A polypeptide, a polypeptide variant, a method for preparing the same, a composition thereof, and its application.

Abstract

This invention discloses a polypeptide, a polypeptide variant, its preparation method, composition, and application. The amino acid sequence of the polypeptide (CS2201) is shown in SEQ ID NO: 1, and it was isolated and purified from Pu-erh tea. CS2201 contains a cell membrane penetration core fragment KRMMEKK (SEQ ID NO: 2) rich in positively charged amino acids, which can efficiently penetrate the cell membrane and enter the cell to exert anti-inflammatory effects. In vitro experiments have confirmed that CS2201 can significantly inhibit LPS-induced release of NO and TNF-α, IL-6, and IL-1β inflammatory factors, the mechanism of which is to block NF-κB p65 nuclear translocation after penetrating the cell membrane. This invention also provides a truncated polypeptide variant of CS2201, retaining at least amino acids 10-22; both the polypeptide and the polypeptide variant can be prepared using the Fmoc solid-phase polypeptide synthesis method. This invention also provides compositions containing the above-mentioned polypeptide or polypeptide variant active ingredients. The polypeptide, polypeptide variant, and composition of this invention have anti-inflammatory, soothing, and skin barrier repair effects, and can be used to prepare drugs or cosmetics with anti-inflammatory, soothing, and skin barrier repair effects.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, and specifically relates to a polypeptide with cell membrane penetration ability isolated from Pu-erh tea (Camellia sinensis var. assamica (JW Mast.) Kitam.), polypeptide variants, preparation methods, compositions and applications thereof. Background Technology

[0002] Inflammation is the body's defensive response to infection, injury, or external stimuli. Excessive or chronic inflammation is closely related to various diseases such as atopic dermatitis, psoriasis, rheumatoid arthritis, and inflammatory bowel disease. In the field of dermatology, inflammation is a core pathological element in problems such as sensitive skin, acne, and photoaging. Currently, commonly used anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids are effective, but long-term use often brings side effects such as gastrointestinal damage, immunosuppression, and skin atrophy. In the cosmetics field, commonly used anti-inflammatory ingredients such as dipotassium glycyrrhizate and bisabolol have limitations such as limited activity and single target. Developing anti-inflammatory active substances that are naturally derived, highly safe, and have novel mechanisms is an important direction for the current research and development of functional raw materials.

[0003] Bioactive peptides have become a research hotspot for novel anti-inflammatory raw materials due to their high activity, high specificity, and low toxicity. Currently reported anti-inflammatory peptides are mostly derived from animals or microorganisms, such as frog skin Magainin, melittin (a bee venom peptide), and human endogenous BPC-157. However, animal-derived peptides face risks of viral transmission, allergenicity, and ethical controversies, while microbial fermentation peptides suffer from limitations such as low yield and high purification costs. Plant-derived peptides are increasingly valued due to their high safety, sustainability, and consumer acceptance. In recent years, peptides with anti-inflammatory activity have been isolated from plants such as soybeans, rice, and flaxseed. However, these peptides are mostly protease hydrolysis products, and anti-inflammatory peptides with complete structures and well-defined sequences obtained directly from natural plant tissues are still relatively rare.

[0004] Tea is one of the world's most consumed beverages, and tea leaves are rich in proteins and polypeptides. Pu-erh tea is a post-fermented tea made from the fresh leaves of the Yunnan large-leaf tea tree (Camellia sinensis var. assamica (JW Mast.) Kitam.) using a specific processing technique. Studies have shown that it has significant effects in lowering lipids, anti-oxidation, and regulating gut microbiota, but research on its polypeptide active components is very limited. Currently reported tea-derived active polypeptides are mostly obtained through in vitro enzymatic hydrolysis of tea proteins; naturally occurring anti-inflammatory polypeptides isolated directly from tea, especially Pu-erh tea, are rarely reported, and tea-derived anti-inflammatory polypeptides with cell membrane penetration capabilities have yet to be identified.

[0005] Peptides with cell membrane-penetrating capabilities can directly cross the cell membrane and enter the cell to act on intracellular targets. Among them, cationic peptides rich in arginine and lysine exhibit excellent membrane-penetrating ability due to electrostatic interactions with the negatively charged components of the cell membrane. Most reported cell-penetrating peptides are artificially designed sequences or derived from animal toxins, while peptides from natural plants that possess both cell-penetrating and anti-inflammatory activities are extremely rare. Compared to existing technologies, such peptides from natural plants possess both the high safety and efficient intracellular delivery capabilities of natural products, thus holding significant scientific research value and promising industrial application prospects. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a polypeptide, a polypeptide variant, a method for preparing the same, a composition thereof, and an application thereof, which possess the high safety and efficient intracellular delivery capability of natural products and have anti-inflammatory activity.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1 (CS2201):

[0009] Asn-Thr-Phe-Ala-Val-Ile-Ser-Met-Ile-Lys-Arg-Met-Met-Glu-Lys-Lys-Cys-Arg-Glu-Leu-Tyr-Lys, the single-letter abbreviation is NTFAVISMIKRMMEKKCRELYK.

[0010] It is an anti-inflammatory polypeptide derived from Pu-erh tea that has the ability to penetrate cell membranes.

[0011] Furthermore, the polypeptide is a polypeptide with anti-inflammatory, soothing and / or skin barrier repair effects.

[0012] In a second aspect, the present invention provides a polypeptide variant whose amino acid sequence is an N-terminal truncated variant of SEQ ID NO: 1, wherein the truncated variant retains at least amino acids 10-22 of SEQ ID NO: 1 and has anti-inflammatory activity.

[0013] Furthermore, the amino acid sequence of the polypeptide variant is as follows:

[0014] (a) As shown in SEQ ID NO: 3: MIKRMMEKKCRELYK (truncated peptide C1).

[0015] Or (b) as shown in SEQ ID NO: 4: KRMMEKKCRELYK (truncated peptide C2).

[0016] Thirdly, the present invention also provides a method for preparing the above-mentioned polypeptide or polypeptide variant, which is prepared by Fmoc solid-phase polypeptide synthesis method;

[0017] Using Rink Amide resin as a solid support, deprotection and coupling reactions were carried out sequentially according to the amino acid sequence of the above-mentioned polypeptide or polypeptide variant.

[0018] After synthesis, the target peptide or peptide variant is obtained by cleavage with a cleavage reagent, purification by reversed-phase high-performance liquid chromatography, and lyophilization.

[0019] Fourthly, the present invention also provides a composition in which the active ingredient comprises the above-described polypeptide or polypeptide variant, and the composition further comprises a cosmetic or pharmaceutically acceptable matrix, carrier, excipient or other inactive ingredient.

[0020] Furthermore, the polypeptide or polypeptide variant has a mass percentage content of 0.001%-5%;

[0021] And / or, the dosage form of the composition is a serum, lyophilized powder, facial mask liquid, gel, spray or cream.

[0022] Further, the composition is an anti-inflammatory and soothing essence containing 0.05 wt% of the polypeptide described in claim 1 or 2, 5.0 wt% of glycerin, 3.0 wt% of butylene glycol, 0.1 wt% of sodium hyaluronate, 0.2 wt% of allantoin, 0.5 wt% of p-hydroxyacetophenone, 0.5 wt% of 1,2-hexanediol, and deionized water to 100 wt%.

[0023] Fifthly, the present invention also provides an application of the above-mentioned polypeptide, polypeptide variant, or composition, characterized in that the application is:

[0024] Application in the preparation of cosmetics or pharmaceuticals with anti-inflammatory, soothing and / or skin barrier repair effects.

[0025] Furthermore, the anti-inflammatory effect is achieved by penetrating the cell membrane into the cytoplasm, inhibiting p65 nuclear translocation, blocking the NF-κB signaling pathway, and reducing the release of inflammatory factors NO, TNF-α, IL-6, and IL-1β.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) Novel source: The anti-inflammatory polypeptide CS2201 with cell membrane penetration ability was discovered for the first time in Pu'er tea, filling the gap in cell-penetrating anti-inflammatory polypeptides from Pu'er tea.

[0028] (2) Mechanism innovation: It reveals the mechanism by which CS2201 exerts its intracellular anti-inflammatory effect by inhibiting the NF-κB signaling pathway after penetrating the cell membrane. Unlike conventional anti-inflammatory peptides that only act on membrane receptors, this breakthrough is achieved from "extracellular signal blocking" to "direct intervention of intracellular targets".

[0029] (3) Significant activity: In vitro experiments confirmed that CS2201 inhibited NO by 86.2% at a concentration of 25 μg / mL, which was significantly better than the positive control dexamethasone, and could broadly inhibit the release of multiple inflammatory factors such as TNF-α, IL-6, and IL-1β.

[0030] (4) Reliable application: The serum containing CS2201 can significantly improve the skin barrier function, reduce skin inflammation, increase skin moisture content, and has good safety.

[0031] (5) Industrial applicability: It can be prepared on a large scale through Fmoc solid-phase synthesis method. The process is mature and can be linearly scaled up to meet the needs of industrialization. Attached Figure Description

[0032] 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.

[0033] Figure 1 This is a comparison of the cell membrane penetration efficiency of the full-length CS2201 polypeptide and its various truncated peptides.

[0034] Figure 2 The effects of CS2201 and its truncated peptide on the inhibition rate of NO release in LPS-induced RAW 264.7 cells.

[0035] Figure 3 The effect of CS2201 on the inhibition rate of LPS-induced TNF-α release in RAW 264.7 cells.

[0036] Figure 4 The effect of CS2201 on the inhibition rate of LPS-induced IL-6 release in RAW 264.7 cells.

[0037] Figure 5 The effect of CS2201 on the inhibition rate of LPS-induced IL-1β release in RAW 264.7 cells.

[0038] Figure 6 The effect of CS2201 on LPS-induced NF-κB p65 nuclear translocation in RAW 264.7 cells.

[0039] Figure 7 The changes in transepidermal moisture loss in the skin after 4 weeks of use of an anti-inflammatory and soothing serum containing CS2201.

[0040] Figure 8 Changes in skin erythema index after 4 weeks of human use of an anti-inflammatory and soothing serum containing CS2201.

[0041] Figure 9 Changes in skin hydration after 4 weeks of human use of an anti-inflammatory and soothing serum containing CS2201. Detailed Implementation

[0042] 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 examples 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.

[0043] This invention marks the first time a novel natural polypeptide, CS2201 (amino acid sequence shown in SEQ ID NO: 1), has been isolated from Pu-erh tea leaves (Camellia sinensis var. assamica (JW Mast.) Kitam.). Polypeptide CS2201 was isolated from Pu-erh tea leaves through the following steps: hot water extraction, ultrafiltration fractionation, gel filtration chromatography, and reversed-phase high-performance liquid chromatography purification, followed by sequence identification by de novo MALDI-TOF / TOF mass spectrometry sequencing. Database searches and comparisons confirmed CS2201 as a newly discovered natural polypeptide.

[0044] CS2201 contains a cell membrane penetration core fragment KRMMEKK (SEQ ID NO: 2) rich in positively charged amino acids. Because it contains a unique positively charged amino acid enriched fragment KRMMEKK, which is rich in positively charged lysine (K) and arginine (R), it can bind to the cell membrane phospholipid bilayer through electrostatic interaction, mediating the peptide to penetrate the cell membrane and enter the cytoplasm. Therefore, it can efficiently penetrate the cell membrane and enter the cell to exert an anti-inflammatory effect.

[0045] In vitro experiments confirmed that CS2201 significantly inhibits LPS-induced NO release and the expression of TNF-α, IL-6, and IL-1β inflammatory factors by blocking the NF-κB signaling pathway through inhibiting the translocation of the NF-κB p65 subunit from the cytoplasm to the nucleus. This mechanism of action differs from conventional anti-inflammatory peptides that only act on membrane receptors, achieving a breakthrough from "extracellular signal blocking" to "direct intervention at intracellular targets." It combines the dual advantages of natural product safety and efficient intracellular delivery, providing a novel source and mechanism of action for the development of tea-derived anti-inflammatory peptides, and possesses significant scientific research value and promising industrial application prospects.

[0046] This invention also provides truncated polypeptide variants of CS2201. The polypeptide CS2201 and its truncated polypeptide variants can be prepared on a large scale using the Fmoc solid-phase synthesis method, a mature process that can be linearly scaled up, meeting industrialization requirements.

[0047] This invention also provides compositions comprising the above-mentioned polypeptides or polypeptide variants as active ingredients. Human efficacy studies have shown that serums containing CS2201 can significantly improve skin barrier function, reduce inflammation, and increase skin hydration, exhibiting anti-inflammatory, soothing, and skin barrier repair effects. The polypeptides and polypeptide variants, and compositions of this invention possess anti-inflammatory, soothing, and skin barrier repair effects, and can be used to prepare drugs or cosmetics with anti-inflammatory, soothing, and skin barrier repair effects.

[0048] The following is a detailed explanation through specific embodiments:

[0049] Example 1: Isolation, purification, identification and structural confirmation of CS2201, a natural polypeptide from Pu-erh tea.

[0050] 1.1 Raw Materials and Extraction

[0051] Take one bud and two leaves of fresh Pu-erh tea (Camellia sinensis var. assamica (JW Mast.) Kitam.), flash-freeze with liquid nitrogen, and grind into powder. Weigh 100 g of tea powder, add deionized water at a material-to-liquid ratio of 1:15 (w / v), and extract in a 90℃ water bath for 2 hours with intermittent stirring. Filter the extract through four layers of gauze, centrifuge at 4℃ and 10000g for 20 minutes, and collect the supernatant.

[0052] 1.2 Ultrafiltration Stage

[0053] The supernatant was sequentially passed through ultrafiltration membranes (Millipore, Pellicon system) with molecular weight cutoffs of 30 kDa, 10 kDa, and 3 kDa. The 3–10 kDa fraction was collected, which showed the strongest NO release inhibition activity in the preliminary activity screening. This fraction was freeze-dried to obtain crude peptide powder.

[0054] 1.3 Gel Filtration Chromatography

[0055] Crude peptide powder was dissolved in deionized water (50 mg / mL) and loaded onto a Sephadex G-25 gel filter column (2.6 cm × 60 cm). Deionized water was used as the mobile phase, and the flow rate was 1 mL / min. 5 mL was collected per tube. The absorbance was monitored at 280 nm, and each peak component was collected. After concentration under reduced pressure, the activity was tracked using a RAW 264.7 cell NO inhibition model, and the activity peak (Fr-3) was combined.

[0056] 1.4 Purification by Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)

[0057] The active component Fr-3 was further purified using a semi-preparative C18 reversed-phase column (10 mm × 250 mm, 5 μm). Mobile phase A was water containing 0.1% trifluoroacetic acid (TFA), and mobile phase B was acetonitrile containing 0.1% TFA. The gradient elution program was: 0–5 min, 5% B; 5–45 min, 5%–45% B; 45–50 min, 45%–95% B. The flow rate was 2 mL / min, and the detection wavelengths were 214 nm and 280 nm. A single main peak was collected (retention time 28.3 min), and the purified peptide CS2201 (approximately 12.3 mg) was obtained by lyophilization.

[0058] 1.5 Purity Identification

[0059] Purity was determined by analytical RP-HPLC. Column: C18 column (4.6 mm × 150 mm, 3.5 μm); gradient: 5%–60% B, 25 min. Purity was calculated using the peak area normalization method and was above 98.5%.

[0060] 1.6 Molecular weight determination

[0061] The precise molecular weight was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / TOF MS). The matrix was α-cyano-4-hydroxycinnamic acid (CHCA), and the positive ion reflectance mode was used. The measured quasi-molecular ion peak [M+H]⁺ was 2689.4 m / z, consistent with the theoretical calculation (calculated value: 2689.2 Da).

[0062] 1.7 De novo sequencing of amino acid sequences

[0063] MS / MS fragmentation was performed using collision-induced dissociation (CID) mode. The b-ion and y-ion sequences were clearly defined, and the complete amino acid sequence was obtained through complementary analysis of both.

[0064] H2N-Asn-Thr-Phe-Ala-Val-Ile-Ser-Met-Ile-Lys-Arg-Met-Met-Glu-Lys-Lys-Cys-Arg-Glu-Leu-Tyr-Lys-COOH (SEQ ID NO: 1), the single-letter abbreviation is NTFAVISMIKRMMEKKCRELYK.

[0065] The positions of each amino acid residue were confirmed by the mass difference of the b / y ions, and the sequence assignment was clear.

[0066] 1.8 Sequence Database Retrieval and Alignment

[0067] The identified sequence was searched using BLASTp in the NCBI non-redundant protein database and the tea proteome database. No completely matching known protein fragments or bioactive peptide sequences were found, confirming that CS2201 is a newly discovered natural polypeptide.

[0068] 1.9 Synthesis Verification

[0069] To further verify the accuracy of the natural sequence, a peptide with the same sequence was synthesized using the Fmoc solid-phase synthesis method. After purification by RP-HPLC, the synthesized peptide was co-injected with the naturally isolated peptide for analysis. The two peptides showed consistent retention times, identical molecular weights as determined by MALDI-TOF MS, and comparable in vitro anti-inflammatory activity (NO inhibition rate), confirming the correct sequence identification of CS2201.

[0070] Example 2: Determination of CS2201 cell membrane penetration ability and identification of core transmembrane fragments

[0071] 2.1 Quantitative determination of cell membrane permeability by flow cytometry

[0072] FITC-labeled CS2201 cells (10 μM) were co-incubated with human immortalized keratinocytes (HaCaT) at 37°C for 2 hours. After washing with PBS, the cells were washed twice with PBS containing heparin (0.5 mg / mL) to remove adsorbed peptides from the cell surface. Cells were collected by trypsin digestion, and intracellular fluorescence intensity was detected by flow cytometry. 10,000 cells were counted for each sample, and the membrane penetration efficiency was quantified as mean fluorescence intensity (MFI). The classic membrane penetration peptide TAT (amino acid sequence GRKKRRQRRRPPQ, 10 μM) was used as a positive control, and an equal concentration of FITC solution was used as a negative control. A separate 4°C incubation group, with all other conditions identical, was included to investigate the temperature dependence of the membrane penetration process.

[0073] 2.2 Identification of the core transmembrane segment

[0074] To identify the core region responsible for cell membrane penetration in the CS2201 sequence, the following FITC-labeled truncated peptides were designed and synthesized based on the cationic amino acid enrichment characteristics of the KRMMEKK fragment in the sequence:

[0075] Deleted peptide: FITC-NTFAVISMICRELYK (This sequence is obtained by deleting amino acids KRMMEKK from positions 10-16 of SEQ ID NO: 1, that is, by directly splicing NTFAVISMI at the N-terminus and CRELYK at the C-terminus of the original sequence, corresponding to SEQ ID NO: 5).

[0076] Shortened peptide C1: FITC-MIKRMMEKKCRELYK (SEQ ID NO: 3, retaining the transmembrane core region and part of the N-terminal sequence);

[0077] Shortened peptide C2: FITC-KRMMEKKCRELYK (SEQ ID NO: 4, retaining only the transmembrane core region and C-terminal sequence).

[0078] Following method 2.1, the intracellular MFI of each truncated peptide was determined after incubation at 37°C for 2 hours with HaCaT cells.

[0079] 2.3 Results and Conclusions

[0080] The membrane penetration efficiency of each experimental group is as follows: Figure 1 As shown, the relative membrane penetration efficiency of CS2201 under incubation at 37℃ was (97.4 ± 4.5)% (normalized with TAT as 100%), which was not significantly different from the positive control TAT (p > 0.05), indicating that CS2201 has a high cell membrane penetration capacity comparable to the classic membrane-penetrating peptide TAT. Under incubation at 4℃, the relative membrane penetration efficiency of CS2201 significantly decreased to (19.2 ± 2.7)% (p < 0.01), indicating that its cellular uptake process is highly temperature-dependent. Low temperature can inhibit cell membrane fluidity and energy metabolism, blocking the endocytic pathway. This temperature-dependent characteristic confirms that the membrane penetration mechanism of CS2201 is an energy-dependent active endocytosis process, rather than passive physical diffusion, consistent with the cellular uptake mechanism of the classic membrane-penetrating peptide TAT. The approximately 19% signal remaining at 4℃ may originate from a small amount of non-energy-dependent direct membrane penetration pathway or trace amounts of adsorbed fluorescence remaining on the membrane surface after washing. The relative cell penetration efficiency of the negative control group (FITC) was only (2.8 ± 0.9)%, confirming that the fluorescent label itself does not confer cell penetration ability.

[0081] The relative membrane penetration efficiencies of the truncated peptides C1 and C2 were (89.8 ± 4.5)% and (82.6 ± 4.2)%, respectively, which were not statistically different from those of the full-length CS2201 peptide (p > 0.05). The relative membrane penetration efficiency of the peptide lacking the KRMMEKK fragment was only (3.5 ± 1.0)%, which was not significantly different from that of the negative control group (2.8 ± 0.9)% (p > 0.05), indicating that the membrane penetration function was completely lost after the KRMMEKK fragment was deleted.

[0082] The above results collectively confirm that:

[0083] (1) CS2201 has a high cell membrane penetration ability comparable to the classic transmembrane peptide TAT, and its transmembrane penetration process depends on the energy-dependent endocytosis pathway.

[0084] (2) The KRMMEKK fragment shown in SEQ ID NO: 2 is a core region essential for the cell membrane penetration function of CS2201 cells. Deletion of this fragment results in complete loss of membrane penetration function, while this core fragment alone is sufficient to endow the peptide with cell membrane penetration ability. Its membrane penetration mechanism can be attributed to the KRMMEKK fragment being rich in positively charged lysine (K) and arginine (R) residues, which bind to negatively charged phospholipids and proteoglycans on the cell membrane surface through electrostatic interactions, triggering cellular uptake via the endocytic pathway.

[0085] Example 3: In vitro cytotoxicity evaluation and determination of safe concentration range of CS2201

[0086] 3.1 Test Methods

[0087] The cytotoxicity of CS2201 against human immortalized keratinocytes (HaCaT) and human skin fibroblasts (HSF) was detected using the CCK-8 assay. HaCaT and HSF cells in logarithmic growth phase were seeded at 5 × 10³ cells / well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator. The culture medium was discarded, and DMEM complete medium containing different concentrations of CS2201 (0, 10, 25, 50, 100, 200 μg / mL) was added, with three replicates per group. The cells were cultured for another 24 hours. 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance (OD value) was then measured at 450 nm using a microplate reader. Cell viability was calculated as follows: Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%.

[0088] 3.2 Results

[0089] The effect of CS2201 on the survival rate of HaCaT and HSF cells was concentration-dependent. Within the concentration range of 0–50 μg / mL, the survival rate of both cell types was greater than 90%, indicating that CS2201 has good biocompatibility within this concentration range. At a concentration of 100 μg / mL, the survival rates of HaCaT and HSF cells were (88.7 ± 3.2)% and (85.4 ± 4.1)%, respectively, still greater than 85%. When the concentration was increased to 200 μg / mL, the survival rates of HaCaT and HSF cells decreased to (72.3 ± 5.6)% and (65.8 ± 6.3)%, respectively, showing significant cytotoxicity. Cell survival data at each concentration are detailed in Table 1.

[0090] Table 1. Cytotoxicity of CS2201 against HaCaT and HSF cells (24 h)

[0091] Concentration (μg / mL) HaCaT survival rate (%, mean ± SD) HSF survival rate (%, mean ± SD) 0 (Control) 100 100 10 98.5 ± 2.1 97.2 ± 2.8 25 96.3 ± 3.0 94.6 ± 3.5 50 92.1 ± 3.4 90.3 ± 4.0 100 88.7 ± 3.2 85.4 ± 4.1 200 72.3 ± 5.6 65.8 ± 6.3

[0092] 3.3 Determination of the safe concentration range

[0093] Based on the above results, the survival rates of both HaCaT and HSF cells exceeded 90% within the concentration range of 1-50 μg / mL. Subsequent evaluations of anti-inflammatory activity within this concentration range could eliminate the interference of cytotoxicity on the experimental results. Considering both safety and activity screening needs, subsequent experiments selected 5, 25, and 50 μg / mL as low, medium, and high dose groups, respectively.

[0094] Example 4: Evaluation of the in vitro anti-inflammatory activity of CS2201 and its truncated peptide variants—NO release inhibition

[0095] 4.1 Test Methods

[0096] RAW 264.7 mouse macrophages in the logarithmic growth phase were harvested and processed at a concentration of 1×10⁻⁶ cells / mL. 5 Inoculate 10 cells / well into a 96-well plate and incubate overnight at 37°C with 5% CO2. Discard the culture medium and divide the cells into groups as follows:

[0097] Blank control group: serum-free DMEM medium was added;

[0098] LPS model group: serum-free DMEM medium containing 1 μg / mL LPS was added;

[0099] Positive control group: serum-free DMEM medium containing 1 μg / mL LPS + 10 μg / mL dexamethasone was added;

[0100] Low-dose CS2201 group: serum-free DMEM medium containing 1 μg / mL LPS + 5 μg / mL CS2201 was added;

[0101] Medium-dose group of CS2201: serum-free DMEM medium containing 1 μg / mL LPS + 25 μg / mL CS2201 was added;

[0102] High-dose CS2201 group: Serum-free DMEM medium containing 1 μg / mL LPS + 50 μg / mL CS2201 was added.

[0103] Core fragment group: serum-free DMEM medium containing 1 μg / mL LPS + 25 μg / mL KRMMEKK (SEQ ID NO: 2) was added;

[0104] Shortened peptide C1 group: serum-free DMEM medium containing 1 μg / mL LPS + 25 μg / mL MIKRMMEKKCRELYK (SEQ ID NO: 3) was added;

[0105] Shortened peptide C2 group: Serum-free DMEM medium containing 1 μg / mL LPS + 25 μg / mL KRMMEKKCRELYK (SEQ ID NO: 4) was added.

[0106] Each group was divided into 3 replicates, and cultured for 24 hours at 37℃ in a 5% CO2 incubator. Cell culture supernatant was collected, and NO content was detected using the Griess method: 50 μL of supernatant was mixed with 50 μL of Griess reagent (1% sulfonamide, 0.1% N-1-naphthylethylenediamine hydrochloride, 2.5% phosphoric acid), and reacted at room temperature in the dark for 10 minutes. The absorbance was measured at 540 nm, and the NO content was calculated using a sodium nitrite standard curve. The NO inhibition rate was calculated using the formula: NO inhibition rate (%) = [(NO content in the model group - NO content in the experimental group) / (NO content in the model group - NO content in the blank group)] × 100%.

[0107] 4.2 Results and Conclusions

[0108] NO inhibition rates in each experimental group are as follows: Figure 2 As shown, the NO content in the model group was significantly higher than that in the blank control group after LPS stimulation (p < 0.001), indicating that the inflammation model was successfully established. The NO inhibition rate of the positive control drug dexamethasone (10 μg / mL) was 72.5%.

[0109] CS2201 showed an inhibition rate of 26.7% at 5 μg / mL (p < 0.01), 86.2% at 25 μg / mL (p < 0.001), and 75.7% at 50 μg / mL (p < 0.001). The inhibitory effect of 25 μg / mL CS2201 was significantly better than that of the positive control dexamethasone (p < 0.01), and was the best among the three dosage groups, exhibiting a non-monotonic dose-response characteristic.

[0110] At a concentration of 25 μg / mL, the NO inhibition rate of the core fragment KRMMEKK (SEQ ID NO: 2) was 18.5%, that of the truncated peptide C1 (SEQ ID NO: 3) was 79.8%, and that of the truncated peptide C2 (SEQ ID NO: 4) was 72.3%. The NO inhibition rates of the full-length CS2201 peptide compared to the truncated peptides C1 and C2 were ranked as follows: full-length CS2201 (86.2%) > truncated peptide C1 (79.8%) > truncated peptide C2 (72.3%) > core fragment KRMMEKK (18.5%).

[0111] The above results indicate that CS2201 possesses potent anti-inflammatory activity, with 25 μg / mL being the optimal effective concentration. The truncated peptides C1 and C2 retained most of the anti-inflammatory activity of the full-length peptide, while the core transmembrane fragment KRMMEKK alone exhibited weaker anti-inflammatory activity. This suggests that the KRMMEKK fragment primarily confers membrane-penetrating ability to the peptide, but the C-terminal amino acid residues such as CRELYK in the full-length sequence significantly contribute to the full expression of anti-inflammatory activity. The relationship between transmembrane penetration and anti-inflammatory activity is not a simple linear one; the peptide needs to possess both transmembrane penetration capability and intracellular target binding ability to achieve optimal anti-inflammatory effects.

[0112] Example 5: Evaluation of the in vitro anti-inflammatory activity of CS2201 – Detection of inflammatory factors

[0113] 5.1 Test Methods

[0114] RAW 264.7 mouse macrophages in the logarithmic growth phase were harvested and processed at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded per well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. The culture medium was discarded, and the cells were grouped according to Example 4: blank control group (serum-free DMEM medium), LPS model group (1 μg / mL LPS), positive control group (1 μg / mL LPS + 10 μg / mL dexamethasone), low-dose CS2201 group (1 μg / mL LPS + 5 μg / mL CS2201), medium-dose CS2201 group (1 μg / mL LPS + 25 μg / mL CS2201), and high-dose CS2201 group (1 μg / mL LPS + 50 μg / mL CS2201). Each group was divided into three replicates, and the cells were incubated for another 24 hours at 37°C in a 5% CO2 incubator. The cell culture supernatant was collected, and the levels of TNF-α, IL-6, and IL-1β in the supernatant were detected using an ELISA kit, strictly following the kit instructions. The formula for calculating the inhibition rate of each inflammatory factor is: Inhibition rate (%) = [(Model combination amount - Experimental combination amount) / (Model combination amount - Blank combination amount)] × 100%.

[0115] 5.2 Results and Conclusions

[0116] The inhibition rates of the three inflammatory factors in each experimental group are as follows: Figure 3-5 As shown in the figure, after LPS stimulation, the levels of TNF-α, IL-6, and IL-1β in the model group were significantly higher than those in the blank control group (p < 0.001), indicating that the inflammation model was successfully established. The positive control dexamethasone (10 μg / mL) inhibited the levels of TNF-α, IL-6, and IL-1β by 74.4%, 69.1%, and 71.9%, respectively.

[0117] At a concentration of 5 μg / mL, CS2201 inhibited the release of three inflammatory factors by 19.9%, 19.2%, and 22.0% (p < 0.05), respectively; at 25 μg / mL, the inhibition rates reached 55.8%, 49.7%, and 59.5% (p < 0.001), the best among the three dosage groups; at 50 μg / mL, the inhibition rates decreased to 42.8%, 36.8%, and 44.1% (p < 0.001). Among the three inflammatory factors, CS2201 showed the most significant inhibitory effect on IL-1β (59.5% inhibition rate at 25 μg / mL), and all factors exhibited a non-monotonic dose-response characteristic optimal at 25 μg / mL, consistent with the NO inhibition results in Example 4. These results indicate that CS2201 can broadly inhibit the release of multiple inflammatory factors induced by LPS, with particularly significant inhibition of IL-1β, and 25 μg / mL is the preferred effective concentration.

[0118] Example 6: Intracellular anti-inflammatory mechanism of CS2201—inhibition of NF-κB signaling pathway

[0119] 6.1 Test Methods

[0120] Using LPS-stimulated RAW 264.7 mouse macrophages as a model, the regulatory role of CS2201 on the NF-κB signaling pathway was investigated. Logarithmically growing RAW 264.7 cells were harvested and treated at a concentration of 2 × 10⁻⁶ cells / cells. 6 Cells were seeded at 1 / well in 6-well plates and cultured overnight. After pretreatment with CS2201 (25 μg / mL) for 2 hours, cells were stimulated with LPS (1 μg / mL) for 30 minutes. A blank control group, an LPS model group (LPS stimulation without CS2201), and a positive control group (LPS + 10 μg / mL dexamethasone) were also established. Cytoplasmic and nuclear proteins were extracted separately, and protein concentrations were determined using the BCA method. Equal volumes of proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk for 1 hour, anti-NF-κB p65 antibody, anti-Lamin B antibody (nuclear control), and anti-β-actin antibody (cytoplasmic control) were added, and the membranes were incubated overnight at 4°C. After washing with TBST, HRP-labeled secondary antibody was added, and the membranes were incubated at room temperature for 1 hour. Chemiluminescence imaging was performed using ECL, and images were acquired using a gel imaging system. ImageJ software was used to perform grayscale quantitative analysis of protein bands, and the relative expression level of the protein was expressed as the grayscale ratio of the target protein to the corresponding internal control protein.

[0121] 6.2 Results and Conclusions

[0122] The results of protein band and gray-scale quantitative analysis of cells in each group are as follows: Figure 6 As shown.

[0123] Nuclear p65 protein expression: After LPS stimulation, the relative expression level of nuclear p65 protein in the model group was significantly higher than that in the blank control group (p < 0.001), indicating that p65 translocated extensively from the cytoplasm to the nucleus, and the NF-κB pathway was activated. After pretreatment with CS2201 (25 μg / mL), the nuclear p65 level was significantly lower than that in the model group (p < 0.001), similar to the effect of the positive control dexamethasone.

[0124] Cytoplasmic p65 protein expression: In contrast to the nuclear trend, the cytoplasmic p65 level in the LPS model group was significantly lower than that in the blank control group (p < 0.001), indicating a large loss of p65 in the cytoplasm. After CS2201 pretreatment, the cytoplasmic p65 level rebounded significantly compared with the model group (p < 0.001), indicating that p65 was effectively retained in the cytoplasm.

[0125] The mirror-image changes in the nucleus and cytoplasm of p65, exhibiting a "give and take" relationship, jointly confirm that CS2201 directly blocks the translocation of p65 from the cytoplasm to the nucleus after entering the cell. Its site of action is located at or upstream of p65 nuclear translocation, rather than indirectly exerting its effect through competition for cell surface receptors. Combined with the cell membrane penetration ability of CS2201 in Example 2, this example establishes a complete intracellular anti-inflammatory chain of CS2201: "penetrating the cell membrane into the cytoplasm → inhibiting p65 nuclear translocation → blocking the NF-κB signaling pathway → reducing the release of inflammatory factors."

[0126] Example 7: Solid-phase synthesis and purification of CS2201 and its variants

[0127] To demonstrate that CS2201 and its variants can be prepared on a large scale through chemical synthesis to meet industrialization needs, this embodiment provides a solid-phase synthesis and purification method for Fmoc.

[0128] 7.1 Test Methods

[0129] CS2201 (SEQ ID NO: 1), the core transmembrane fragment KRMMEKK (SEQ ID NO: 2), and truncated peptide derivatives (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5) were prepared using the Fmoc solid-phase peptide synthesis method. 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 minutes. Deprotection and coupling reactions were performed sequentially: deprotection was performed using a 20% piperidine / DMF solution for 10 minutes; coupling was performed by adding 4 molar excesses of Fmoc-protected amino acids, HBTU, HOBt, and DIEA, and reacting for 60 minutes. After coupling, the resin was washed alternately with DMF and DCM. The coupling efficiency was monitored using the ninhydrin colorimetric method; if coupling was incomplete, the coupling steps were repeated. After all amino acids were sequentially coupled, the resin was shrunk with methanol and dried under vacuum.

[0130] 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 hours. Filter to remove the resin, add the filtrate to pre-cooled diethyl ether to precipitate, centrifuge (4000 rpm, 10 minutes), collect the precipitate, wash three times with diethyl ether, and freeze-dry to obtain the crude peptide.

[0131] The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Chromatographic conditions: C18 preparative column (250 mm × 10 mm, 10 μm); mobile phase A: 0.1% TFA-water; mobile phase B: 0.1% TFA-acetonitrile; gradient elution (10%-50% B, 30 min); flow rate: 3.0 mL / min; detection wavelength: 214 nm. The main peak was collected, lyophilized, and the purified product was obtained.

[0132] 7.2 Results

[0133] Analytical RP-HPLC analysis showed that the purity of purified CS2201 and the core fragment KRMMEKK was 98.5%, and the purity of each truncated peptide derivative was ≥98%. MALDI-TOF mass spectrometry determined the molecular weight of CS2201 to be 2689.4 Da, consistent with the theoretical value of 2689.2 Da (error <0.01%). The measured molecular weights of the core fragment and each truncated peptide derivative were also consistent with their respective theoretical values. These results indicate that the Fmoc solid-phase synthesis method combined with RP-HPLC purification can efficiently prepare high-purity CS2201 and its derivative variants. The process is mature, linearly scalable, and capable of large-scale production, providing a reliable foundation for the industrial application of CS2201.

[0134] Example 8: Anti-inflammatory and soothing serum containing CS2201 and its efficacy evaluation

[0135] 8.1 Formulation Composition

[0136] CS2201 0.05 wt%, Glycerin 5.0 wt%, Butylene Glycol 3.0 wt%, Sodium Hyaluronate 0.1 wt%, Allantoin 0.2 wt%, P-hydroxyacetophenone 0.5 wt%, 1,2-Hexanediol 0.5 wt%, Deionized water to 100 wt%.

[0137] 8.2 Preparation method

[0138] Dissolve sodium hyaluronate in deionized water and stir until completely dissolved. Add glycerin, butylene glycol, and allantoin sequentially, stirring until dissolved and homogeneous. Add CS2201, p-hydroxyacetophenone, and 1,2-hexanediol, stirring until well mixed. Filter the mixture for sterilization, then fill into vials to obtain the final product.

[0139] 8.3 Evaluation of Human Efficacy

[0140] 8.3.1 Subjects

[0141] 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.

[0142] 8.3.2 Test Method

[0143] A self-controlled design was used. Subjects applied an anti-inflammatory and soothing serum containing 0.05% CS2201 to the left side of their face (sample group), while the right side received a blank matrix serum without CS2201 (placebo group). The serum was applied twice daily, morning and evening, for four weeks. Skin parameters were measured before the experiment (week 0) and after the experiment (weeks 2 and 4).

[0144] The test indicators include: transepidermal water loss (TEWL), measured using a water loss tester, which reflects the skin barrier function; the lower the value, the better the barrier function. Erythema index (EI), measured using a skin colorimeter, reflects the degree of skin inflammation; the lower the value, the milder the inflammation. Skin hydration, measured using a skin moisture tester, reflects the skin's hydration status; the higher the value, the higher the hydration.

[0145] 8.3.3 Test Results

[0146] The test results of transepidermal water loss of the skin are as follows Figure 7As shown in the figure, after 4 weeks of use, the transdermal water loss value in the sample group decreased from 18.3 g / h·m² to 10.5 g / h·m², with an improvement rate of 42.6%, while the placebo group only decreased from 18.5 g / h·m² to 16.5 g / h·m², with an improvement rate of 10.8%. The difference between the two groups was extremely significant (p < 0.01), indicating that CS2201 can effectively repair the skin barrier function.

[0147] The results of the skin erythema index test are as follows: Figure 8 As shown, after 4 weeks of use, the erythema index in the sample group decreased from 12.8 to 7.5, with an improvement rate of 41.4%, while the placebo group only decreased from 12.6 to 11.2, with an improvement rate of 11.1%. The difference between the two groups was extremely significant (p < 0.01), indicating that CS2201 can significantly reduce skin inflammation.

[0148] Skin moisture content test results as follows Figure 9 As shown in the figure, after 4 weeks of use, the skin 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 from 38.2 au to 41.5 au, an increase of 8.6%. The difference between the two groups was extremely significant (p < 0.01), indicating that CS2201 can significantly improve skin hydration.

[0149] The above results indicate that CS2201, when added as an active ingredient to serum, can significantly improve skin barrier function, reduce skin inflammation, and increase skin hydration. It has comprehensive effects of anti-inflammatory, soothing, and skin barrier repair, and is suitable for the development of sensitive skin care and daily anti-inflammatory and soothing skin care products.

[0150] 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 polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein, The polypeptide is a polypeptide with anti-inflammatory, soothing and / or skin barrier repair effects.

3. A polypeptide variant, characterized in that, The amino acid sequence of the polypeptide variant is an N-terminal truncated variant of SEQ ID NO: 1, wherein the truncated variant retains at least amino acids 10-22 of SEQ ID NO: 1 and has anti-inflammatory activity.

4. The polypeptide variant of claim 3, wherein, The amino acid sequence of the polypeptide variant is as follows: (a) As shown in SEQ ID NO: 3; Or (b) as shown in SEQ ID NO:

4.

5. A method for the production of a polypeptide according to any of claims 1 to 2 or a variant of a polypeptide according to any of claims 3 to 4, characterized in that, Prepared using the Fmoc solid-phase peptide synthesis method; Using Rink Amide resin as a solid support, the amino acid sequences of the polypeptides according to any one of claims 1-2 or the polypeptide variants according to any one of claims 3-4 are sequentially subjected to deprotection and coupling reactions. After synthesis, the target peptide or peptide variant is obtained by cleavage with a cleavage reagent, purification by reversed-phase high-performance liquid chromatography, and lyophilization.

6. A composition characterized in that, Its active ingredient comprises a polypeptide as described in any one of claims 1-2 or a polypeptide variant as described in any one of claims 3-4, and the composition further comprises a cosmetic or pharmaceutically acceptable matrix, carrier, excipient or other inactive ingredient.

7. The composition of claim 5, wherein, The polypeptide or polypeptide variant has a mass percentage content of 0.001%-5%; And / or, the dosage form of the composition is a serum, lyophilized powder, facial mask liquid, gel, spray or cream.

8. The composition of claim 6, wherein, The composition is an anti-inflammatory and soothing essence containing 0.05 wt% of the polypeptide described in claim 1 or 2, 5.0 wt% of glycerin, 3.0 wt% of butylene glycol, 0.1 wt% of sodium hyaluronate, 0.2 wt% of allantoin, 0.5 wt% of p-hydroxyacetophenone, 0.5 wt% of 1,2-hexanediol, and deionized water to 100 wt%.

9. The use of a polypeptide according to any one of claims 1-2, a polypeptide variant according to any one of claims 3-4, or a composition according to any one of claims 6-8, characterized in that, The application is as follows: Application in the preparation of cosmetics or pharmaceuticals with anti-inflammatory, soothing and / or skin barrier repair effects.

10. Use according to claim 9, characterized in that, The anti-inflammatory effect is achieved by penetrating the cell membrane into the cytoplasm, inhibiting p65 nuclear translocation, blocking the NF-κB signaling pathway, and reducing the release of inflammatory factors NO, TNF-α, IL-6, and IL-1β.

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