a concentration.

[0204] 6) Results

[0205] Lipopolysaccharide can activate cells to synthesize and release various cytokines and inflammatory mediators through the cell signal transduction system, and the aggregation of inflammatory factors can cause vasodilation, redness, stinging, itching, and other symptoms, thereby causing the skin to be sensitive to stinging. In this experiment, 25 ng / mL LPS was selected to induce the establishment of an anti-inflammatory activity evaluation model.

[0206] The experimental results are shown in Table 1. Figures 9-11 Compared with the blank control, the expression levels of NO, TNF-a, and IL-6 in Raw264.7 cells were significantly increased after LPS induction, indicating that the Raw264.7 cell inflammation model was successfully established;

[0207] In the sample group, NP1706 at a concentration of 20 μg / mL can significantly inhibit the expression levels of TNF-α, IL-6 and NO, and exhibit good anti-inflammatory activity; except for NP1717, the remaining 16 polypeptides can inhibit the expression level of TNF-α; in addition, the 17 polypeptides can inhibit the expression level of IL-6; and except for NP1715-NP1717, the remaining 14 polypeptides can inhibit the expression level of NO.

[0208] As can be seen from the above, NP1701-NP1717 can all inhibit skin inflammation to a certain extent, and have the potential to relieve skin redness and itching.

[0209] In combination with the above Examples 1-7, by testing the 17 polypeptides (NP1701-NP1717) derived from R. spinosa in the same in vitro model in parallel, representative data of safety, promotion of cell migration and anti-inflammatory were obtained. As shown in Table 1 below:

[0210] Table 1: Comparison results of parallel testing of NP1701-NP1717 in the same in vitro model

[0211]

[0212]

[0213] As can be seen from the above table:

[0214] (1) In the cytotoxicity test (Raw264.7 and THP-1 cells): NP1706, relative to the parent peptide NP1711, the CV75 value thereof is increased from 63.779 g / mL, 163.779 g / mL of the parent peptide to 300 g / mL, 300 g / mL, respectively; it can be seen that the toxicity of NP1706 is significantly reduced, and it requires a higher concentration (about 5 or 2 times) than the parent peptide NP1711 to produce the same toxicity; the potential therapeutic window of NP1706 is expanded, and it can maintain low cytotoxicity at a higher dose, greatly improving the safety of drug use.

[0215] (2) In the HaCat cell scratch repair activity experiment: NP1706, relative to the blank control, has a 12h HaCaT migration promotion rate value of 92.82, and it can be seen that it has obvious repair activity; and it is not much different from NP1711, and both can achieve scratch repair.

[0216] (3) In the in vitro anti-inflammatory activity detection: NP1706, relative to the parent peptide NP1711, the LPS-induced RAW264.7 TNF-α inhibition rate is increased from 28.33% to 55.90%; that is, NP1706 can achieve a doubling of the inhibition rate at the same concentration.

[0217] It is worth noting here that the above data show that NP1706, relative to the parent peptide, through the mutation of the three specific sites without knowing the structure and function of the corresponding relationship, realizes the synergistic improvement of the effect, such as greatly reducing the toxicity while improving the TNF-α inhibition rate under the condition that the repair function is basically unchanged, and achieves the unexpected synergistic effect.

[0218] In addition, as can be seen from the above table, NP1706 has a comprehensive performance significantly better than the other 16 comparison peptides in various important indicators (such as cytotoxicity, cell migration promotion, TNF-α inhibition rate, etc.), highlighting its superior activity and safety in anti-inflammatory and tissue repair.

[0219] Example 8: Application method in cosmetic composition

[0220] This example shows the application of NP1706 in three typical cosmetic carriers - repair serum, repair mask and repair emulsion, as well as its use method and efficacy evaluation idea. The target population is those with damaged skin barrier, sensitive skin or those in need of soothing repair care.

[0221] 1) Repair serum

[0222] A. Main ingredients and concentrations

[0223] NP1706: 0.05% (w / w), glycerol: 5%, sodium hyaluronate: 0.1%, butylene glycol: 4%, PEG-40 hydrogenated castor oil: 1%, emulsifying / thickening agent (such as carbomer or light non-ionic emulsifier): 0.2%, pH adjuster (citric acid / sodium citrate buffer): appropriate amount to pH 5.5, preservative: according to regulations (such as phenoxyethanol + ethylhexylglycerin): 0.5%, purified water: to 100%.

[0224] B. Preparation process flow

[0225] a) Active peptide dissolution: weigh the NP1706 freeze-dried powder, dissolve it with a small amount of purified water or butylene glycol / water mixed solvent at 25-30°C, and gently assist with ultrasonic, to ensure complete dissolution without high temperature.

[0226] b) Water phase mixing: add purified water and butylene glycol to the main stirring container, stir uniformly, then add the peptide solution; then add glycerol and sodium hyaluronate, and dissolve uniformly.

[0227] c) Oil phase or emulsifier addition: if it is an emulsion type serum, the oil phase such as PEG-40 hydrogenated castor oil is pre-mixed with part of the water phase to form a microemulsion; add it to the main tank and use shearing or homogenizing equipment to mix; if it is a water-soluble gel, this step can be omitted, and only the thickening agent is used to adjust the viscosity.

[0228] d) pH adjustment and preservative: after the system temperature drops to room temperature, measure the pH and adjust it to about 5.0-6.0; add preservatives, antioxidants, essences, etc., and stir evenly below 40°C.

[0229] e) filtration and filling: filter through appropriate pore size (such as 0.22-0.45 μm) to reduce microbial risk; fill in sterile or clean environment.

[0230] 2) Repairing mask

[0231] A. Main ingredients and carriers

[0232] a) Matrix: bio-cellulose film or hyaluronic acid gel sheet;

[0233] b) Active liquid: repairing liquid containing NP1706, ingredients can refer to the peptide concentration range (0.02-0.1%) in repairing serum and moisturizing synergistic ingredients (glycerin 3-5%, butylene glycol 3-5%, hyaluronic acid 0.1%, etc.);

[0234] c) pH adjustment and preservative system according to the principles of serum.

[0235] B. Preparation process

[0236] a) Active solution preparation: prepare an aqueous solution containing NP1706, moisturizing agent, soothing synergistic ingredients, adjust the pH to 5.0-6.0, and ensure that the formula is clear and free of precipitates.

[0237] b) Carrier impregnation: dry film or pre-made gel sheet is fully immersed in active solution (such as 2-4 hours at room temperature or overnight), until the carrier is saturated. Remove excess liquid and keep moist.

[0238] c) Packaging: individually sealed packaging to avoid contamination and moisture evaporation; label storage conditions (such as 4-25°C, avoid light).

[0239] 3) Repairing emulsion

[0240] A. Main ingredients and concentrations

[0241] NP1706: 0.02-0.05%, moisturizing agent: glycerin 3-5%, sodium hyaluronate 0.05-0.1%, oil phase: a small amount of vegetable oil or light oil (such as caprylic / capric acid triglyceride) 1-5%, emulsifier: non-ionic emulsifier or polymer emulsifier 1-2%, solvent: purified water, butylene glycol, etc., pH adjuster, preservative, antioxidant, etc. added according to regulations.

[0242] B. Preparation process

[0243] a) Oil phase and water phase separation preparation: heating separately (not more than 40℃), oil phase contains emulsifier and light oil, water phase contains peptide solution and humectant.

[0244] b) Homogeneous emulsification: slowly add oil phase to water phase or vice versa, shear or high pressure homogenization to form a homogeneous emulsion.

[0245] c) Post-processing: add preservatives and antioxidants after cooling to room temperature, adjust pH to 5-6; filter and fill.

[0246] 4) Method of use

[0247] A. Repair serum: after cleansing / toning, take an appropriate amount of serum and apply it to the face or local damaged area, gently massage until completely absorbed; use once a day in the morning and evening, or adjust according to skin condition, continue to use for several weeks to observe the effect.

[0248] B. Repair mask: after cleansing the face, apply the mask to the skin and keep it for 15-20 minutes, then gently massage the remaining essence until it is absorbed; use 1-3 times a week, or as needed; complete the repair care process with serum and emulsion.

[0249] C. Repair emulsion: use after serum or alone, apply to the face / neck, gently massage until absorbed; use in the morning and evening, or in the daytime with sunscreen.

[0250] 5) Efficacy evaluation and safety

[0251] A. Efficacy evaluation

[0252] a) In vitro or ex vivo: test the formula on skin reconstruction models or exfoliated skin tissue for skin barrier repair (such as TEWL test), soothing anti-inflammatory (inflammatory factor expression detection), etc.

[0253] b) Small sample test on humans: after ethical approval, conduct patching or trial use on volunteers, observe moisture retention, redness subsidence, comfort, skin barrier recovery, etc.; can use instruments for measurement (such as skin resistance, infrared thermography, skin moisture content, etc.);

[0254] c) Long-term use: continue to use for several weeks or months to evaluate anti-aging, fine line improvement, skin elasticity, etc.

[0255] B. Safety evaluation

[0256] a) Skin irritation and sensitization test: in vitro reconstructed skin model or human patch test to confirm no significant irritation;

[0257] b) Periocular or sensitive area test: if the formula is intended for the eye, conduct the corresponding safety evaluation;

[0258] c) Microbial limit and preservative challenge: Ensure formulation is stable, safe;

[0259] d) Active peptide stability: HPLC or LC-MS to determine NP1706 content and degradation products in the formulation, ensure activity is maintained over shelf life.

[0260] Example 9: Application method in pharmaceutical composition

[0261] This example demonstrates the application of NP1706 in two medical administration forms: topical gel, dressing, for medical purposes such as promoting wound repair, ulcer healing, burn repair, or ischemic injury recovery.

[0262] 1) Topical gel application

[0263] A. Main ingredients and concentrations

[0264] a) NP1706: 0.1-0.2% (w / w) (optimized according to activity test, can be adjusted within the range of 0.05-0.5%);

[0265] b) Excipients: sodium carboxymethyl cellulose or polymer hydrogel matrix 1-3% (adjust viscosity, maintain a moist environment);

[0266] c) Moisturizing / penetration aid: glycerol 3%, butylene glycol 3%;

[0267] d) Gelling agent / thickening agent: carbomer or related gelling agent 0.2-0.5%;

[0268] e) pH adjuster: citric acid / sodium citrate buffer to pH 6.0-7.0 (slightly neutral or weakly acidic suitable for wound administration);

[0269] f) Preservative: according to medical device or topical drug regulations (if disposable, long-term preservation may not be required);

[0270] g) Other synergistic ingredients (optional): a small amount of antibacterial agent (such as silver ions, povidone iodine, etc., which must be confirmed to be compatible with the peptide and not affect activity), antioxidants, small molecule growth factor auxiliary fragments, etc.

[0271] B. Preparation method

[0272] a) Peptide dissolution: Dissolve NP1706 in sterile physiological saline or buffer, concentration according to formulation requirements; maintain low temperature (4-25°C) and sterile operation.

[0273] b) Gel matrix preparation: Disperse sodium carboxymethyl cellulose or gelling agent in physiological saline / buffer under sterile conditions, stir until uniform and lump-free, adjust pH to form a transparent gel matrix.

[0274] c) Mixing the active solution: mix the peptide solution with the gel base, stir until homogeneous; add the moisturizing adjuvants and other synergistic ingredients, keeping sterile.

[0275] d) Filling and packaging: fill the gel into sterile soft tubes or dressing syringes in a clean / sterile environment, seal.

[0276] e) Sterilization / disinfection: if the formulation and packaging allow, use non-thermal methods (filtration, irradiation) for disinfection; avoid high temperatures to prevent peptide degradation.

[0277] C. Method of use

[0278] a) Indications: acute and chronic wounds (trauma, incision, ulcer), superficial burn wounds, postoperative skin repair, adjunctive therapy for diabetic foot ulcers, promotion of angiogenesis in ischemic wounds, etc.

[0279] b) Administration:

[0280] After debridement (if necessary), apply the gel evenly to the wound surface and surrounding skin; cover with sterile dressing or leave open; change daily or every other day, adjust according to wound healing;

[0281] For superficial wounds, apply a thin layer; for deep or larger wounds, increase the peptide concentration or application thickness as needed;

[0282] During use, observe the progress of wound closure, inflammation reduction, and regenerative tissue formation, and adjust the treatment method accordingly.

[0283] c) Evaluation criteria: wound closure rate (area change), histological analysis (biopsy or animal model sampling: regenerative tissue thickness, angiogenesis marker CD31 staining, etc.), inflammation factor level detection, change in sterility or bacterial load, healing time and pain / discomfort of patients or animal models, etc.

[0284] 2) Dressing application

[0285] A. Preparation example

[0286] d) Process the gel base containing NP1706 (same as the external gel formulation) into a thin film or a peptide-containing hydrogel patch; or combine the peptide with biocellulose or other medical dressing materials to make a dressing patch.

[0287] e) Ensure sustained drug release of the dressing under physiological conditions; maintain sterility during preparation.

[0288] B. Method of use

[0289] a) After wound debridement, cover the dressing directly on the wound, maintaining a moderately moist environment; the dressing can be changed daily or every 2-3 days as needed;

[0290] b) Observe wound healing, secretion changes, infection risks; dressings can be combined with negative pressure dressing systems or used alone, depending on specific clinical needs.

[0291] C. Performance evaluation

[0292] a) In vitro release kinetics: test NP1706 release curve in simulated wound environment (simulated exudate);

[0293] b) Biocompatibility and cell interaction: in vitro cell patch model test (such as fibroblast and endothelial cell proliferation and migration in the presence of dressing);

[0294] c) Animal model verification: compare healing speed, tissue reconstruction quality, etc. in wound models with / without NP1706 dressing.

[0295] 3) Quality and safety control

[0296] A. Raw material quality: NP1706 must have a purity of ≥95%, endotoxin, heavy metals and related impurities meet pharmaceutical standards;

[0297] B. Sterile preparation: topical gel, dressing, microneedle patch and injection must be prepared in an environment that meets GMP / sterile specifications;

[0298] C. Stability test: each dosage form is subjected to accelerated and long-term stability evaluation, including activity retention, appearance, pH, viscosity (gel), carrier structure integrity (microneedle), injection clarity, etc.

[0299] D. Biocompatibility and safety: topical / patch preparations need to be tested for skin irritation and allergy; injections need to be subjected to preliminary toxicological evaluation (such as local tolerance, immunogenicity, etc.);

[0300] E. Efficacy verification: test the biological activity and efficacy of different dosage forms in in vitro cell models and animal models to ensure the effectiveness of the formula.

[0301] 4) Application process

[0302] A. Topical gel example: apply 0.1% NP1706 gel to model animals (such as mouse full-thickness skin wounds) once a day, cover with dressing, and observe wound closure curve and histological indicators;

[0303] B. Dressing example: apply peptide-containing hydrogel dressing to the wound, replace it every 48 hours according to the release curve, and monitor the regeneration effect.

[0304] Example 10: Skin wound healing test

[0305] 1) Experimental method

[0306] Animal model establishment: In order to test the effect of NP1706 on promoting wound healing in vivo, we constructed a mouse skin wound model for testing. In this study, 5-week-old SPF male ICR mice (25-32 g) were randomly divided into 3 groups, including a blank control group, an endothelial growth factor group, and an NP1706 group (n = 6). The mice were anesthetized with 4.00% w / v chloral hydrate (0.01 mL / g). The dorsal hair was removed with a shaving razor and depilatory cream, and an 8-mm excisional wound was made on the back with a biopsy punch.

[0307] Dosing regimen: The drug was administered topically once a day, (recombinant human epidermal growth factor, 10 μg / cm 2 ), NP1706 group (concentration of 20 μg / mL, 10 μg / cm 2 ), and the blank control group was given an equal volume of normal saline. The wound healing was observed and photographed for 14 consecutive days.

[0308] 2) Experimental results

[0309] As Figure 12 shown, compared with the blank control group, NP1706 in the present application can significantly accelerate the healing process of the mouse dorsal skin wound, and the effect is comparable to that of the clinically commonly used positive drug recombinant human epidermal growth factor. No adverse reactions such as weight loss or local tissue necrosis were observed during the experiment, and the regenerated skin tissue had no excessive contraction or scar hyperplasia phenomenon, indicating that NP1706 can effectively promote wound healing in vivo without obvious toxic side effects.

[0310] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Those skilled in the art can make some simple modifications, equivalent changes or modifications using the disclosed technical content, which are all within the protection scope of the present application.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is the amino acid sequence shown in SEQ ID NO:

6.

2. A polypeptide derivative, characterized in that, The polypeptide derivative comprises a sequence having at least 70% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 11, and the sequence is a polypeptide derivative with original biological activity obtained by site-directed amino acid mutation at the site of an active non-essential residue at position 5, 9 and / or 14 in the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO:

11. The original biological activity is the activity of promoting cell proliferation and migration, antioxidant activity and / or anti-inflammatory activity.

3. The polypeptide derivative according to claim 2, characterized in that, The polypeptide derivative is a polypeptide derivative with original biological activity obtained by modification based on the amino acid sequence shown in SEQ ID NO: 6 or the modified polypeptide derivative.

4. The polypeptide derivative according to claim 3, characterized in that, The modification is acylation at the N- or C-terminus, or PEGylation or fatty acid coupling modification.

5. A method for the preparation of an optimized polypeptide derivative, characterized in that, It comprises: site-directed amino acid mutation at the site of an active non-essential residue at position 5, 9 and / or 14 in the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 11; safety evaluation and biological activity detection of the modified polypeptide derivative, thereby obtaining an optimized polypeptide derivative with original biological activity; The original biological activity is the activity of promoting cell proliferation and migration, antioxidant activity and / or anti-inflammatory activity.

6. A composition characterized in that, The composition is a cosmetic composition or a pharmaceutical composition; The active ingredient of the composition comprises the polypeptide of claim 1 or the polypeptide derivative of any one of claims 2-4 or the optimized polypeptide derivative prepared by the preparation method of claim 5; The composition further comprises a cosmetically or pharmaceutically acceptable base, carrier and / or adjuvant.

7. The composition of claim 6, wherein, The cosmetic composition is an external emulsion, serum or mask; The pharmaceutical composition is an external gel, dressing, topical drug delivery system or injection; The composition has the activity of promoting cell proliferation and migration, antioxidant activity and / or anti-inflammatory activity.

8. The composition of claim 6, wherein, The cosmetic composition has the effects of anti-inflammatory, soothing, promoting skin barrier repair and / or antioxidant aging; The pharmaceutical composition has the effects of anti-inflammatory, promoting wound repair, ulcer healing, burn repair, ischemic injury and / or other tissue regeneration.

9. Use of the polypeptide according to claim 1, of the polypeptide derivative according to any one of claims 2 to 4, of the optimized polypeptide derivative prepared according to the method of claim 5 or of the composition according to any one of claims 6 to 8, characterized in that, The application is: The application is in the preparation of a cosmetic or a pharmaceutical with the activity of promoting cell proliferation and migration, antioxidant activity and / or anti-inflammatory activity.

10. Use of the polypeptide according to claim 1, of the polypeptide derivative according to any one of claims 2 to 4, of the optimized polypeptide derivative prepared according to the method of claim 5 or of the composition according to any one of claims 6 to 8, characterized in that, The application is: The application is in the preparation of a cosmetic with the effects of anti-inflammatory, soothing, promoting skin barrier repair and / or antioxidant aging; Or, the application is in the preparation of a pharmaceutical with the effects of anti-inflammatory, promoting wound repair, ulcer healing, burn repair, ischemic injury and / or other tissue regeneration.

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