Skin-moisturizing short peptide and application thereof

Abstract

This invention discloses a skin-moisturizing short peptide and its applications, belonging to the field of bioactive short peptide technology. The amino acid sequence of the short peptide is shown in SEQ ID NO: 3, derived from lotus seed protein, and the molecular weight of the short peptide is less than 1000 Da. This invention precisely screened a novel moisturizing active short peptide, QEMGR (SEQ ID NO: 3), from lotus seed protein. It consists of only 5 amino acids, has a molecular weight of less than 1000 Da, and has the advantages of simple synthesis, low cost, and high transdermal absorption efficiency.

Description

Technical Field

[0001] This invention relates to the field of bioactive short peptide technology, specifically to a skin moisturizing short peptide and its applications. Background Technology

[0002] Developing small-molecule peptides with moisturizing activity from natural plant proteins is a research hotspot in the field of skincare ingredients. Short peptides, due to their small molecular weight, high transdermal absorption efficiency, and low chemical synthesis cost, have broad prospects for cosmetic applications. Lotus seed protein is rich in various amino acids and is a potential source for developing moisturizing active peptides.

[0003] However, existing technologies for obtaining short peptides from lotus seed proteins that possess both ultra-short sequences and highly efficient moisturizing activity face several challenges. The excessively short amino acid sequences of these peptides may result in the loss of key pharmacological groups, making it difficult to form stable binding bonds with moisturizing-related target proteins. While sequence lengthening can enhance target affinity, it increases molecular weight, raises synthesis costs, and reduces transdermal efficiency. Furthermore, current techniques for screening moisturizing peptides from lotus seed proteins primarily focus on peptides of conventional length, lacking systematic screening for short peptides. Therefore, we propose a short skin moisturizing peptide and its applications to alleviate or resolve the aforementioned problems.

[0004] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a skin-moisturizing short peptide and its application, thereby solving the problem of the lack of lotus seed-derived short peptides that possess both ultra-short sequences and highly efficient moisturizing activity in the prior art.

[0006] To achieve the above objectives, the present invention provides a skin moisturizing short peptide, wherein the amino acid sequence of the lotus seed short peptide is QEMGR (SEQ ID NO: 3).

[0007] The present invention also provides the application of the lotus seed short peptide in the preparation of skin moisturizing or anti-dryness products.

[0008] Compared with the prior art, the beneficial effects of the present invention are:

[0009] This invention precisely screened a novel moisturizing active short peptide, QEMGR (SEQ ID NO: 3), from lotus seed protein. Composed of only 5 amino acids with a molecular weight less than 1000 Da, it boasts advantages such as simple synthesis, low cost, and high transdermal absorption efficiency. Experiments confirmed that this short peptide can bind with high affinity to six human skin barrier-related enzyme proteins, with binding free energies all exceeding -6 kcal / mol. Furthermore, it significantly improved the survival rate of immortalized human keratinocytes (HaCaT) in a cell desiccation stress model, demonstrating superior moisturizing activity.

[0010] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0011] Figure 1 This is a diagram showing the three-dimensional structure and amino acid residue confidence distribution of the short peptide Pep#002 (SEQ ID NO: 3) predicted using AlphaFold in Example 1 of this invention.

[0012] Figure 2 The figure shows the results of three protective experiments on the survival rate of human immortalized keratinocytes (HaCaT) after extreme dryness stress using the short peptide Pep#002 in Example 3 of this invention. Detailed Implementation

[0013] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be noted that the drawings are schematic and not illustrated to scale. For clarity and convenience, the relative sizes and proportions of the parts shown in the drawings have been exaggerated or reduced in size. Any size is only exemplary and not limiting.

[0014] Example 1: Virtual screening and preparation of lotus seed short peptides

[0015] Lotus seed protein sequences were obtained from the Nelumbo nucifera genome database. Using the virtual hydrolysis tool in the BIOPEP-UWM database, the amino acid sequences of lotus seed proteins were independently virtually hydrolyzed using pepsin (pH 1.3), trypsin, chymotrypsin A, papain, bromelain, and alcalase, respectively. All peptides generated by the six enzymes were collected, and after removing repetitive sequences, an initial candidate peptide library was constructed.

[0016] All peptides obtained from virtual enzyme digestion were screened using a multi-dimensional approach, with peptide lengths defined as 4-10 amino acid residues; molecular weight less than 1000 Da; PeptideRanker score greater than 0.31; and Boman index greater than 3.0. After initial screening, the core sequence Pep#002:QEMGR (SEQ ID NO: 3) meeting the screening criteria was obtained from the bromelain digestion product of lotus seed protein. The PeptideRanker score was 0.315, and the Boman index was 4.79 kcal / mol.

[0017] Pep#002 was prepared using the Fmoc solid-phase peptide synthesis method (SPPS). Purification was performed by reversed-phase high-performance liquid chromatography (RP-HPLC) using an acetonitrile / water system (containing 0.1% trifluoroacetic acid) as the mobile phase for gradient elution. ESI-MS analysis showed that the measured molecular weight was consistent with the theoretical value, and the HPLC purity was greater than 98%. The product was then lyophilized under vacuum and stored at -20°C.

[0018] The three-dimensional structure of Pep#002 (SEQ ID NO: 3) was predicted using AlphaFold 3. (See attached image.) Figure 1 As shown, Pep#002 exhibits a certain flexible conformation in solution, and the confidence distribution of its amino acid residues shows that the prediction results have high confidence, giving the short peptide good conformational adaptability and target adaptation ability.

[0019] Example 2: Molecular docking simulation of active peptides with multiple skin targets

[0020] Crystal structures of six human skin barrier-related enzyme proteins (CEIP2, TYRO, MMP13, MMP1, NEP, GSHR) were obtained from the PDB database. Water molecules and ligands were removed using PyMOL software, and hydrogenation, charge calculation, and atom type assignment were performed using AutoDockTools. The zinc ion region of the MMP1 active site was designated as the docking pocket (grid box), with center coordinates set at (36.1, 14.5, 34.2) and a size of 60×60×60 Å. Flexible docking was performed using AutoDock Vina, and binding energies were calculated. As shown in Table 1, Pep#002 binds well to all six skin targets, with binding free energies all below -6 kcal / mol, meeting the evaluation criteria for high-affinity binding. The docking parameters for the other five target proteins were independently set according to the reported active site coordinates and pocket sizes in the PDB database.

[0021] This binding posture indicates that Pep#002 occupies the substrate binding regions of six target sites, forming steric hindrance and possessing good moisturizing potential.

[0022] Table 1. Results of Pep#002 short peptide molecular docking

[0023] Human skin barrier-related enzymes Binding affinity (kcal / mol) CEIP2 -8.0 TYRO -6.5 MMP13 -6.1 MMP1 -7.1 NEP -7.2 GSHR -6.4

[0024] Example 3: Protective experiment of Pep#002 against keratinocyte dryness damage

[0025] (1) Preparation of short peptide samples

[0026] The short peptide powder shown in SEQ ID NO: 3 obtained in Example 1 was accurately weighed, thoroughly dissolved in sterile PBS buffer, and prepared into a high-concentration stock solution. After sterilization by filtration through a 0.22 µm microporous membrane, it was aliquoted and stored at -80°C. Before the experiment, the stock solution was diluted to 10, 25, 50, 100, 200, 500, and 1000 µM using complete culture medium.

[0027] (2) Cell culture and grouping

[0028] The cell line was human immortalized keratinocytes (HaCaT), cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0029] (3) Grouping

[0030] Normal control group: cultured under normal humidity conditions.

[0031] Dry model group: cultured under extremely dry conditions (RH~11%).

[0032] Short peptide intervention group: pretreated with 25, 50, 100, and 200 µM Pep#002.

[0033] Positive control group: 100 µg / mL hyaluronic acid (HA) was added.

[0034] (4) Construction of the extreme dryness model

[0035] A saturated barium chloride (BaCl2) solution was added to the bottom of a sealed desiccator to create a microenvironment with a constant relative humidity (RH) of approximately 11%. The pretreated HaCaT cells were then aspirated of their culture medium and placed in the desiccator for 18 hours of drying stress.

[0036] (5) Cell viability assay

[0037] Healthy cells in the logarithmic growth phase were selected, digested, and the cell density was adjusted to 1×10⁴ cells / well using complete culture medium. Cells were seeded into 96-well plates, with 100 µL of cell suspension added to each well. Cells were cultured for 24 h to allow adhesion. The old culture medium was then discarded, and 100 µL of pre-diluted recombinant protein working solution (concentration gradient: 25, 50, 100, 200, 300, 400, 500 µg / mL) was added to each well. Different concentrations of the test sample were added, and cells were cultured for 24 h. A zero-entry group (cell-free but containing culture medium) and a control group (containing cells but no drug) were established. After 24 h of culture, the old culture medium was discarded and replaced with fresh complete culture medium. 10 µL of CCK-8 reagent was added to each well, and the mixture was gently mixed. The cells were incubated at 37°C in a 5% CO₂ incubator for 3 h. The absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated for each treatment group.

[0038] The results are attached. Figure 2 As shown, the cell survival rate of the drying model group decreased significantly, indicating that extreme dryness stress caused cell damage. Lotus seed short peptides can significantly improve the survival rate of human immortalized keratinocytes (HaCaT) under extreme dryness at low concentrations, with the 100 μM concentration showing the best effect, indicating that lotus seed short peptide Pep#002 has superior moisturizing activity.

[0039] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0040] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A short peptide for skin moisturizing, characterized in that: The amino acid sequence of the short peptide is shown in SEQ ID NO: 3, which is derived from lotus seed protein, and the molecular weight of the short peptide is less than 1000 Da.

2. The use of the short peptide according to claim 1 in the preparation of skin moisturizing or anti-dryness products.

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