A modified periplaneta americana polypeptide derivative, a preparation method thereof, a polypeptide composition and application thereof

Abstract

This invention discloses a modified American cockroach polypeptide derivative, its preparation method, polypeptide composition, and applications. The amino acid sequence of the polypeptide derivative is shown in SEQ ID NO: 1 (PA1702); it is modified by replacing the asparagine at position 13 of the natural polypeptide PA1701 with glutamine, and can be prepared by Fmoc solid-phase polypeptide synthesis. Network pharmacology predicts that it acts on 59 core anti-inflammatory targets, enriched in pathways such as PI3K-Akt and MAPK. Molecular docking shows that PA1702 has a strong binding affinity for TNF-α and AKT1. In vitro experiments confirm that PA1702 is non-cytotoxic, can inhibit LPS-induced release of NO, TNF-α, IL-6, and IL-1β, and promote cell migration. The above-mentioned polypeptide derivative can be used as an active ingredient in cosmetics or pharmaceutical compositions; it has anti-inflammatory, skin barrier repair, and wound healing promotion effects, and can be used to prepare anti-inflammatory, skin barrier repair, and wound healing-promoting cosmetics or drugs.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, and in particular to a modified American cockroach polypeptide derivative, its preparation method, polypeptide composition, and applications. Background Technology

[0002] The American cockroach (Periplaneta americana) is an insect belonging to the order Blattodea in the class Insecta. Its extracts (such as Kangfuxin liquid) are widely used clinically in the treatment of gastric ulcers, burns, and wound repair, exhibiting good healing-promoting and anti-inflammatory effects. The American cockroach body is rich in various small-molecule active peptides, possessing potential medicinal and skincare value.

[0003] However, conventionally prepared American cockroach extracts are complex in composition, containing a variety of small-molecule active peptides, as well as a large number of impurities such as proteins and polysaccharides, leading to large batch-to-batch variations and difficulties in quality control. At the same time, they may contain residual allergenic proteins or insect antigens, which may cause allergic reactions or immunogenic risks, limiting their safe application in the cosmetics and pharmaceutical fields. More importantly, it is currently impossible to determine which small-molecule active peptides are the true active ingredients, requiring creative work to identify and verify them.

[0004] In addition, the inventors have found that the natural polypeptides directly isolated from American cockroaches have the following technical defects: (1) poor sequence stability: some amino acid residues in natural polypeptides are prone to deamidation or oxidation modification under physiological conditions, resulting in decreased activity; (2) limited activity: natural polypeptides usually act on a single target or a single pathway, and their anti-inflammatory and repair-promoting activities are limited, making it difficult to meet the needs of clinical applications; (3) unclear mechanism of action: the molecular target spectrum and signaling pathway network of natural polypeptides are not yet clear, and there is a lack of systematic understanding, which limits their further optimization and application.

[0005] To address the aforementioned shortcomings, while some researchers have attempted to obtain more potent anti-inflammatory peptide derivatives from the American cockroach through truncation modification, these methods remain limited to a single structural truncation strategy, failing to incorporate systematic target prediction and multi-dimensional modification techniques, and their specific sequences are not publicly available. Furthermore, existing research largely focuses on screening for single targets or pathways, lacking a systematic understanding of the synergistic effects of peptides across multiple targets and pathways.

[0006] Network pharmacology, an analytical method based on systems biology, can systematically predict drug mechanisms of action, screen core targets, and evaluate multi-target synergistic effects by constructing "compound-target-pathway" networks. However, there are currently no reports of combining network pharmacology with peptide structure optimization for screening peptides derived from American cockroaches.

[0007] Therefore, developing a highly active American cockroach polypeptide derivative with a clear mechanism of action based on network pharmacology target prediction guidance has significant scientific research value and promising industrial application prospects. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a novel, stable, well-defined, and highly active modified American cockroach polypeptide derivative, its preparation method, polypeptide composition, and applications.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a modified American cockroach polypeptide derivative, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0011] Furthermore, the polypeptide derivative is obtained by replacing the asparagine at position 13 of the natural polypeptide SEQ ID NO: 2 with glutamine.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned modified American cockroach polypeptide derivative, which is prepared by Fmoc solid-phase polypeptide synthesis and purified by HPLC to a purity of ≥98%.

[0013] Thirdly, the present invention provides a polypeptide composition whose active ingredient comprises the above-mentioned modified American cockroach polypeptide derivative.

[0014] The composition is a cosmetic composition or a pharmaceutical composition;

[0015] The composition also includes a cosmetic or pharmaceutically acceptable matrix, carrier, excipient or other inactive ingredient.

[0016] Furthermore, the modified American cockroach polypeptide derivative has a mass percentage content of 0.001%-5%.

[0017] Furthermore, the composition is a composition having anti-inflammatory, skin barrier repair, and / or wound healing promotion effects.

[0018] Furthermore, the dosage form of the composition is an essence, lyophilized powder, facial mask liquid, gel, spray, or cream.

[0019] Furthermore, the composition is an anti-inflammatory and repairing facial mask liquid, comprising 0.03 wt% of the modified American cockroach polypeptide derivative, 5.0 wt% of glycerin, 3.0 wt% of propylene glycol, 0.05 wt% of sodium hyaluronate, 0.1 wt% of xanthan gum, 0.5 wt% of p-hydroxyacetophenone, 0.5 wt% of 1,2-hexanediol, and deionized water to 100 wt%.

[0020] Fourthly, the present invention also provides an application of the above-mentioned modified American cockroach polypeptide derivative or polypeptide composition, wherein the application is as follows:

[0021] Applications in the preparation of cosmetics or pharmaceuticals that are anti-inflammatory, repair the skin barrier, and / or promote wound healing.

[0022] Fifthly, the present invention also provides an application of the above-mentioned modified American cockroach polypeptide derivative or polypeptide composition, wherein the application is:

[0023] Applications in the preparation of cosmetics or drugs that inhibit NO release and the expression of inflammatory factors TNF-α, IL-6, and IL-1β, or promote HaCaT cell migration.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) Novel structure: This invention provides a novel polypeptide derivative sequence from American cockroach, which has not been reported before.

[0026] (2) The mechanism is clear: network pharmacology revealed that the peptide acts on 59 core anti-inflammatory targets and is enriched in inflammation and repair-related pathways such as PI3K-Akt and MAPK.

[0027] (3) Significant activity: In vitro experiments have confirmed that the polypeptide has no cytotoxicity, can significantly inhibit the release of NO and TNF-α, IL-6, IL-1β inflammatory factors, and promote cell migration.

[0028] (4) Reliable application: Mask liquid containing this peptide can significantly improve skin barrier function, reduce inflammation and increase water content.

[0029] (5) Industrial applicability: It can be synthesized on a large scale, meets GMP requirements, and is applicable to the fields of cosmetics and pharmaceuticals. Attached Figure Description

[0030] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Figure 1 Venn diagram of potential targets and inflammatory targets of PA1702 shows 59 core anti-inflammatory targets.

[0032] Figure 2 A network diagram of protein-protein interactions (PPIs) for 59 core anti-inflammatory targets.

[0033] Figure 3The bar chart shows the GO functional enrichment analysis of the core anti-inflammatory targets of this invention (top 10 in each dimension).

[0034] Figure 4 Bubble chart (Top 20) showing the KEGG pathway enrichment analysis of the core anti-inflammatory targets of this invention.

[0035] Figure 5 Results of PA1702 cytotoxicity assay on HSF and HaCaT cells.

[0036] Figure 6 Bar chart showing the effect of PA1702 on LPS-induced TNF-α release in RAW 264.7 cells.

[0037] Figure 7 Bar chart showing the effect of PA1702 on LPS-induced IL-6 release in RAW 264.7 cells.

[0038] Figure 8 Bar chart showing the effect of PA1702 on LPS-induced IL-1β release in RAW 264.7 cells. Detailed Implementation

[0039] Several typical embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be particularly noted that the embodiments shown in the drawings are merely illustrative representations of the present invention and are not intended to limit the scope of protection of the present invention. The present invention can be implemented through various methods, and the embodiments described herein are intended to fully illustrate the technical principles of the present invention and ensure that those skilled in the art can fully understand the technical boundaries of the present invention. Specific embodiments are described below:

[0040] Example 1: Isolation, Identification and Structural Modification of Natural Polypeptides from American Cockroaches

[0041] 1.1 Isolation and sequencing of natural peptides

[0042] Dried American cockroach bodies were collected, pulverized, and extracted with purified water by ultrasonication. The supernatant was collected by centrifugation. The supernatant was then separated using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The filtrate was collected, freeze-dried, and the crude peptide lyophilized powder was obtained.

[0043] The crude peptide lyophilized powder was dissolved in 0.1% trifluoroacetic acid aqueous solution, and purified by C18 reversed-phase high-performance liquid chromatography (RP-HPLC). The main chromatographic peaks were collected, and the purified peptide sample was freeze-dried. Sequence identification was performed using electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS / MS). Data analysis yielded a natural peptide sequence, named PA1701, with the following amino acid sequence:

[0044] Met-Lys-Thr-Phe-Val-Arg-Leu-Tyr-Arg-Ser-Leu-Ile-Asn-Lys-Val-Leu-His (SEQ ID NO: 2).

[0045] The above sequence is a novel natural polypeptide sequence isolated and purified from a complex extract of the American cockroach. Experimental verification showed that although it possesses certain activity, its stability is poor and its activity level is limited, making it difficult to use directly as a cosmetic or pharmaceutical ingredient. To address the stability defects of this natural polypeptide, conventional solutions in the field include: (1) structural modifications such as terminal amidation, cyclization, or deletion of easily degradable residues; and (2) abandoning the sequence and re-isolating and purifying other natural polypeptides. However, in solution (1), due to the short length of the polypeptide sequence and unclear structure-activity relationship, the above conventional modifications often fail to improve stability while preserving its original conformation and activity, presenting unpredictable technical obstacles. Solution (2) requires a large amount of repetitive separation and screening work, resulting in low efficiency and uncertain results.

[0046] 1.2 Design Basis for Peptide Structure Modification

[0047] To address the issues of poor stability and limited activity inherent in natural peptides, the inventors have implemented the following structural modification design:

[0048] (1) Substitution of conserved amino acids

[0049] The inventors discovered that asparagine (Asn, N) at position 13 of a natural polypeptide sequence is prone to deamidation under physiological conditions, generating aspartic acid or isoaspartic acid, leading to conformational changes and decreased activity. To overcome this deamidation problem, conventional modification methods known in the art include: mutating Asn to a non-amide amino acid (such as Ala, Leu, Val) to completely eliminate the amide group, introducing a non-natural amino acid, or performing cyclization modification to limit conformational changes. However, it is generally believed in the art that replacing Asn with glutamine (Gln, Q), which has the most similar structure, cannot effectively solve the deamidation problem. The reason is that both Asn and Gln contain free amide groups in their side chains, have the same deamidation reaction mechanism, and possess highly similar chemical properties. Those skilled in the art generally expect Gln to also readily undergo deamidation, and even have a higher reaction rate due to its longer side chain. Therefore, those skilled in the art lack the motivation to replace this residue with Gln when facing the Asn deamidation problem. Surprisingly, after numerous trials, the inventors discovered that replacing Asn with Gln significantly reduced the deamidation rate of the polypeptide compared to the natural polypeptide.

[0050] (2) Design basis of network pharmacology guidance

[0051] To verify the rationality of the modification strategy and predict the mechanism of action of the modified peptides, this invention introduces network pharmacology into the modification design for the first time. By predicting targets and analyzing pathways of the modified peptides, the molecular mechanisms of their anti-inflammatory and repair-promoting effects are systematically elucidated, avoiding the blindness of traditional "trial and error" modification methods.

[0052] 1.3 Obtaining Modified Peptide Derivatives

[0053] Based on the above design, the natural polypeptide PA1701 was modified by conservative amino acid substitution: the asparagine (N) at position 13 was replaced with glutamine (Q) to obtain a modified polypeptide derivative, named PA1702, whose amino acid sequence is shown in SEQ ID NO: 1: Met-Lys-Thr-Phe-Val-Arg-Leu-Tyr-Arg-Ser-Leu-Ile-Gln-Lys-Val-Leu-His, which is abbreviated as MKTFVRLYRSLIQKVLH.

[0054] 1.4 Comparison and prediction of peptide properties before and after modification

[0055] The physicochemical properties of the peptides before and after modification were predicted and compared using peptide property prediction tools. The results showed that the molecular weight, isoelectric point, hydrophobicity, and other physicochemical properties of the peptides before and after modification were basically the same, indicating that the conservative substitution did not significantly change the basic physicochemical properties of the peptides. However, the predicted elimination of the deamidation site could significantly improve the stability of the peptides under physiological conditions and prolong their half-life in serum and skin environments. These prediction results were experimentally verified in subsequent examples.

[0056] Example 2: Screening of modified peptide core anti-inflammatory targets and PPI network analysis based on network pharmacology

[0057] 2.1 Intersection screening of target prediction and inflammatory targets

[0058] Potential targets for the modified peptide derivative (PA1702) of this invention were predicted using peptide-protein interaction databases (PPIKB, PEPBI), resulting in 118 potential targets. Intersection analysis was performed between these targets and the top 1000 inflammation-related targets (ranked by relevance score) in the GeneCards database (https: / / www.genecards.org / ), and the results were visualized using a Venn diagram. Figure 1 A total of 59 common targets were obtained, which are considered to be the core targets for the anti-inflammatory effect of the modified peptide derivatives of this invention.

[0059] The 59 core anti-inflammatory targets are: AKT1, MAPK1, MAPK3, EGFR, VEGFA, STAT3, CASP3, SRC, JAK1, JAK2, PTGS1, TLR4, MYD88, RELA, CCND1, BCL2, ADAM17, KEAP1, GSK3B, PIK3CA, PIK3R1, MTOR, BRAF, HSPA5, FGFR1, FGFR2, KDR, FLT1, and ITG. B1, ITGB3, PTEN, FOXO1, FOXO3, TP53, FOS, JUN, CREB1, TRAF6, RIPK1, BCL10, CARD11, MALT1, STAT1, STAT5 A. STAT5B, SOCS1, SOCS3, HIF1A, EPO, NOS2, NOS3, PTPN11, KRAS, CDC42, RAC1, RHOA, PRKCA, PRKCD, SMAD3.

[0060] 2.2 Construction and Analysis of Protein-Protein Interaction (PPI) Networks

[0061] The 59 core anti-inflammatory targets were imported into the STRING database (https: / / string-db.org / ), with the species set to "Homo sapiens" and the confidence threshold set to 0.9, to construct a protein-protein interaction network. Statistical analysis of the network was performed using the "Analysis" function of the STRING database to obtain the PPI network diagram. Figure 2 ).

[0062] Network analysis results show that the network contains 59 nodes and 280 edges, with an average node degree of 9.49 and an average local clustering coefficient of 0.56. The expected number of edges was 51, while the actual observed number was 280, indicating that the network's interactions are significantly higher than expected. The PPI enrichment p-value is less than 1.0e-16, indicating that the interactions in the network are highly statistically significant, meaning that the observed protein-protein interactions are significantly higher than in the random background.

[0063] The above results demonstrate that there are close interactions among the 59 core anti-inflammatory targets of the modified peptide derivatives of this invention, forming a highly interconnected protein-protein interaction network, suggesting that these targets may synergistically participate in anti-inflammatory related biological processes. Compared with the linear action mode of natural peptides acting through only a single target in the prior art, this invention achieves a fundamental shift in the dimension of action through a multi-target synergistic network mechanism, significantly enhancing the synergistic effect of anti-inflammatory regulation.

[0064] Example 3: Joint analysis of GO function and KEGG pathway based on core anti-inflammatory targets

[0065] 3.1 Test Methods

[0066] 3.1.1 Data Source

[0067] The 59 core anti-inflammatory targets obtained in Example 2 were used as the analysis objects.

[0068] 3.1.2 GO Functional Enrichment Analysis

[0069] GO functional enrichment analysis was performed using the DAVID database (https: / / david.ncifcrf.gov / ), covering three dimensions: biological process (BP), cellular component (CC), and molecular function (MF). Using p < 0.05 as a significance threshold, the top 10 GO entries with the highest gene enrichment in each dimension were selected for analysis.

[0070] 3.1.3 KEGG pathway enrichment analysis

[0071] KEGG pathway enrichment analysis was performed using the DAVID database. The top 20 signaling pathways with the highest gene enrichment were selected for analysis using p < 0.05 as the significance threshold.

[0072] 3.2 Results and Analysis

[0073] 3.2.1 GO Functional Enrichment Analysis

[0074] GO functional enrichment analysis showed that 59 core anti-inflammatory targets were significantly enriched in multiple functional items related to inflammation regulation, cell survival, and tissue repair. Figure 3 ).

[0075] Key findings related to anti-inflammatory activity:

[0076] Inflammation regulation: Significant enrichment of cytokine-mediated signaling pathways (14 genes) and response tolipopolysaccharide (14 genes) confirms that the peptides of this invention can regulate inflammatory signaling pathways. Enriched targets include core inflammatory factors and signal transduction molecules such as TNF-α, IL-6, TLR4, MYD88, RELA, and TRAF6.

[0077] Cell survival and anti-apoptosis: The negative regulation of apoptotic process (24 genes) was highly enriched, involving targets such as AKT1, BCL2, CASP3, and TP53, indicating that the peptides can exert tissue protection by inhibiting apoptosis.

[0078] Key findings related to repair activity:

[0079] Cell migration and proliferation: positive regulation of cell migration (16 genes) and positive regulation of cell population proliferation (19 genes) were significantly enriched, involving targets such as EGFR, VEGFA, AKT1, MAPK1, SRC, RAC1, and CDC42, which are highly consistent with the function of peptides in promoting the migration of keratinocytes and fibroblasts and accelerating wound healing.

[0080] Signaling pathway regulation: positive regulation of PI3K-Akt signal transduction (15 genes) and positive regulation of ERK1 and ERK2 cascade (13 genes) were significantly enriched, which is consistent with the mechanism of peptide activation to promote survival signaling pathways.

[0081] Cellular localization: Focal adhesion (16 genes) was significantly enriched, indicating that the target site was located in the focal adhesion structure, which plays a central role in cell adhesion, migration and signal transduction; receptor complex (11 genes) was enriched, suggesting that the peptide may exert signal transduction regulation function by acting on the receptor complex.

[0082] Molecular function: protein kinase activity (18 genes) and protein tyrosine kinase activity (11 genes) were significantly enriched, confirming the mechanism by which peptides regulate kinase signaling pathways such as MAPK and PI3K-Akt.

[0083] 3.2.2 KEGG pathway enrichment analysis

[0084] KEGG pathway enrichment analysis showed that 59 core anti-inflammatory targets were significantly enriched in multiple signaling pathways related to inflammation regulation, cell survival, and tissue repair. Figure 4 ).

[0085] Key findings related to anti-inflammatory activity:

[0086] Inflammation and Metabolism: Lipid and atherosclerosis (25 genes) involves inflammatory responses triggered by lipid metabolism, with enriched targets including AKT1, MAPK1, TNF-α, IL-6, TLR4, etc.; AGE-RAGE signaling pathway indiabetic complications (24 genes) is a core inflammatory pathway for diabetic complications, with enriched targets including AKT1, MAPK1, RELA, JAK2, STAT3, etc., suggesting that peptides may alleviate inflammatory responses by inhibiting the AGE-RAGE signaling axis, which is particularly suitable for diabetic wound repair.

[0087] Infection and Immunity: Infectious disease pathways such as Hepatitis B (26 genes), Shigellosis (26 genes), and Measles (19 genes) are enriched with a large number of anti-inflammatory immune-related targets, such as SRC, JAK1, STAT3, TRAF6, and MYD88, further supporting the potential role of peptides in immune regulation.

[0088] Key findings related to repair activity:

[0089] Cell migration and adhesion: Focal adhesion (22 genes) is a cell-matrix connection structure that plays a core role in cell adhesion, migration and survival. Enriched targets include AKT1, MAPK1, SRC, RAC1, RHOA, CDC42, ITGB1, ITGB3, etc., indicating that peptides can promote the migration and adhesion of keratinocytes and fibroblasts by regulating focal adhesion signaling.

[0090] Survival-promoting signaling pathways include: the MAPK signaling pathway (21 genes), which regulates inflammatory responses, cell proliferation, and differentiation, with enriched targets including MAPK1, MAPK3, EGFR, KRAS, RAC1, CDC42, FOS, and JUN; the Rap1 signaling pathway (20 genes), which participates in cell adhesion and cell polarity regulation, with enriched targets including EGFR, KRAS, RAC1, CDC42, ITGB1, and ITGB3; and the Neurotrophin signaling pathway (19 genes), which regulates cell survival, with enriched targets including AKT1, MAPK1, MAPK3, RAC1, and CDC42. These pathways collectively mediate the role of peptides in promoting cell survival, migration, and proliferation.

[0091] Other enriched pathways: The enrichment of some cancer-related pathways (such as Proteoglycans in cancer, Chemical carcinogenesis, PD-L1 expression, etc.) reflects the broad role of the target in the regulation of cell cycle, apoptosis and oxidative stress. These mechanisms are also involved in the inflammation and repair process, but they are not the core focus of this invention, so they will not be elaborated on.

[0092] 3.3 Conclusion

[0093] In summary, through joint analysis of the GO function and KEGG pathway of 59 core anti-inflammatory targets, the following conclusions were drawn:

[0094] Anti-inflammatory mechanism: The polypeptide derivatives of this invention act on core inflammatory targets such as TNF-α, IL-6, TLR4, MYD88, RELA, and TRAF6 by regulating biological processes such as cytokine-mediated signaling pathway, response to lipopolysaccharide, and inflammatory response. They are also enriched in inflammation-related pathways such as Lipid and atherosclerosis and AGE-RAGE signaling pathway, thus exerting broad-spectrum anti-inflammatory activity.

[0095] Mechanism of action for promoting repair: The polypeptide derivatives of this invention regulate biological processes such as positive regulation of cell migration, positive regulation of cell population proliferation, and negative regulation of apoptotic process, acting on targets such as EGFR, VEGFA, AKT1, MAPK1, SRC, RAC1, CDC42, and ITGB1, and are enriched in repair-related pathways such as Focal adhesion, MAPK signaling pathway, Rap1 signaling pathway, and Neurotrophin signaling pathway, thereby promoting cell migration, adhesion, proliferation, and survival, and accelerating wound healing.

[0096] Molecular localization and function: The target is mainly located in the cytoplasm, nucleus, plasma membrane and focal adhesion, and has protein kinase activity and protein binding function, which is consistent with the mechanism of polypeptide regulation of protein interaction network, kinase cascade reaction and gene transcription.

[0097] The above combined analysis results of GO function and KEGG pathway corroborate each other, revealing the molecular mechanism by which the polypeptide derivative of this invention exerts a comprehensive anti-inflammatory and repair-promoting effect by synergistically regulating inflammatory response, cell survival, cell migration and adhesion-related signaling pathways through multiple targets.

[0098] Example 4: Molecular docking verification

[0099] 4.1 Molecular docking method

[0100] To verify the binding affinity between the modified polypeptide derivative of this invention and the core anti-inflammatory targets, molecular docking studies were conducted on the core targets TNF-α (PDB ID: 2AZ5) and AKT1 (PDB ID: 3CQW), which ranked high in the Degree value in network pharmacology analysis.

[0101] The three-dimensional structure of the target protein was downloaded from the RCSB PDB database, and preprocessed using AutoDock Tools software for dehydration, hydrogenation, and charge distribution. The three-dimensional structure of the modified polypeptide derivative (SEQ ID NO: 1) of this invention was predicted using the PEP-FOLD3 server. Molecular docking was performed using AutoDock Vina software, and the docking results were evaluated using binding energy (∆G, kcal / mol), with lower binding energy indicating stronger binding affinity.

[0102] 4.2 Results and Analysis

[0103] The molecular docking results are shown in Table 1. The binding energy of the modified polypeptide derivative PA1702 of this invention to TNF-α is -8.6 kcal / mol, and the binding energy to AKT1 is -7.9 kcal / mol, both of which are significantly lower than the strong binding threshold of -5.0 kcal / mol, indicating that it has a strong binding affinity to both core targets.

[0104] Table 1. Molecular docking binding energy results

[0105] sample target Binding energy (kcal / mol) PA1702 TNF-α -8.6 PA1702 AKT1 -7.9 PA1701 TNF-α -5.3 PA1701 AKT1 -4.9 Positive control (TNF-α inhibitor) TNF-α -8.5 Positive control (AKT1 inhibitor) AKT1 -8.1

[0106] Compared to the natural peptide PA1701, the binding energy of PA1702 to TNF-α increased from -5.3 kcal / mol to -8.6 kcal / mol, an increase of 62.3%. It is well known to those skilled in the art that a binding energy ≤ -5.0 kcal / mol only indicates that binding can occur, while ≤ -7.0 kcal / mol is considered a generally accepted functional threshold for good binding ability. While the natural peptide PA1701's -5.3 kcal / mol reached a binding level, it did not cross the functional threshold of -7.0 kcal / mol; however, PA1702's -8.6 kcal / mol was significantly lower than -7.0, crossing this generally accepted threshold and leaping from "binding capable" to the category of "good binding ability," achieving a qualitative improvement.

[0107] Furthermore, compared to the natural peptide PA1701, the binding energy of PA1702 with AKT1 increased from -4.9 kcal / mol to -7.9 kcal / mol, an increase of 61.2%. It is well known to those skilled in the art that a more negative binding energy value indicates stronger binding affinity; typically, ≤-5.0 kcal / mol is considered the baseline level for binding, while ≤-7.0 kcal / mol is widely regarded as a reference threshold for good binding ability. The natural peptide PA1701's -4.92 kcal / mol did not even reach the baseline binding level, while PA1702's -7.93 kcal / mol was significantly lower than -7.0 kcal / mol, entering the range of good binding ability, achieving a qualitative improvement from "ineffective binding" to "strong binding."

[0108] Furthermore, the binding energy of PA1702 to TNF-α (-8.6 kcal / mol) is comparable to that of the positive control of TNF-α inhibitor (-8.5 kcal / mol), and its binding energy to AKT1 (-7.9 kcal / mol) is close to that of the positive control of AKT1 inhibitor (-8.1 kcal / mol), indicating that it has excellent target binding ability.

[0109] Analysis of the molecular docking mode between PA1702 and TNF-α revealed that the peptide stably binds to the active pocket of TNF-α through multiple hydrogen bonds and hydrophobic interactions. Key hydrogen bonds involve Tyr-59 and Gln-61 residues of TNF-α. Simultaneously, aromatic amino acid residues in the peptide form π-π stacking interactions with the hydrophobic region of TNF-α. This binding mode is similar to the binding characteristics of known TNF-α inhibitors, further confirming the potential of the modified peptide derivative of this invention to target TNF-α and inhibit its activity.

[0110] 4.3 Conclusion

[0111] Molecular docking verification results show that the modified polypeptide derivative of this invention has strong binding affinity to the core anti-inflammatory targets TNF-α and AKT1. This result is consistent with the network pharmacology prediction results, providing a theoretical basis for subsequent in vitro and in vivo activity evaluation.

[0112] Example 5: Solid-phase synthesis and purification of the modified polypeptide derivative PA1702

[0113] 5.1 Test Methods

[0114] The modified polypeptide derivative of this invention (SEQ ID NO: 1) was prepared using the Fmoc solid-phase polypeptide synthesis method (SPPS). Rink Amide resin with a degree of substitution of 0.5 mmol / g was used as the carrier. The resin was placed in a synthesis tube and swollen with DMF for 30 min. Deprotection and coupling reactions were performed sequentially: deprotection was performed using a 20% piperidine / DMF solution for 10 min; coupling was performed by adding 4 molar excesses of Fmoc-protected amino acids, HBTU, HOBt, and DIEA, and reacting for 60 min. After coupling, the resin was washed alternately with DMF and DCM. After all amino acids were sequentially coupled, the resin was shrunk with methanol and dried under vacuum.

[0115] Add the cleavage reagent (TFA / TIS / H2O = 95:2.5:2.5, v / v / v) to the dried resin and stir at room temperature for 2 h. Filter to remove the resin, add the filtrate to pre-cooled diethyl ether to precipitate, centrifuge (4000 rpm, 10 min), collect the precipitate, wash three times with diethyl ether, and freeze-dry to obtain the crude peptide.

[0116] The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Chromatographic conditions: C18 column (250 mm × 10 mm, 10 μm); mobile phase A: 0.1% TFA-water; mobile phase B: 0.1% TFA-acetonitrile; gradient elution; flow rate: 3.0 mL / min; detection wavelength: 214 nm. The main peak was collected, lyophilized, and the purified product was obtained.

[0117] 5.2 Results

[0118] HPLC analysis showed that the purity of the purified peptide was ≥98%; mass spectrometry (MS) analysis determined the molecular weight, and the measured value matched the theoretical value, confirming that the synthesized peptide was the target peptide.

[0119] Example 6: Evaluation of in vitro cell viability

[0120] 6.1 Cell Culture

[0121] Human skin fibroblasts (HSF), human immortalized keratinocytes (HaCaT), and mouse macrophages (RAW264.7) were all purchased from ATCC. Cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) and routinely cultured in a 37°C, 5% CO2 incubator.

[0122] 6.2 Cytotoxicity assay (CCK-8 assay)

[0123] HSF and HaCaT cells in logarithmic growth phase were seeded at 5 × 10³ cells / well in 96-well plates and cultured overnight. Different concentrations (0, 10, 25, 50, 100, 200 μg / mL) of a modified polypeptide derivative (SEQ ID NO: 1) were added, with three replicates per group, and cultured for another 24 h. 10 μL of CCK-8 solution was added to each well, and after incubation for 2 h, absorbance was measured at 450 nm. Cell viability was calculated using the formula: Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%.

[0124] The results are as follows Figure 5 As shown, the modified peptide derivative 1702 had a concentration-dependent effect on the survival rate of HSF and HaCaT cells. HSF cells were more sensitive to peptide toxicity, while HaCaT cells showed relatively stronger tolerance. Within the concentration range of 1-25 μg / mL, the survival rate of both cell types was greater than 85%, indicating good biocompatibility within this concentration range. Subsequent anti-inflammatory activity experiments used 1, 5, and 25 μg / mL as low, medium, and high dose groups, respectively.

[0125] 6.3 Evaluation of anti-inflammatory activity (NO release inhibition)

[0126] RAW 264.7 cells were harvested and treated at a concentration of 1×10⁻⁶ cells. 5 100 cells / well were inoculated into 96-well plates and incubated overnight. Grouping was as follows:

[0127] Blank control group: Normal culture;

[0128] LPS model group: 1 μg / mL LPS was added;

[0129] Positive control group: 1 μg / mL LPS + 10 μmol / L dexamethasone were added;

[0130] Low-dose group: Add 1 μg / mL LPS + 1 μg / mL PA1702;

[0131] Medium-dose group: 1 μg / mL LPS + 5 μg / mL PA1702 were added;

[0132] High-dose group: Add 1 μg / mL LPS + 25 μg / mL PA1702.

[0133] Each group had 3 replicates. After 24 h of culture, the supernatant was collected, and the NO content was measured using the Griess method. NO inhibition rate (%) = [(NO content in model group - NO content in drug group) / (NO content in model group - NO content in blank group)] × 100%.

[0134] The results, as shown in Table 2, indicate that the NO content in the LPS model group was significantly increased. The modified peptide derivative inhibited NO release in a concentration-dependent manner, with the high-dose group (25 μg / mL) showing an inhibition rate of 65.6%, significantly better than the model group, and no significant difference from the positive control group (dexamethasone, inhibition rate 72.3%).

[0135] Table 2. Inhibitory effect of PA1702 on LPS-induced NO release in RAW 264.7 cells.

[0136] Group NO content (μmol / L, mean ± SD) Inhibition rate (%) Blank control group 2.1 ± 0.1 - LPS model group 26.8 ± 0.3 - Dex-10 μg / mL 8.9 ± 0.1 72.5 PA1702-1 μg / mL 26.6 ± 0.2 0.8 PA1702-5 μg / mL 23.3 ± 0.2 14.2 PA1702-25 μg / mL 10.6 ± 0.2 65.6

[0137] 6.4 Evaluation of anti-inflammatory activity (detection of inflammatory factors)

[0138] RAW 264.7 cells were harvested and treated according to the above grouping for 24 h. The cell culture supernatant was then collected. The levels of key inflammatory factors TNF-α, IL-6, and IL-1β in the supernatant were detected using an ELISA kit, following the kit instructions.

[0139] The results are as follows Figure 6-8 The results showed that after LPS stimulation, the levels of TNF-α, IL-6, and IL-1β were significantly higher than those in the blank control group (p < 0.001). The modified peptide derivatives exhibited concentration-dependent inhibitory effects on the three inflammatory factors within the concentration range of 1-25 μg / mL. The low-dose group (1 μg / mL) showed inhibition rates of 2.4%, 5.8%, and 5.0% for each inflammatory factor, respectively, which were not significantly different from the LPS model group (p > 0.05), indicating no significant anti-inflammatory effect. The medium-dose group (5 μg / mL) showed inhibition rates of 7.6%, 14.7%, and 14.1%, respectively, with significant inhibition of IL-6 and IL-1β (p < 0.05 or p < 0.001), but no significant difference was observed for TNF-α. The high-dose groups (25 μg / mL) showed inhibition rates of 34.0%, 30.8%, and 43.5%, respectively, all of which were highly significant compared with the LPS model group (p < 0.01 or p < 0.001), with the most significant inhibitory effect on IL-1β. The positive control group (dexamethasone, 10 μg / mL) showed inhibition rates of 74.3%, 69.0%, and 71.3%, respectively, demonstrating a strong inhibitory effect.

[0140] 6.5 Evaluation of Repair-Promoting Activity (Scratch Test)

[0141] HaCaT cells were collected at a concentration of 5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured until 90% confluence. A straight line was drawn at the bottom of each well using a 200 μL sterile pipette tip, and floating cells were removed by washing with PBS. Serum-free medium containing different concentrations of the modified peptide derivative (1, 5, 25 μg / mL) was added, and scratch healing rate was calculated after photographing under an inverted microscope at 0 h, 24 h, and 48 h. Scratch healing rate (%) = (0 h scratch area - post-drug administration scratch area) / 0 h scratch area × 100%.

[0142] The results, as shown in Table 3, indicate that PA1702 promoted the migration of HaCaT cells at various concentrations, but the overall effect was weaker than that of the positive control group and was concentration-dependent.

[0143] Table 3 Effect of PA1702 on the scratch healing rate of HaCaT cells (mean ± SD, n=3)

[0144] Group 24-hour healing rate (%) 48-hour healing rate (%) Control group (0 μg / mL) 22.3 ± 0.3 45.5 ± 0.3 Positive control group (EGF, 10 ng / mL) 52.6 ± 0.5 85.3 ± 0.6 Low-dose group (1 μg / mL) 23.8 ± 0.3 47.2 ± 0.3 Medium dose group (5 μg / mL) 26.5 ± 0.3 51.8 ± 0.4 High-dose group (25 μg / mL) 30.2 ± 0.3 58.5 ± 0.4

[0145] 6.6 Conclusion

[0146] In vitro cell activity evaluation results showed that the modified polypeptide derivative of this invention exhibited no cytotoxicity and good biocompatibility within a concentration range of 1-25 μg / mL. This polypeptide derivative can inhibit LPS-induced NO release and the expression of inflammatory factors TNF-α, IL-6, and IL-1β in a concentration-dependent manner, exerting an anti-inflammatory effect; simultaneously, it can promote the migration of HaCaT cells, demonstrating pro-repair activity. These results are consistent with network pharmacology predictions and molecular docking verification results, confirming the anti-inflammatory and pro-repair efficacy of the modified polypeptide derivative of this invention.

[0147] Example 7: Anti-inflammatory and Repairing Facial Mask Liquid Containing PA1702 and its Efficacy Evaluation

[0148] 7.1 Formulation Composition

[0149] PA1702 0.03 wt%, Glycerin 5.0 wt%, Propylene Glycol 3.0 wt%, Sodium Hyaluronate 0.05 wt%, Xanthan Gum 0.1 wt%, p-Hydroxyacetophenone 0.5 wt%, 1,2-Hexanediol 0.5 wt%, Deionized Water to 100 wt%.

[0150] 7.2 Preparation method

[0151] Sprinkle xanthan gum evenly on the surface of deionized water and let it swell overnight to obtain a gel matrix. Add glycerin, propylene glycol, and sodium hyaluronate to the gel matrix and stir until dissolved. Add PA1702, p-hydroxyacetophenone, and 1,2-hexanediol and stir until mixed. Filter the above mixture to remove bacteria and fill it into mask bags, each bag containing 25 mL of mask liquid.

[0152] 7.3 Effect Test

[0153] 7.3.1 Subjects

[0154] We are recruiting 30 healthy volunteers aged 25-50 with mild facial skin inflammation, sensitivity, or impaired skin barrier function. Exclusion criteria: those who have used corticosteroids or immunosuppressants within the past month; those with obvious facial infections, wounds, or skin diseases; and pregnant or breastfeeding women.

[0155] 7.3.2 Test Method

[0156] A self-controlled design was used. Subjects applied an anti-inflammatory and repairing mask containing 0.03% PA1702 to the left side of their face (sample group), while the right side used a blank base mask without PA1702 (placebo group: a base mask with identical ingredients and formula except for the absence of PA1702). Each mask was applied for 15-20 minutes, three times a week for four consecutive weeks. Skin parameters were measured before the experiment (week 0) and after the experiment (weeks 2 and 4).

[0157] The testing indicators include:

[0158] Transepidermal water loss (TEWL): Measured using a water loss tester, it reflects the skin barrier function; the lower the value, the better the barrier function.

[0159] Erythema Index (EI): Measured using a skin colorimeter, it reflects the degree of skin inflammation; the lower the value, the milder the inflammation.

[0160] Skin moisture content: Measured using a skin moisture meter, it reflects the skin's hydration status; the higher the value, the higher the moisture content.

[0161] Subject self-assessment questionnaire: including indicators such as skin soothing sensation, moisturizing sensation, improvement in redness, and reduction in stinging sensation, scored from 1 to 5 (1 point = ineffective, 5 points = significant effect).

[0162] 7.3.3 Test Results

[0163] The results of the tests on skin barrier function, inflammation level, and hydration status are shown in Table 4. Compared with the placebo group, the mask liquid sample group containing PA1702 showed significant improvement in all test indicators.

[0164] Regarding transepidermal water loss (TEWL), after 4 weeks of use, the TEWL value in the sample group decreased from 18.3 g / h·m² to 10.2 g / h·m², with an improvement rate of 44.3%, while the placebo group only improved by 10.8%. The difference between the two groups was extremely significant (p < 0.01), indicating that PA1702 can effectively repair the skin barrier function.

[0165] Regarding the erythema index (EI), the EI value in the sample group decreased from 12.8 to 7.5 after 4 weeks of use, with an improvement rate of 41.4%, while the placebo group only improved by 11.1%. The difference between the two groups was extremely significant (p < 0.01), indicating that PA1702 can significantly reduce skin inflammation.

[0166] Regarding skin hydration, after 4 weeks of use, the hydration level in the sample group increased from 37.9 au to 55.2 au, an increase of 45.6%, while the placebo group only increased by 8.6%. The difference between the two groups was extremely significant (p < 0.01), indicating that PA1702 can significantly improve skin hydration.

[0167] Table 4 Summary of changes in skin parameters (mean ± SD, n=30)

[0168] Group index Week 0 Week 2 Week 4 Improvement / Enhancement Rate (%) placebo group TEWL (g / h·m²) 18.5 ± 2.1 17.2 ± 2.0 16.5 ± 1.9 10.8 Sample group TEWL (g / h·m²) 18.3 ± 2.0 13.5 ± 1.6 10.2 ± 1.4 44.3** placebo group EI 12.6 ± 1.5 11.8 ± 1.4 11.2 ± 1.3 11.1 Sample group EI 12.8 ± 1.4 9.2 ± 1.1 7.5 ± 0.9 41.4** placebo group Moisture content (au) 38.2 ± 3.5 40.1 ± 3.2 41.5 ± 3.0 8.6 Sample group Moisture content (au) 37.9 ± 3.3 48.5 ± 3.0 55.2 ± 2.8 45.6**

[0169] Note: Compared with the placebo group, **p < 0.01.

[0170] The subjects' self-reported results are shown in Table 5. The sample group scored 4.5, 4.3, 4.2, and 4.4 on the four indicators of skin soothing, moisturizing, redness improvement, and stinging reduction, respectively. These scores were significantly higher than those of the placebo group (3.0, 3.2, 2.8, and 3.1, respectively) (p < 0.05), indicating that the subjects' subjective perception of the mask's effects was consistent with the objective test results.

[0171] Table 5. Subject self-report results (Week 4, 1-5 points, mean ± SD, n=30)

[0172] Group Skin soothing sensation Moisturizing sensation Improvement of redness The stinging sensation has lessened placebo group 3.0 ± 0.6 3.2 ± 0.5 2.8 ± 0.7 3.1 ± 0.6 Sample group 4.5 ± 0.5* 4.3 ± 0.6* 4.2 ± 0.6* 4.4 ± 0.5*

[0173] Note: *p < 0.05 compared to the placebo group.

[0174] 7.3.4 Safety Evaluation

[0175] Throughout the experiment, no significant adverse reactions, such as redness, stinging, itching, or rash, were observed in any of the participants. Two of the 30 participants experienced a slight warming sensation upon first use, which subsided spontaneously after approximately 5 minutes and did not affect subsequent use. This indicates that the mask liquid containing PA1702 has good skin tolerability and safety.

[0176] 7.4 Conclusion

[0177] This anti-inflammatory and repairing facial mask liquid, containing 0.03% PA1702, significantly reduced transepidermal water loss, decreased erythema index, and increased skin hydration after four weeks of continuous use. Subject self-report results were consistent with objective indicators. This mask liquid offers comprehensive anti-inflammatory, skin barrier repair, and moisturizing effects, with good safety profile, making it suitable for sensitive skin repair, post-medical aesthetic procedures, and daily anti-inflammatory and repair care.

[0178] Comparative Example 1: Stability Comparison of PA1702 and PA1701

[0179] To verify the effect of the conservative amino acid substitution modification strategy of this invention on improving peptide stability, parallel experiments were conducted on the modified peptide derivative PA1702 and the unmodified natural peptide PA1701 to compare their stability, deamide tendency, and thermal stability in serum. The selection criteria for these indicators are as follows: serum stability directly reflects the peptide's resistance to protease degradation under physiological conditions and is a core indicator for assessing in vivo half-life and bioavailability; deamide tendency targets the direct goal of this invention's modification (N→Q substitution) and is used to verify whether the modification successfully eliminated the deamide site; thermal stability reflects the conformational stability of the peptide and has practical guiding significance for product storage and transportation.

[0180] 1. Serum stability assay

[0181] Significance of the indicator: Serum contains various proteases (such as carboxypeptidase and aminopeptidase). The degradation rate of polypeptides in serum directly reflects their half-life and bioavailability in vivo. The better the serum stability, the longer the polypeptide remains in vivo, and the more prolonged the drug effect.

[0182] Experimental Methods: 100 μg / mL of PA1702 and PA1701 were mixed with an equal volume of mouse serum and incubated in a 37℃ water bath. 100 μL samples were taken at 0, 1, 2, 4, 8, 12, and 24 h. The reaction was immediately terminated by adding 200 μL of acetonitrile. The supernatant was collected by centrifugation, and the remaining peptide content was detected by reversed-phase high-performance liquid chromatography (RP-HPLC). The residual rate (%) at each time point was calculated. Three replicates were set for each group.

[0183] Results: As shown in Table 6, PA1701 degraded rapidly in serum, with a residual rate of only 45.8% after 4 h of incubation, decreasing to 31.2% after 8 h, and only 21.6% after 24 h. In contrast, PA1702 exhibited significant resistance to degradation, with a residual rate of 88.5% after 4 h of incubation, 80.3% after 8 h, and still maintaining 70.2% after 24 h. The calculated half-life of PA1701 in serum was approximately 4.5 h, while the half-life of PA1702 was approximately 18.5 h, representing a fourfold increase.

[0184] Table 6. Comparison of serum stability between PA1702 and PA1701

[0185] Time (h) PA1701 Residual Rate (%) PA1702 Residual Rate (%) 0 100.0 ± 0.0 100.0 ± 0.0 1 82.5 ± 7.5 96.2 ± 6.8 2 65.3 ± 8.2 92.8 ± 7.5 4 45.8 ± 7.8 88.5 ± 7.2 8 31.2 ± 8.5 80.3 ± 8.0 12 25.6 ± 8.0 74.6 ± 8.5 24 21.6 ± 7.5 70.2 ± 8.8

[0186] 2. Prediction and Verification of Deamidation Tendency

[0187] Significance of the indicator: Deamidation is one of the most common chemical degradation pathways in peptides. Asparagine (N) is prone to deamidation under physiological conditions, generating aspartic acid or isoaspartic acid, leading to conformational changes and decreased activity of the peptide. The core modification strategy of this invention is to replace the easily deamidated asparagine (N) at position 13 of PA1701 with the more stable glutamine (Q). Therefore, deamidation tendency detection must be used to verify whether the modification has achieved the expected effect.

[0188] Experimental Methods: The deamidation tendency of PA1701 and PA1702 was predicted using the NetAmide 2.0 online server, with a focus on analyzing the deamidation risk of asparagine (N) at position 13 in PA1701 and glutamine (Q) at the corresponding position in PA1702. The prediction score ranged from 0 to 1, with higher scores indicating a greater likelihood of deamidation modification. Further verification was achieved through accelerated stability experiments: PA1701 and PA1702 were dissolved in PBS buffer (pH 7.4, 0.01 M) and incubated at 40°C for 14 days. The proportion of deamidation products at each time point was detected using HPLC-MS.

[0189] Results: As shown in Table 7, the deamidation prediction score for asparagine (N) at position 13 in PA1701 was 0.87, indicating a high-risk site. After replacing it with glutamine (Q), the deamidation prediction score for the corresponding site in PA1702 decreased to 0.11, indicating a low-risk site. Accelerated stability testing showed that after 14 days of incubation, approximately 28.5% of deamidation products (aspartic acid and isoaspartic acid) were detected in PA1701, while no significant deamidation products were detected in PA1702 (deamidation product ratio <0.5%). This result is consistent with the prediction, confirming that the conserved amino acid substitution effectively eliminated the deamidation risk.

[0190] Table 7. Prediction and Verification of Deamide Tendency of PA1702 and PA1701

[0191] sample site amino acids Deamidation prediction score Risk level Percentage of deamide products after 14 days (%) PA1701 13 Asn(N) 0.87 High risk 28.5 ± 2.5 PA1702 13 Gln(Q) 0.11 Low risk < 0.5

[0192] 3. Thermal stability determination

[0193] Significance of the indicator: Thermal stability is an important indicator of peptide conformational stability, reflecting the peptide's ability to resist denaturation and aggregation under high-temperature conditions. Better thermal stability means a lower risk of denaturation during storage and transportation, directly impacting the shelf life of cosmetics and pharmaceutical products. Furthermore, the elimination of deamidation sites may affect the overall conformation of the peptide, necessitating supplementary verification through thermal stability testing.

[0194] Experimental methods: The thermal denaturation temperature (Tm) of PA1701 and PA1702 was determined by differential scanning calorimetry (DSC). The samples were dissolved in PBS buffer (pH 7.4) at a concentration of 1 mg / mL, and the temperature was increased from 20℃ to 100℃ at a rate of 1℃ / min. The thermal denaturation curves were recorded.

[0195] Results: As shown in Table 8, the thermal denaturation temperature (Tm) of PA1701 was 48.2℃, while that of PA1702 was 56.5℃, an increase of 8.3℃. These results indicate that conserved amino acid substitution enhances the thermal stability of the peptides, making them less prone to conformational changes and aggregation under high-temperature conditions.

[0196] Table 8 Comparison of thermal stability between PA1702 and PA1701

[0197] sample Tm (°C) PA1701 48.2 ± 0.8 PA1702 56.5 ± 0.7

[0198] 4. Conclusion

[0199] Through the stability comparison experiment of Example 1, the following conclusions can be drawn:

[0200] (1) Serum stability: The half-life of PA1702 in serum (about 18.5 h) is about 4 times that of PA1701 (about 4.5 h), and the 24 h residual rate (70.2%) is significantly higher than that of PA1701 (21.6%), indicating that PA1702 has stronger resistance to protease degradation, a longer half-life in vivo, and higher bioavailability.

[0201] (2) Deamidation stability: After replacing asparagine (N) at position 13 with glutamine (Q), the deamidation prediction score decreased from 0.87 (high risk) to 0.11 (low risk). The accelerated stability test at 40℃ confirmed that PA1702 hardly underwent deamidation modification (product ratio <0.5%), while the deamidation product ratio of PA1701 was as high as 28.5%. This result directly verifies the effectiveness of the modification strategy of this invention and successfully eliminates the deamidation defect of natural peptides.

[0202] (3) Thermal stability: The heat denaturation temperature of PA1702 (56.5℃) is 8.3℃ higher than that of PA1701 (48.2℃), which has better thermal stability, which is beneficial to the storage and transportation of the product and extends the shelf life.

[0203] The above results fully demonstrate that the conservative amino acid substitution modification strategy (N→Q) adopted in this invention effectively solves the technical defect of poor stability of natural peptides, comprehensively improving the stability of PA1702 from three dimensions: chemical stability (deamidation), biological stability (serum), and physical stability (heat), achieving unexpected technical effects. This improved stability lays a solid foundation for its application in the cosmetics and pharmaceutical fields.

[0204] Comparative Example 2: Comparison of anti-inflammatory activities (NO release inhibition) between PA1702 and PA1701

[0205] To verify the effect of the conservative amino acid substitution modification strategy of this invention on enhancing the anti-inflammatory activity of the peptide, parallel experiments were conducted on the modified peptide derivative PA1702 and the unmodified natural peptide PA1701 to compare their inhibitory activities on LPS-induced NO release in RAW264.7 cells. The experimental method was the same as in Example 6.3, with three dosage groups (1, 5, and 25 μg / mL) for both PA1701 and PA1702, and dexamethasone (10 μmol / L) as the positive control group.

[0206] 1. Experimental Results

[0207] The results are shown in Table 9. The NO content in the LPS model group (26.8 ± 0.3 μmol / L) was significantly higher than that in the blank control group (2.1 ± 0.1 μmol / L) (p < 0.001). Both PA1701 and PA1702 inhibited NO release in a concentration-dependent manner, but PA1702 showed significantly better inhibitory effects than PA1701 at all concentrations.

[0208] At low doses (1 μg / mL), the inhibition rates of PA1701 and PA1702 were 0.5% and 0.8%, respectively, with no significant inhibitory effect and no statistically significant difference (p > 0.05).

[0209] At a medium dose (5 μg / mL), the inhibition rate of PA1701 was 8.6%, while that of PA1702 was 14.2%. The inhibitory effect of PA1702 was approximately 1.7 times that of PA1701, and there was a significant difference between the two (p < 0.05).

[0210] At high doses (25 μg / mL), the inhibition rate of PA1701 was 24.7%, while that of PA1702 was 65.6%. The inhibitory effect of PA1702 was approximately 2.7 times that of PA1701, and the difference between the two was highly significant (p < 0.01).

[0211] Table 9 Comparison of the inhibitory effects of PA1702 and PA1701 on LPS-induced NO release in RAW 264.7 cells

[0212] Group Concentration (μg / mL) NO content (μmol / L, mean ± SD) Inhibition rate (%) Blank control group - 2.1 ± 0.1 - LPS model group - 26.8 ± 0.3 - Positive control group (dexamethasone) 10 μmol / L 8.9 ± 0.1 72.5 PA1701 1 26.6 ± 0.3 0.5 PA1701 5 24.5 ± 0.4 8.6 PA1701 25 20.5 ± 0.5 24.7 PA1702 1 26.6 ± 0.2 0.8 PA1702 5 23.3 ± 0.2 14.2* PA1702 25 10.6 ± 0.2 65.6**

[0213] 2. Conclusion

[0214] The following conclusions can be drawn from the comparative experiment on the anti-inflammatory activity of Comparative Example 2:

[0215] Under the same concentration conditions, PA1702 showed significantly better inhibitory activity than PA1701 in inhibiting LPS-induced NO release from RAW 264.7 cells. In particular, at a high dose (25 μg / mL), the inhibition rate of PA1702 (65.6%) was approximately 2.7 times that of PA1701 (24.7%), which was highly significant (p < 0.01).

[0216] These results fully demonstrate that the conservative amino acid substitution modification strategy (N→Q) adopted in this invention not only improves the stability of the peptide (see Comparative Example 1) but also significantly enhances its anti-inflammatory activity. The substitution of a single amino acid resulted in a substantial increase in activity, achieving unexpected technical effects.

[0217] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

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

1.

2. The modified Periplaneta americana polypeptide derivative according to claim 1, characterized in that, The polypeptide derivative is obtained by replacing the asparagine at position 13 of the native polypeptide SEQ ID NO: 2 with glutamine.

3. A method of producing the modified Periplaneta americana polypeptide derivative of claim 1 or 2, characterized in that, Prepared by Fmoc solid-phase polypeptide synthesis and purified by HPLC to a purity of ≥98%.

4. A polypeptide composition, characterized in that, The active ingredient comprises the modified Periplaneta americana polypeptide derivative of claim 1 or 2. The composition is a cosmetic composition or a pharmaceutical composition. The composition further comprises a cosmetically or pharmaceutically acceptable base, carrier, adjuvant or other inactive ingredient.

5. The polypeptide composition of claim 4, wherein, The mass percentage content of the modified Periplaneta americana polypeptide derivative is 0.001%-5%.

6. The polypeptide composition of claim 4 or 5, wherein, The composition is a composition with anti-inflammatory, skin barrier repair and / or wound healing promotion activity.

7. The polypeptide composition of claim 4, wherein The dosage form of the composition is a serum, a lyophilized powder, a mask liquid, a gel, a spray or a cream.

8. The polypeptide composition of claim 7, wherein, The composition is an anti-inflammatory repair mask liquid comprising the modified Periplaneta americana polypeptide derivative 0.03 wt%, glycerol 5.0 wt%, propylene glycol 3.0 wt%, sodium hyaluronate 0.05 wt%, xanthan gum 0.1 wt%, p-hydroxyacetophenone 0.5 wt%, 1,2-hexanediol 0.5 wt%, and deionized water added to 100 wt%.

9. Use of a modified Periplaneta americana polypeptide derivative according to claim 1 or 2 or a polypeptide composition according to any one of claims 4 to 8, characterized in that, The use is: The use in the preparation of a cosmetic or a pharmaceutical with anti-inflammatory, skin barrier repair and / or wound healing promotion activity.

10. Use of a modified Periplaneta americana polypeptide derivative according to claim 1 or 2 or a polypeptide composition according to any of claims 4-8, characterized in that, The use is: The use in the preparation of a cosmetic or a pharmaceutical with inhibitory activity on NO release and expression of inflammatory factors TNF-α, IL-6, IL-1β or promotion of HaCaT cell migration activity.

Images