Modified melittin peptide, product comprising modified melittin peptide and application of modified melittin peptide

Abstract

The invention discloses a modified melittin peptide, a product containing the modified melittin peptide and application, and belongs to the technical field of biological medicine. The amino acid sequence of the modified melittin is shown as Seq ID No: 2, the molecular weight of the modified melittin is 3123.5 Da, and the isoelectric point of the modified melittin is 12.64. The invention further discloses application of the modified melittin peptide in preparation of drugs or cosmetics for inhibiting inflammation and relieving skin erythema and itching. The invention also discloses application of the modified melittin peptide in preparation of drugs or cosmetics for enhancing cell activity and promoting tissue repair. The melittin modified peptide can significantly reduce the cytotoxicity of natural melittin and significantly enhance the anti-inflammatory activity and tissue repair activity of the melittin, has no sensitization and phototoxicity, and can provide key technical support for research and development of new-generation efficient and safe anti-inflammatory repair drugs, cosmetic products and related biological materials.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a melittin modified peptide with anti-inflammatory and repair activities, and products and applications thereof. Background Art

[0002] Medical research believes that the occurrence of sensitive skin is a complex process involving the skin barrier-neurovascular-immune inflammatory network. Exogenous stimuli first activate keratinocytes, triggering the release of cytokines (such as IL-1, IL-6 and TNF-a), chemokines (such as IL-8) and antimicrobial peptides (such as defensins), causing an inflammatory response. In response to inflammatory / stress stimuli, inflammatory pathways such as NF-κB are activated to produce pro-inflammatory mediators, thereby maintaining and exacerbating the inflammatory process. At the same time, barrier damage increases the sensitivity of sensory nerves, and stimulants act on the transient receptor potential (TRP) family, such as TRPV1, inducing a large amount of Ca 2+ Influx activates sensory nerve endings, causing burning and itching sensations in the skin. TRPV1 activation further triggers the release of endothelin and vascular endothelial growth factor, leading to vasodilation and increased vasoactive responses. The continuous release of inflammatory factors interferes with barrier repair, causing the allergic reaction to persist and worsen, creating a vicious cycle.

[0003] In recent years, a surge of ingredients with allergy-relief properties have emerged on the market. These ingredients, primarily derived from natural products or analogs of the human extracellular matrix, work through barrier repair, moisture retention, or anti-inflammatory mechanisms. However, existing allergy-relief natural botanical ingredients lack effective targets and pharmacological mechanisms. Therefore, there is an urgent need for products with mild, non-irritating ingredients and superior anti-inflammatory, antipruritic, and allergy-relief effects to meet consumer needs for improving skin sensitivity and repairing damaged skin barriers.

[0004] Melittin, a natural biotoxin, is the main active ingredient in bee venom. It is an amphiphilic polypeptide composed of 26 amino acids. The amino acid residue sequence of its primary structure is Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2 (Seq ID No: 1). It has a wide range of pharmacological effects, such as anti-inflammatory, antibacterial, antiviral, anti-radiation, and anti-tumor. Its mechanism of action involves cell membrane penetration, inhibition of inflammatory factors (such as TNF-α and IL-6), and activation of repair signaling pathways such as PI3K / Akt. The anti-inflammatory activity of melittin is 100 times that of hydrocortisone, and its analgesic potency is 40% that of morphine. It has significant inhibitory effects on a variety of Gram-negative and Gram-positive pathogens, and is particularly effective against penicillin-resistant Staphylococcus aureus. In addition, melittin has a selective killing effect on tumor cells, inducing tumor cell apoptosis and inhibiting tumor angiogenesis. In clinical applications, melittin has been studied for the treatment of diseases such as rheumatoid arthritis, neuralgia, hypertension, tumors, and herpes zoster, showing significant therapeutic potential. However, natural melittin has certain limitations in practical applications, such as strong hemolytic activity and nonspecific cytotoxicity, as well as volatility, rapid in vivo degradation, and a short half-life, which seriously restrict its promotion and use in clinical treatment and biomaterials.

[0005] Therefore, existing natural melittin still has obvious inconveniences and defects in its application, and further improvement is urgently needed. The development of a novel melittin-modified peptide with anti-inflammatory and repairing activity, as well as products and applications thereof, that can mitigate the toxic effects of melittin on normal tissue cells while enhancing its anti-inflammatory and repairing effects, achieving a toxicity-reducing and efficacy-enhancing effect, is crucial for promoting the clinical application of melittin and its application in anti-inflammatory and repairing cosmetic products. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the limitations of natural melittin and provide a melittin modified peptide to reduce the toxic effects of melittin on normal tissue cells, while enhancing its anti-inflammatory and repair activity effects, thereby achieving the effect of reducing toxicity and increasing efficacy.

[0007] In order to solve the above technical problems, the present invention provides a melittin modified peptide, the amino acid sequence of which is shown in Seq ID No: 2.

[0008] The molecular weight of the melittin modified peptide is 3123.5 Da, and the isoelectric point is 12.64.

[0009] Another technical problem to be solved by the present invention is to provide a use of the melittin modified peptide in the preparation of medicines or cosmetics for inhibiting inflammation and relieving skin erythema and itching.

[0010] Furthermore, the bee venom modified peptide can inhibit the expression levels of NO, IL-6, TNF-α and IL-8, reduce neutrophils or inhibit the secretion of IL-1a, and is used to inhibit inflammation of sensitive skin and relieve skin erythema and itching.

[0011] Another technical problem to be solved by the present invention is to provide an application of the melittin modified peptide in the preparation of medicines or cosmetics for enhancing cell activity and promoting tissue repair.

[0012] Furthermore, the melittin modified peptide can enhance the activity of BJ cells and improve the cell healing rate of HaCat cells, and is used to enhance cell activity and promote tissue repair.

[0013] In addition, the present invention also provides a cosmetic comprising the aforementioned melittin modified peptide, which can reduce neutrophils or inhibit the secretion of IL-1a by inhibiting the expression levels of NO, IL-6, TNF-α, and IL-8, and is used to inhibit inflammation and relieve skin erythema and itching for sensitive skin.

[0014] And / or, the melittin modified peptide can also enhance BJ cell viability and improve the cell healing rate of HaCat cells, and is used to enhance cell activity and promote tissue repair.

[0015] The present invention also provides a medicine comprising the aforementioned modified melittin peptide and pharmaceutically acceptable excipients or auxiliary ingredients. The modified melittin peptide can reduce neutrophils or inhibit the secretion of IL-1a by inhibiting the expression levels of NO, IL-6, TNF-α, and IL-8, and is used to inhibit inflammation and relieve skin erythema and itching in sensitive skin.

[0016] And / or, the melittin modified peptide can enhance BJ cell viability and improve the cell healing rate of HaCat cells, and is used to enhance cell activity and promote tissue repair.

[0017] After adopting such a design, the present invention has at least the following advantages:

[0018] The modified melittin peptide of the present invention is obtained by optimizing the amino acid sequence and modifying the functional groups of natural melittin. This modified melittin peptide can reduce the cytotoxicity and sensitization of natural melittin, and is significantly superior to natural melittin in terms of anti-inflammatory and tissue repair activities. Compared with natural melittin, it has the effect of reducing toxicity and increasing efficacy. Experiments have shown that the present invention can effectively inhibit inflammatory responses and accelerate skin wound healing at micromolar concentrations, and has excellent stability. It provides key technical support for the development of a new generation of highly effective and safe anti-inflammatory and repair drugs, cosmetics, and related biomaterials, and is used to meet consumer needs for improving skin sensitivity and repairing damaged skin barriers. It is of vital significance for promoting the application of melittin in clinical pharmaceutical or cosmetic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0020] Figure 1-3 The cytotoxicity test results of Example 1 of the melittin modified peptide of the present invention are shown in FIG.

[0021] Figure 4-6 The anti-inflammatory activity test results of the Raw 264.7 cells of Example 2 of the melittin modified peptide of the present invention are shown in FIG.

[0022] Figure 7 This is the anti-inflammatory and soothing activity test result of HaCaT cells of Example 3 of the melittin modified peptide of the present invention;

[0023] Figure 8 The results of the BJ cell proliferation activity test of Example 4 of the melittin modified peptide of the present invention are as follows;

[0024] Figure 9-10 This is the HaCaT cell scratch repair activity test result of Example 5 of the melittin modified peptide of the present invention;

[0025] Figure 11 The zebrafish anti-inflammatory and soothing test results of Example 6 of the melittin modified peptide of the present invention;

[0026] Figure 12-14 This is the 3D skin soothing test result of Example 7 of the melittin modified peptide of the present invention;

[0027] Figure 15 The sensitization performance test results of Example 8 of the melittin modified peptide of the present invention are shown in FIG.

[0028] Figure 16-17 This is the phototoxicity test result of Example 9 of the melittin modified peptide of the present invention. DETAILED DESCRIPTION

[0029] Exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0030] Melittin, a natural biotoxin, is an amphiphilic polypeptide composed of 26 amino acids. Its primary structure has the following amino acid sequence: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2. It is abbreviated as GIGAVLKVLTTGLPALISWIKRKRQQ (Seq ID No: 1). It has a molecular weight of 2847.43 Da and an isoelectric point of 12.43. It is referred to herein as NWT01AM2601.

[0031] Based on the structure of natural melittin, the present invention uses conventional solid-phase synthesis methods to obtain the following two melittin modified peptides:

[0032] The molecular weight of the melittin modified peptide NWT01AM2602 is 3123.5 Da, the isoelectric point is 12.64, and the primary structure of the entire sequence is GIGAVWKMLRTGLPARWSWIKRKRQQ (Seq ID No: 2).

[0033] The molecular weight of the melittin modified peptide NWT01AM2603 is 3236.75 Da, the isoelectric point is 13.01, and the primary structure of the full sequence is GIGARWKMLRTGWPARWSWSKRKRQH (Seq ID No: 3).

[0034] This application conducts the following experiments on NWT01AM2601, NWT01AM2602, and NWT01AM2603, and the specific examples are as follows:

[0035] Example 1 Cytotoxicity Test

[0036] 1.1 Reagents and Materials

[0037] Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), DPBS, dimethyl sulfoxide (DMSO), and CCK-8.

[0038] 1.2 Instruments

[0039] CO2 incubator, biological safety cabinet, inverted microscope, and microplate reader.

[0040] 1.3 Cell lines

[0041] Mouse mononuclear macrophage leukemia cells (Raw264.7).

[0042] 1.4 Samples to be tested

[0043] Sample groups: NWT01AM2601, NWT01AM2602, NWT01AM2603, test concentrations: 300 μg / mL, 100 μg / mL, 30 μg / mL, 10 μg / mL, 3 μg / mL, 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL.

[0044] Solvent control group: DMEM complete medium;

[0045] Positive control group: DMEM complete medium containing 10% DMSO;

[0046] Zero adjustment group: DMEM complete medium (no cells).

[0047] 1.5 Experimental Methods

[0048] Raw264.7 cells were cultured at a density of 2.5 × 10 5 100 μL of cell suspension (100 cells / mL) was seeded into each well of a 96-well plate and cultured. When the cell confluence reached approximately 40%, the supernatant was removed and samples of varying concentrations were added sequentially for treatment with bioactive substances. After 24 hours of treatment, CCK-8 reagent was added at a ratio of 1:10. After 40 minutes, the absorbance of each well at 450 nm was measured using a microplate reader. Cell viability was calculated according to the following formula:

[0049]

[0050] 1.6 Results

[0051] The CCK-8 assay is a method for detecting cell survival and growth. The measured OD value is proportional to cell activity.

[0052] The results of the effects of the test samples on the cytotoxicity of Raw264.7 cells are shown in Table 1. Figures 1 to 3 The results showed that NWT01AM2601, NWT01AM2602, and NWT01AM2603 maintained cell viability greater than 90% in RAW 264.7 cells at concentrations of 5.7 μg / mL, 30.2 μg / mL, and 100 μg / mL, respectively, showing no significant cytotoxicity. Furthermore, the cytotoxicity of NWT01AM2602 and NWT01AM2603 was significantly lower than that of NWT01AM2601.

[0053] Table 1 Results of relative cell viability test of samples

[0054]

[0055] Example 2 Anti-inflammatory activity test of Raw264.7 cells

[0056] 2.1 Reagents and Materials

[0057] High-glucose medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, lipopolysaccharide (LPS), IL-1α (Mouse) ELISA kit, IL-6 (Mouse) ELISA kit, NO detection kit.

[0058] 2.2 Instruments

[0059] CO2 incubator, biological safety cabinet, inverted microscope, and microplate reader.

[0060] 2.3 Cell lines

[0061] Mouse mononuclear macrophage leukemia cells (Raw264.7).

[0062] 2.4 Samples to be tested

[0063] Sample group: NWT01AM2601 and NWT01AM2602, tested at concentrations of 0.5 μg / mL, 2.5 μg / mL, and 5 μg / mL; NWT01AM2603, tested at concentrations of 50 μg / mL and 100 μg / mL;

[0064] Blank control group: DMEM medium containing 1% FBS and 1% P / S;

[0065] Model group: 25 ng / mL LPS;

[0066] Positive control group: 0.5 μg / mL dexamethasone.

[0067] 2.5 Experimental Methods

[0068] Raw264.7 cells were cultured at a density of 1.5 × 10 5 500 μL of a 1.5-cell / mL cell suspension was seeded into each well of a 48-well plate and cultured until the cell confluence reached approximately 40%. The supernatant was removed. The test samples were then added sequentially for bioactive substance treatment. After 24 hours of treatment, the supernatant was collected and centrifuged at 1000 rpm for 20 minutes. The supernatant was then assayed for NO and cytokine (IL-6, TNF-α) concentrations according to the ELISA kit instructions.

[0069] 2.6 Results

[0070] Lipopolysaccharide (LPS) can activate the synthesis and release of various cytokines and inflammatory mediators through the cell signaling system. The accumulation of inflammatory factors can cause vasodilation, redness, swelling, stinging, and itching, resulting in skin sensitivity. In this experiment, 25 ng / mL LPS was used to induce the establishment of an anti-inflammatory activity evaluation model.

[0071] The experimental results are as follows Figure 4-6 As shown in the figure, compared with the blank control, the expression levels of NO, TNF-α and IL-6 in Raw264.7 cells were significantly increased after LPS induction, indicating that the Raw264.7 cell inflammation model was successfully established.

[0072] In the sample group, NWT01AM2601 at a concentration of 0.5 μg / mL could significantly inhibit the expression levels of IL-6 and TNF-α.

[0073] In the sample group, NWT01AM2602 at a concentration of 2.5 μg / mL could significantly inhibit the expression level of IL-6; NWT01AM2602 at a concentration of 5 μg / mL could significantly inhibit the expression levels of NO, IL-6 and TNF-α, showing strong anti-inflammatory activity.

[0074] In the sample group, NWT01AM2603 at a concentration of 50 μg / mL could significantly inhibit the expression level of IL-6; NWT01AM2603 at a concentration of 100 μg / mL could significantly inhibit the expression levels of NO and IL-6.

[0075] In summary, NWT01AM2602 has shown strong anti-inflammatory activity at micromolar concentrations, is non-cytotoxic, and has anti-inflammatory and soothing effects.

[0076] Example 3 HaCat cell anti-inflammatory and soothing activity test

[0077] 3.1 Reagents and Materials

[0078] Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), dual-phosphate-buffered saline (DPBS), lipopolysaccharide (LPS), PolyI:C, and IL-8 (Human) ELISA kit.

[0079] 3.2 Instruments

[0080] CO2 incubator, biological safety cabinet, inverted microscope, and microplate reader.

[0081] 3.3 Cell lines

[0082] Human immortalized keratinocytes (HaCaT).

[0083] 3.4 Samples to be tested

[0084] Dosage group: NWT01AM2602, tested at concentrations of 2.5 μg / mL, 5 μg / mL, and 10 μg / mL;

[0085] Model group: 30 μg / mL LPS + 30 μg / mL Poly (I:C);

[0086] Blank control group: DMEM medium containing 1% FBS.

[0087] 3.5 Experimental methods

[0088] HaCaT cells were cultured at a density of 5 × 10 4 500 μL of a cell suspension (100 cells / mL) was seeded into each well of a 48-well plate and cultured until the cell confluence reached approximately 50%. The supernatant was removed. The test samples were then added sequentially for bioactive substance treatment. After 24 hours of treatment, the supernatant was collected and centrifuged at 1000 rpm for 20 minutes. The supernatant was then assayed for IL-8 concentration according to the ELISA kit instructions.

[0089] 3.6 Results

[0090] IL-8 is a specific inflammatory factor for eczema, a typical inflammatory skin disease. The release of IL-8 further activates specific immune responses, mediating the inflammatory cascade and ultimately leading to symptoms such as erythema and itching. Cosmetics that inhibit IL-8 release can alleviate these symptoms. This method uses a polyI:C and LPS-induced keratinocyte (HaCaT) in vitro model to evaluate anti-inflammatory activity.

[0091] The experimental results are as follows Figure 7 As shown, compared with the blank control, the combined induction of PolyI:C and LPS significantly increased IL-8 expression in HaCaT cells, indicating a successful inflammatory model. In the treatment group, NWT01AM2602 significantly suppressed IL-8 expression at a concentration of 2.5 μg / mL, demonstrating strong anti-inflammatory activity. This suggests that the melittin-modified peptide NWT01AM2602 can be used to inhibit inflammation and relieve skin erythema and itching.

[0092] Example 4 Proliferation Activity Test

[0093] 4.1 Reagents and Materials

[0094] High glucose medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, CCK-8.

[0095] 4.2 Instruments

[0096] CO2 incubator, biological safety cabinet, inverted microscope, and microplate reader.

[0097] 4.3 Cell lines

[0098] Human skin fibroblasts (BJ).

[0099] 4.4 Samples to be tested

[0100] Sample group: NWT01AM2602, tested concentrations: 0.1μg / mL, 0.3μg / mL, 1μg / mL, 3μg / mL, 10μg / mL, 30μg / mL;

[0101] Positive control group: DMEM medium with 10% FBS;

[0102] Blank control group: DMEM medium containing 1% FBS.

[0103] 4.5 Experimental Methods

[0104] BJ cells were cultured at a density of 1×10 4 100 μL of cell suspension per well of cells / mL was inoculated into a 96-well cell culture plate and cultured. When the cell fusion rate reached 20%, the supernatant was removed. The samples to be tested were added sequentially for treatment with bioactive substances. After administration, the 96-well plate was placed in a CO2 incubator (37°C, 5% CO2) and cultured for 72 hours. After 72 hours of treatment, CCK-8 reagent was added at a ratio of 1:10. After 40 minutes, the absorbance of each well at 450 nm was measured using a microplate reader, and the cell viability was calculated according to the following formula:

[0105]

[0106] 4.6 Experimental Results

[0107] This experiment selected the BJ proliferation activity evaluation model. Figure 8 As shown, NWT01AM2602 at a concentration of 1 μg / mL in the sample group can significantly enhance the relative viability of BJ cells in a concentration-dependent manner, exhibiting strong proliferation activity.

[0108] Example 5 HaCat cell scratch repair activity

[0109] 5.1 Reagents and Materials

[0110] Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, and epidermal growth factor (EGF).

[0111] 5.2 Instruments

[0112] CO2 incubator, biological safety cabinet, inverted microscope, microplate reader, 2 Well Culture-inserts for self-insertion (Ibidi, 80209).

[0113] 5.3 Cell lines

[0114] Human immortalized keratinocytes (HaCaT).

[0115] 5.4 Samples to be tested

[0116] Sample group: NWT01AM2602, tested at concentrations of 1 μg / mL, 3 μg / mL, 10 μg / mL, and 30 μg / mL;

[0117] model group, 5 ng / mL EGF;

[0118] Blank control group: DMEM medium containing 0.1% FBS.

[0119] 5.5 Experimental Methods

[0120] HaCaT cells were cultured at a density of 6 × 10 5 70 μL of cells / mL cell suspension was inoculated into the scratch plug in each well for culture. When the cell fusion rate reached 100%, the supernatant was removed. The samples to be tested were added in sequence for treatment with bioactive substances. After the administration was completed, the 6-well plate was placed in a CO2 incubator (37°C, 5% CO2) for culture. At 0 h, 24 h and 48 h after administration, a 5× microscope was used to select the same field of view to take pictures and record the scratch spacing at the corresponding time. Image J was used to count the area of ​​the scratch in the photo, and the scratch healing rate was calculated using the following formula:

[0121]

[0122] 5.6 Results

[0123] The differentiation ability of keratinocytes plays a vital role in the healing process of skin wounds. The cell scratch test is a common method to detect cell migration ability.

[0124] The experimental results are as follows Figure 9 and 10 As shown, compared with the blank control, the sample group NWT01AM2602 at concentrations of 3μg / mL and 10μg / mL could significantly improve the healing rate of HaCaT cells at 24h and 48h, indicating that NWT01AM2602 has repair activity.

[0125] Example 6 Anti-inflammatory and soothing activity of zebrafish

[0126] 6.1 Reagents and Materials

[0127] Anhydrous copper sulfate, methylcellulose, indomethacin, dimethyl sulfoxide, embryo culture medium, tricaine.

[0128] 6.2 Instruments

[0129] Biochemical incubator, fluorescent microscope, and one-hundred-thousandth balance.

[0130] 6.3 Zebrafish strains

[0131] Transgenic neutrophil red fluorescent strain zebrafish Tg (Lyz: dsRed).

[0132] 6.4 Samples to be tested

[0133] Sample group: NWT01AM2602, tested at concentrations of 1 μg / mL and 2 μg / mL;

[0134] Model group: 0.8 μg / mL copper sulfate solution;

[0135] Positive group: 1 μg / mL indomethacin;

[0136] Blank control group: embryo culture medium.

[0137] 6.5 Experimental Methods

[0138] Normally developed zebrafish 3 days after fertilization (72 hpf) were selected and plated in a 24-well plate, with 12 fish per well and two replicates per group. The above solution was added to the corresponding groups, 2 mL of solution per well, and incubated in a biochemical incubator at 28°C for 1 hour. After incubation, the fish were washed twice with culture medium. After washing, the zebrafish were anesthetized with tricaine solution for 15 minutes, fixed on a slide with 5% methylcellulose, and imaged under a microscope with unified parameters and body positions. The number of neutrophils in the lateral line area of ​​each fish embryo, three-quarters of the way from the anus, was counted, and the inhibition rate was calculated according to the following formula:

[0139]

[0140] 6.6 Results

[0141] Neutrophils are the primary immune white blood cells, responsible for phagocytosis and clearing infections or harmful substances from the skin. Changes in the number of neutrophils migrating to the neuromasts of the zebrafish tail can reflect the inflammatory state in the body, thereby evaluating the anti-inflammatory and soothing effects of the raw materials.

[0142] The experimental results are as follows Figure 11As shown, the number of neutrophils in the sample group was significantly lower than that in the model group at a concentration of 1 μg / mL, and the neutrophil inhibition rate reached 39.53%, further indicating that NWT01AM2602 has anti-inflammatory and soothing activity.

[0143] Example 7 3D skin anti-inflammatory and soothing activity

[0144] 7.1 Reagents and Materials

[0145] 3D skin model kit maintenance medium, test medium, IL-1a ELISA kit, IL-8 ELISA kit, DPBS, sodium dodecyl sulfate (SLS).

[0146] 7.2 Instruments

[0147] Microplate reader (Thermo), biological safety cabinet (Thermo), etc.

[0148] 7.3 3D Skin

[0149] The 3D skin model used in this test is the Episkin large-pore skin model.

[0150] 7.4 Samples to be tested

[0151] Sample set: NWT01AM2602, test concentrations of 1 ppm and 2.5 ppm;

[0152] Model group: SLS;

[0153] Positive group: dexamethasone sodium phosphate;

[0154] Blank control group: culture medium.

[0155] 7.5 Experimental Methods

[0156] A chemically sensitive skin model was constructed using a 3D epidermal model reconstructed in vitro using SLS stimulation. Samples and a positive control were then applied to the 3D skin epidermis and incubated in an incubator at 37°C and 95% humidity. Skin culture fluid was collected and assayed for relative expression of the inflammatory cytokines IL-1a and IL-8 using ELISA kits. Data were analyzed descriptively using mean ± standard deviation, and intergroup comparisons were plotted using GraphPad Prism. One-way analysis of variance was used to compare groups. All statistical analyses were two-tailed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0157] 7.6 Results

[0158] The experimental results are as follows Figures 12 to 14 As shown, NWT01AM2602 at 1 ppm and 2.5 ppm concentrations significantly inhibited SLS-induced IL-1a secretion in a sensitive 3D skin model (p<0.0001) in a dose-dependent manner. NWT01AM2602 at 1 ppm significantly inhibited IL-8 secretion in the sensitive 3D skin model (p<0.05). At 1 ppm and 2.5 ppm, NWT01AM2602 demonstrated significant 3D tissue repair activity (p<0.0001) in a dose-dependent manner. This further demonstrates that NWTO1AM2602 at 1 ppm and 2.5 ppm has anti-inflammatory and soothing effects.

[0159] Example 8 Sensitization Performance Test

[0160] 8.1 Reagents and Materials

[0161] 1640 culture 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 (κ Isotype Ctrl Antibody), PE Mouse IgG1 (κ Isotype Ctrl Antibody).

[0162] 8.2 Instruments

[0163] CO2 incubator, biological safety cabinet, inverted microscope, flow cytometer.

[0164] 8.3 Cell lines

[0165] Human acute monocytic leukemia cells (THP-1).

[0166] 8.4 Samples to be tested

[0167] Dosage group: NWT01AM2602, sample test concentrations were 40μg / mL, 20μg / mL, 10μg / mL, and 5μg / mL. The highest test concentration of NWT01AM2602 was 1.2 times the CV75 value.

[0168] Positive control group: 6.25 μg / mL dinitrochlorobenzene (DNCB);

[0169] Negative control group: 2 mg / mL lactic acid (LA);

[0170] Blank control group: culture medium containing 1% FBS (1% FBS + 1% P / S + 1640 culture medium);

[0171] For the solvent control group, the culture medium contained 16% PBS (1% FBS+1% P / S+1640 culture medium+16% PBS).

[0172] 8.5 Experimental methods

[0173] Adjust the density of THP-1 cell suspension to 1.5 × 10 6 Cells were suspended at a concentration of 10 cells / mL. 200 μL of the cell suspension and 200 μL of the test sample at a 1:1 ratio were then inoculated into 48-well plates. After 24 hours of culture, cells were pipetted to mix thoroughly and aliquoted into two 96-well plates, each containing 150 μL. One aliquot served as the experimental group and the other as the isotype control. Fluorescence staining was then performed. The cells were washed with PBS and stained with 7-AAD staining solution to stain dead cells. The cells were then washed with PBS and stained with FITC-CD86 and PE-CD54 in the experimental group, while 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 the FITC, PE, and 7-AAD dyes was measured for each group using flow cytometry. The relative fluorescence intensity (RFI) of the test compound was calculated using the following formula.

[0174]

[0175] 8.6 Results

[0176] When allergens come into contact with the skin, dendritic cells differentiate and mature during the process of migrating to lymphoid organs, and upregulate the expression of a series of surface molecules. By staining the cell surface molecules CD86 and CD54 with fluorescent antibody dyes and measuring them with flow cytometry, it can be determined whether the test substance is allergenic.

[0177] The experimental results are as follows Figure 15As shown, THP-1 cells expressed positive CD86 and CD54 molecules in response to DNCB (RFICD86>150, RFICD54>200), with a cell viability of >50%. They expressed negative CD86 and CD54 molecules in response to LA (RFICD86<150, RFICD54<200), with a cell viability of >50%. They expressed negative CD86 and CD54 molecules in response to the solvent (RFICD86<150, RFICD54<200), with a cell viability of >90%, indicating that the cells passed the stability test. The cell viability of NWT01AM2602 at concentrations of 20 μg / mL, 10 μg / mL, and 5 μg / mL was greater than 90%, and THP-1 cells showed no positive expression of CD86 and CD54 molecules in response to NWT01AM2602, indicating that the cells were not allergenic. Therefore, it can be seen that NWT01AM2602 of the present invention is non-allergenic.

[0178] Example 9 Phototoxicity Test

[0179] 9.1 Reagents and Materials

[0180] Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, neutral red, glacial acetic acid, anhydrous ethanol, chlorpromazine hydrochloride (CPZ), and HEPES.

[0181] 9.2 Instruments

[0182] CO2 incubator, biological safety cabinet, inverted microscope, and microplate reader.

[0183] 9.3 Cell lines

[0184] Mouse embryonic fibroblasts (BALB / C 3T3).

[0185] 9.4 Samples to be tested

[0186] Sample group: NWT01AM2602, tested concentrations: 1 μg / mL, 3 μg / mL, 10 μg / mL, 30 μg / mL, 100 μg / mL, 300 μg / mL, and 1000 μg / mL;

[0187] Blank control group: DMEM medium containing 10% FBS:

[0188] Positive control group: chlorpromazine hydrochloride, test concentrations were 0.1 μg / mL, 0.3 μg / mL, 1 μg / mL, 3 μg / mL, 10 μg / mL, 30 μg / mL, and 100 μg / mL.

[0189] Background group: DMEM medium containing 10% FBS (no cells).

[0190] 9.5 Experimental Methods

[0191] The cells were grown at a density of 2 × 10 5 100 μL of a 100 cells / mL cell suspension was seeded into each well of a 96-well cell culture plate. When the cell confluence reached approximately 100%, the supernatant was removed. The test samples were then added sequentially and treated with bioactive substances for 1 hour. The cell culture plates containing samples requiring illumination were then irradiated with UVA; the non-illumination groups were placed in an incubator for continued incubation. After UVA irradiation, the culture medium was removed and washed. After adding DMEM medium, the 96-well plates were placed in a CO2 incubator (37°C, 5% CO2) for an additional 18-22 hours.

[0192] After incubation, add neutral red staining solution and continue incubation in the cell culture incubator for 3 hours. After incubation, completely remove the neutral red working solution and wash the cells. Then add neutral red desorption solution (distilled water: ethanol: acetic acid = 49:50:1) to completely lyse the cells. Measure the absorbance at 540 nm using a microplate reader. Calculate the viability of the positive control or sample cells according to the following formula:

[0193]

[0194] Then calculate the half inhibitory concentration IC of cells under UVA irradiation (+Irr) and no irradiation (-Irr) conditions respectively 50 , calculate the photostimulation factor (PIF) according to the following formula:

[0195]

[0196] 9.6 Results

[0197] Certain drugs transition from a stable state to an excited state after exposure to light, potentially causing damage to the body. This test, based on the in vitro 3T3 neutral red uptake phototoxicity test method for cosmetic chemical raw materials in the "Safety Technical Specifications for Cosmetics" (2015 edition), measures the ability of BALB / C 3T3 fibroblasts to absorb neutral red or changes in cytotoxicity after exposure to a test substance and ultraviolet light to determine whether the substance is phototoxic.

[0198] The experimental results are as follows Figures 16 and 17 As shown, the typical phototoxic drug CPZ has a PIF value ≥ 5, predicting "phototoxicity," indicating successful model establishment. Sample NWT01AM2602 has a PIF value ≤ 2, predicting "no phototoxicity." The results indicate that NWT01AM2602 is not phototoxic.

[0199] In summary, the modified melittin peptide NWT01AM2602 of the present invention can significantly reduce the cytotoxicity of natural melittin, effectively inhibit inflammatory responses at micromolar concentrations, accelerate skin wound healing, and has excellent stability. It is also non-sensitizing and non-phototoxic, providing key technical support for the development of a new generation of highly effective and safe anti-inflammatory and repair drugs, cosmetics, and related biomaterials. This melittin modified peptide can overcome the limitations of natural melittin and has the technical effect of reducing toxicity and increasing efficacy, which is of great significance for promoting the application of melittin in clinical pharmaceutical or cosmetic products.

[0200] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art can make some simple modifications, equivalent changes or modifications based on the technical content disclosed above, which all fall within the scope of protection of the present invention.

Claims

1. A modified melittin peptide, characterized in that: Its amino acid sequence is shown in Seq ID No:

2.

2. The modified melittin peptide according to claim 1, characterized in that The molecular weight of the melittin modified peptide is 3123.5 Da, and the isoelectric point is 12.

64.

3. Use of the modified melittin peptide according to claim 1 or 2 in the preparation of medicines or cosmetics for inhibiting inflammation and relieving skin erythema and itching.

4. Use of the modified melittin peptide according to claim 3 in the preparation of medicines or cosmetics for inhibiting inflammation and relieving skin erythema and itching, characterized in that: The melittin modified peptide can inhibit the expression levels of NO, IL-6, TNF-α and IL-8, reduce neutrophils or inhibit the secretion of IL-1a.

5. Use of the modified melittin peptide according to claim 1 or 2 in the preparation of medicines or cosmetics for enhancing cell activity and promoting tissue repair.

6. Use of the modified melittin peptide according to claim 5 in the preparation of medicines or cosmetics for enhancing cell activity and promoting tissue repair, characterized in that: The melittin modified peptide can enhance the activity of BJ cells and improve the cell healing rate of HaCat cells.

7. A cosmetic, characterized in that: The modified melittin peptide according to claim 1 or 2 can inhibit the expression levels of NO, IL-6, TNF-α and IL-8, reduce neutrophils or inhibit the secretion of IL-1a, and is used to inhibit inflammation and relieve skin erythema and itching for sensitive skin; and / or, The melittin modified peptide can enhance the activity of BJ cells and improve the cell healing rate of HaCat cells, and is used to enhance cell activity and promote tissue repair.

8. A medicine, characterized in that The invention comprises the modified melittin peptide according to claim 1 or 2, and pharmaceutically acceptable excipients or auxiliary ingredients, wherein the modified melittin peptide can inhibit the expression levels of NO, IL-6, TNF-α and IL-8, reduce neutrophils or inhibit the secretion of IL-1a, and is used for inhibiting inflammation and relieving skin erythema and itching for sensitive skin; and / or, The melittin modified peptide can enhance the activity of BJ cells and improve the cell healing rate of HaCat cells, and is used to enhance cell activity and promote tissue repair.

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