Bioactive peptides, derivatives and uses thereof

Abstract

The present application relates to the field of biological medicine and daily chemical, and discloses a kind of bioactive peptide and its derivative.The amino acid sequence of bioactive peptide is FKTT;Modification is carried out on its amino terminal, carboxyl terminal or any amino acid side chain group to obtain its derivative.The bioactive peptide and its derivative of the present application have good anti-aging and antioxidant effects, and can be widely used in the field of medicine, daily chemical, health care product or food, etc., and have good application prospect.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a skin care product or pharmaceutical product whose main ingredient is a bioactive peptide with anti-aging activity. Background Technology

[0002] Bioactive peptides are a class of polypeptides that are beneficial to health or have specific biological functions. They are specific protein fragments composed of short amino acid sequences, generally consisting of two or more but less than 20 amino acids linked by peptide bonds, with a molecular weight of less than 6 kDa. They can be obtained from protein hydrolysates through enzymatic hydrolysis, microbial fermentation, and acid and alkali treatment.

[0003] Peptides possess unique biological activities, forming fundamental components of protein structure and function. They exhibit a wide range of biological activities, including antioxidant, ACE inhibitory, antitumor, immunomodulatory, anti-aging, and anti-inflammatory effects. Due to their high specificity, low toxicity, high structural diversity, and low molecular weight, they are widely used in the pharmaceutical, food, and cosmetic industries. Bioactive peptides, with their diverse amino acid sequences and high structural plasticity, are increasingly becoming important candidate molecules for drug design.

[0004] In the field of cell biology, maintaining cellular energy homeostasis is a core element in ensuring the body's health. Cellular regeneration, the process by which energy carriers such as adenosine triphosphate (ATP) are synthesized within cells, mainly depends on mitochondrial oxidative phosphorylation. When cellular energy metabolism is impaired, such as when mitochondrial function is disordered, it leads to insufficient cellular energy supply, which in turn causes cellular dysfunction, aging, and even apoptosis. This is closely related to various metabolic diseases, neurodegenerative diseases, and the aging process.

[0005] Traditional strategies for improving cellular energy metabolism, such as supplementing with basic nutrients like coenzyme Q10 and B vitamins, while effective to some extent, often lack specificity or have limited efficiency. Therefore, there is an urgent need in this field to find novel bioactive substances that can more directly and efficiently activate mitochondrial function and promote regeneration. Recent studies have found that many endogenous and exogenous bioactive peptides can directly or indirectly regulate and promote cellular regeneration through various mechanisms; however, the specific mechanisms are not yet fully understood.

[0006] To address the shortcomings of existing technologies, this application focuses on a strain of *Saccharomyces cerevisiae* originating from the Himalayas (CN114081862A, China General Microbiological Culture Collection Center accession number CGMCC No. 19732). Its fermentation product, HiMurchaSin®, contains various skin-beneficial components such as proteins, peptides, amino acids, minerals, and vitamins, exhibiting excellent repair and cell regeneration capabilities. It can promote skin cell proliferation and collagen regeneration, thereby achieving anti-aging and repair effects. Through research on the protein and peptide information identified in HiMurchaSin® samples, we aim to find bioactive short peptides with better biological activity and the ability to improve cellular energy metabolism. These peptides can serve as important raw materials for applications in cosmetics, pharmaceuticals, food, and health products. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a bioactive peptide and its derivatives with multiple functions.

[0008] In a first aspect, the present invention relates to a bioactive peptide and its derivatives.

[0009] The amino acid sequence of the bioactive peptide (FT-41) is FKTT (SEQ ID No. 1). Its structural formula is shown in formula (A).

[0010] Formula (A) The derivatives of the bioactive peptide are obtained by modifying the amino acid side chain groups, amino terminus, or carboxyl terminus of the above-mentioned bioactive peptide FT-41 with hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification, or glycosylation, etc., without affecting its biological activity.

[0011] In some embodiments, the N-terminus of the bioactive peptide FT-41 is palmitoylated to obtain the modified peptide Pal-FT-41 (or represented by pal FT-41, pal-FT-41, etc.), as shown in Formula (B).

[0012] Formula (B) FT-41 and its derivatives can significantly enhance cell repair capabilities and repair damaged skin, especially damage caused by ultraviolet radiation, peroxides, and oxygen free radicals. They can also increase collagen expression in cells, particularly promoting collagen recovery after ultraviolet damage. Therefore, this suggests that FT-41 and its derivatives possess antioxidant, anti-aging, and sun protection effects, especially against photoaging.

[0013] In addition, FT-41 and its derivatives help maintain and stabilize the skin barrier, protect skin cells, and reduce skin damage.

[0014] FT-41 derivatives, especially Pal-FT-41 with N-terminal palmitoylation modification, can also promote ATP production in cells and increase ATP content. In some embodiments of the present invention, the palmitoylated derivative Pal-FT-41 can increase the ATP content in keratinocytes (such as HaCat), thereby enhancing cell vitality and function, and helping to improve skin condition, repair damage and anti-aging.

[0015] In some embodiments of the present invention, Pal-FT-41 also helps to increase the mitochondrial membrane potential of cells, especially the mitochondrial membrane potential of skin cells after ultraviolet irradiation, maintain mitochondrial function, reduce cell damage, and help delay aging, especially anti-photoaging.

[0016] Therefore, FT-41 and its derivatives have promising applications in pharmaceuticals, food or health products, cosmetics or skincare products.

[0017] In a second aspect, the present invention relates to a pharmaceutical composition comprising the bioactive peptide and a pharmaceutically acceptable carrier.

[0018] In some embodiments, the pharmaceutically acceptable carrier is selected from any one or any combination thereof, water, isotonic saline, ethanol, phosphate buffer, sorbitol, mannitol, starch, modified starch, gum, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, and cellulose.

[0019] Cellulose includes microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), ethyl cellulose (EC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), etc.

[0020] Gum includes acacia gum (gum arabic), astragalus gum, peach gum, propolis, etc.

[0021] In other embodiments, the pharmaceutical composition may be administered orally, inhaled, rectally, nasally, ocularly, transdermally, or otherwise parenterally.

[0022] In other embodiments, the pharmaceutical composition is in the form of an oral, parenteral, spray, topical, or transdermal dosage form. The parenteral dosage form includes, but is not limited to, suppositories, buccal suppositories, sublingual suppositories, nasal suppositories, or inhalers.

[0023] In other embodiments, the pharmaceutical composition further contains other active ingredients that have antioxidant or anti-aging effects.

[0024] Thirdly, the present invention provides a raw material for daily chemical use, containing the above-mentioned bioactive peptides or bioactive peptide derivatives.

[0025] Fourthly, the present invention provides the application of the above-mentioned bioactive peptides, bioactive peptide derivatives, pharmaceutical compositions or daily chemical raw materials in the preparation of skin care products or cosmetics, or the application of the above-mentioned bioactive peptides, bioactive peptide derivatives or daily chemical raw materials in the preparation of food or health products with antioxidant effects.

[0026] Preferably, the skin care product or cosmetic product has anti-aging, and / or antioxidant, and / or skin barrier strengthening, and / or UV protection (sunscreen) effects.

[0027] In some embodiments, the anti-aging effects include: enhancing the ability to repair cellular oxidative damage; and enhancing the skin's ability to repair damage from ultraviolet radiation or sun exposure (anti-photoaging), and increasing collagen expression in cells. The collagen includes type I and type III collagen.

[0028] In some implementation schemes, strengthening the skin barrier means maintaining the skin barrier and reducing skin damage when exposed to external stimuli.

[0029] Skincare or cosmetic products include toners, lotions, serums, facial oils, lotions, creams, facial cleansers, makeup removers / oils, sunscreens, makeup bases, shampoos, body washes, liquid foundations, pressed powders, eyeshadows, lipsticks, etc.

[0030] In some implementations, the drug has anti-aging and / or antioxidant effects.

[0031] In some embodiments, the anti-aging effects include: enhancing the repair capacity of cells from oxidative damage and increasing collagen expression in cells damaged by ultraviolet radiation. The collagen includes type I and type III collagen.

[0032] Fifthly, the present invention provides skin care products or cosmetics containing the above-mentioned bioactive peptides, bioactive peptide derivatives, pharmaceutical compositions or daily chemical raw materials; or food or health products containing the above-mentioned bioactive peptides, bioactive peptide derivatives, pharmaceutical compositions or daily chemical raw materials that have antioxidant effects.

[0033] Sixthly, the present invention provides a medicine comprising the above-mentioned bioactive peptides, bioactive peptide derivatives, pharmaceutical compositions, or raw materials for daily chemical use.

[0034] Molecular docking analysis revealed that both FT-41 and Pal-FT-41 possess the potential for stable binding to SIRT1 and its substrate peptides, potentially exhibiting a similar binding effect to the SIRT1 agonist resveratrol, thereby activating SIRT1. Following N-terminal palmitoylation modification, the binding affinity changed from -10.18 kcal / mol to -13.88 kcal / mol, showing a trend towards tighter binding. This suggests that the N-terminal palmitoylated derivative, Pal-FT-41, exhibits stronger molecular stability and binding advantage, potentially increasing ATP levels in HaCat cells and enhancing mitochondrial function. It may also help improve skin function, such as repairing and strengthening the skin barrier, accelerating damage repair, and anti-aging, indicating its promising application prospects in drug development. Through molecular docking and computational simulation techniques, it was found that the homologous active peptide FT-41 of the Myosin-1 protein fragment and its N-terminal palmitoylated molecule Pal-FT-41 in the sample could form a stable binding with the SIRT1 active site in molecular docking and had potential activity superior to traditional small molecules, suggesting that they may play a unique and important role in regulating the SIRT1-Ki67-PGC1α related pathway.

[0035] The beneficial effects of this invention lie in providing a bioactive peptide FT-41 and its derivative variants, which exhibit excellent anti-aging and antioxidant effects. Furthermore, through modification, derivatives with superior activity and function are obtained. Compared with existing technologies, the bioactive peptides and derivatives of this invention are homologous to yeast fermentation products, have short lengths and small molecular weights, are easy to prepare, and possess good permeability and absorption. They can be used in multiple fields such as pharmaceuticals, health products, food, and daily chemical products (e.g., cosmetics, skincare products), and have broad application prospects. Attached Figure Description

[0036] Figure 1 Example 3 shows the test results of how active peptides FT-41 and pal-FT-41 promote ATP production in HaCaT cells.

[0037] Figure 2 Example 4 shows the test results of how the active peptide pal-FT-41 increases the mitochondrial membrane potential JC-1 in FB cells.

[0038] Figure 3 Example 5 shows the test results of the damage repair model of active peptides FT-41 and pal-FT-41.

[0039] Figure 4 Example 5 shows the results of the antioxidant ROS test of active peptides FT-41 and pal-FT-41.

[0040] Figure 5Example 6 shows the test results of active peptides FT-41 and pal-FT-41 repairing collagen I in UVA-damaged HSF cells.

[0041] Figure 6 Example 6 shows the test results of active peptides FT-41 and pal-FT-41 repairing collagen III in UVA-damaged HSF cells.

[0042] Figure 7 Example 7 shows the predicted molecular docking results of the active peptide FT-41.

[0043] Figure 8 Example 7 shows the results of pal-FT-41 prediction of molecular docking. Detailed Implementation

[0044] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0045] Although the numerical ranges and parameter approximations shown in the broad scope of this application are intended to be as accurate as possible in the specific embodiments, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the stated range “1 to 10” should be considered to include any and all subranges between the minimum value 1 and the maximum value 10 (inclusive); that is, all subranges beginning with a minimum value of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any references marked “incorporated herein” should be understood to be incorporated herein in their entirety.

[0046] Example 1: Acquisition and preparation of FT-41 short peptide CN114081862A (the full text of which is incorporated herein by reference) discloses a brewing yeast derived from barley wine starter (accession number CGMCC No. 19732). Its fermentation product, HiMurchaSin®, can scavenge free radicals, promote cell proliferation, inhibit tyrosinase, protect skin cells from damage caused by oxidation or ultraviolet radiation, and regulate skin flora and microecology. Therefore, it can achieve antioxidant, anti-aging, sun protection, improve skin cell vitality, and protect the skin barrier. The inventors discovered through component analysis that it contains various active ingredients such as organic acids, phenolic vitamins, free amino acids, peptides, and proteins.

[0047] Furthermore, mass spectrometry analysis of the protein components in the fermentation products revealed several candidate protein peptides, one of which has the amino acid sequence FKTT (abbreviated as FT-41, as shown in SEQ ID No. 1, structural formula as shown in formula (A)) and a molecular weight of approximately 700 Da. FT-41 can be prepared using conventional methods such as genetic engineering, enzyme engineering, chemical synthesis, and natural extraction, or by combining these techniques. For example, it can be prepared using solid-phase organic synthesis.

[0048] Example 2: Acquisition and preparation of Pal-FT-41 short peptide The N-terminus of the bioactive peptide FT-41 was modified by palmitoylation to obtain the modified peptide Pal-FT-41, the structure of which is shown in formula (B). The N-terminal palmitoylation product can be prepared by conventional methods such as genetic engineering, enzyme engineering or chemical synthesis, for example, by the more common solid-phase organic synthesis.

[0049] The following examples test the various effects of FT-41 and Pal-FT-41 respectively.

[0050] Example 3: HaCaT cell ATP assay FT-41 and Pal-FT-41 were added to the culture medium to a concentration of 1-5 ppm, and the cells were co-incubated with human immortalized keratinocytes (HaCaT cells) for 24 hours. CellTiter-Glo® reagent (Promega, Cat#G7570) was then added and incubated for 10 minutes. Utilizing the principle that luciferase requires ATP for its luminescence reaction with beetle luciferin substrate, and that the amount of ATP is linearly related to luminescence intensity, the ATP content of each well was obtained by detecting luminescence intensity after incubation. The results are as follows: Figure 1 .

[0051] Experimental results showed that 5 ppm of Pal-FT-41 promoted ATP production in HaCaT. Figure 1A), the ATP content increased by 17.61%. However, FT-41 at concentrations of 1 ppm and 5 ppm had no promoting effect on the ATP content of human immortalized keratinocytes. Figure 1 B).

[0052] Therefore, the palmitoylated derivative Pal-FT-41 can increase the ATP content of HaCat cells, enhance cell vitality and function, maintain cellular energy homeostasis, and help improve skin condition, such as repairing and strengthening the skin barrier, accelerating damage repair, and anti-aging.

[0053] Example 4: JC-1 assay of mitochondrial membrane potential in FB cells (fibroblasts) The mitochondrial membrane potential is the electrochemical gradient formed by the proton concentration difference across the inner mitochondrial membrane. Its normal range is 180-200 mV (positive on the outside, negative on the inside), and it is mainly maintained by the proton pump function of oxidative respiratory chain complexes I, III, and IV. This potential is the energy basis for ATP synthesis and a core indicator for evaluating mitochondrial function. JC-1 is a fluorescent probe widely used to detect changes in mitochondrial membrane potential. Under normal conditions, JC-1 forms a polymer in the mitochondrial matrix, producing orange or red fluorescence at 585 / 590. When the mitochondria are at a low potential, JC-1 becomes free in the cytoplasm, and the monomeric JC-1 produces green fluorescence at 514 / 529. Therefore, the intensity of the red and green fluorescence signals can be used to determine the state of the mitochondrial membrane potential, thus reflecting mitochondrial function.

[0054] Use 2.5J / cm 2 Normal human dermal fibroblasts were irradiated with UVA to induce NHDF, and then Pal-FT-41 was added at 1 ppm and 5 ppm, respectively, and the cells were incubated overnight in an incubator. Probe incubation was performed using the JC-1 kit according to the manufacturer's instructions. Images were captured using a fluorescence microscope, and quantitative analysis of fluorescence signals was performed using ImageJ.

[0055] The results are as follows Figure 2 Analysis showed that Pal-FT-41 significantly enhanced mitochondrial membrane potential, with an increase of 11% at a dosage of 1 ppm. p <0.01), with an increase of 23% at an addition of 5 ppm ( p <0.01), can reduce and repair the damage caused by ultraviolet rays to the mitochondria of skin cells.

[0056] Example 5: Damage Repair Test Experiment of Peptide FT-41 and pal-FT-41 Cell model: SLS (sodium dodecyl sulfate) damage to HaCaT cells Human immortalized keratinocytes (HaCaT cells) were co-incubated with 5-20 ppm of FT-41 and Pal-FT-41 active ingredients, with 1% SLS added, for 24 hours. Subsequently, CCK-8 reagent was added and incubated for 60 minutes. Utilizing the carrier 1-Methoxy PMS, it was reduced by dehydrogenases in the cell mitochondria to a highly water-soluble yellow formazan product. The amount of formazan produced was directly proportional to the number of viable cells. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) scanner, indirectly reflecting the number of viable cells.

[0057] Test groups: 1% Ceramides Mixture; pal-FT-41 (5 / 10 / 20ppm); FT-41 (5 / 10 / 20ppm).

[0058] Experimental results show that... Figure 3 SLS intervention reduced HaCaT cell activity to 54.63%. Both pal-FT-41 and FT-41 promoted HaCaT cell activity after SLS intervention. At a concentration of 20 ppm, cell viability was 67.012% and 73.098%, respectively.

[0059] The results showed that pal-FT-41 and FT-41 help maintain and stabilize the skin barrier and reduce skin damage.

[0060] In addition, similar experiments were conducted on H2O2-damaged HaCaT cells using peptides FT-41 and pal-FT-41, with results as follows: Figure 4 This also indicates that both pal-FT-41 and FT-41 can inhibit ROS production in HaCaT cells induced by hydrogen peroxide. This suggests that in addition to repairing and reducing skin damage, pal-FT-41 and FT-41 also have antioxidant effects, reducing skin damage caused by peroxides or oxygen free radicals.

[0061] Example 6: Test Experiment of FT-41 and pal-FT-41 Anti-UV Damage Collagen Cell model: UVA damage to HSF cells.

[0062] HSF cells were induced by UVA (induction intensity 40 mJ / cm²). 2Human skin fibroblasts (HSF) cells were co-incubated with 5-20 ppm of FT-41 and Pal-FT-41 active ingredients for 24 hours. Cell supernatant was then collected. Type I and type III collagen content was determined using enzyme-linked immunosorbent assay (ELISA). The principle is as follows: Type I collagen specifically binds to collagen antibodies coated on an ELISA plate, which then bind to substrate-labeled anti-type I collagen antibodies. The substrate is catalyzed by the enzyme to generate a colored product. The type I collagen content is positively correlated with the intensity of the colored product. The optical density (OD value) was measured at 450 nm using an ELISA reader to calculate the type I collagen content. The principle for detecting type III collagen is the same.

[0063] Test groups: 100 ng / mL TGF-β1 (transforming growth factor-β); pal-FT-41 (5 / 10 / 20 ppm); FT-41 (5 / 10 / 20 ppm).

[0064] Test results as follows Figure 5 and Figure 6 The experimental results showed that, compared with the blank control group (Control), the levels of Col I and Col III in HSF cells were significantly reduced after UVA intervention (Model). Treatment with pal-FT-41 and FT-41 both promoted the secretion of Col I and Col III in cells after UVA intervention. In other words, FT-41 and pal-FT-41 can significantly promote the recovery of type I and type III collagen in UVA-damaged HSF cells, and both have significant potential for photodamage repair and anti-aging. The results further validated the feasibility and dosage advantage of FT-41 and pal-FT-41 sequences as active peptides in skin repair and anti-aging products.

[0065] Example 7: Mechanism study of the anti-UV, anti-photoaging and repair effects of FT-41 and pal-FT-41 SIRT1 participates in regulating various cellular physiological processes and metabolic pathways, playing an important role in anti-aging. Studies have shown that activation of SIRT1 can improve the survival rate of keratinocytes damaged by ultraviolet radiation.

[0066] Molecular docking and computational simulation techniques have demonstrated that the homologous active peptide FT-41 and its N-terminal palmitoylated molecule Pal-FT-41 of this invention can both form stable binding with the SIRT1 active site during molecular docking. Figure 7 and Figure 8 As shown, both have the potential to bind stably to SIRT1 and its substrate peptides, and may have a similar binding effect to the SIRT1 agonist resveratrol, activating SIRT1.

[0067] FT-41 and Pal-FT-41 possess potential activities superior to traditional small molecules, suggesting that they may play a unique and important role in regulating the SIRT1-Ki67-PGC1α-related pathway.

[0068] The predicted molecular docking binding energy between FT-41 and SIRT1 is -10.18 kcal / mol. After N-terminal palmitoylation modification, the binding affinity between Pal-FT-41 and SIRT1 changes from -10.18 kcal / mol to -13.88 kcal / mol, showing a trend towards a tighter binding.

[0069] Compared to the unmodified peptide FT-41, the N-terminal palmitoyl-modified variant Pal-FT-41 exhibits stronger molecular stability and binding advantages, suggesting its superior application prospects in the development of pharmaceuticals, cosmetics, skincare products, food, and health supplements.

[0070] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. 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 bioactive peptide, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. A bioactive peptide derivative, characterized in that, It is a derivative obtained by palmitoylation modification of the amino terminus of a polypeptide with an amino acid sequence as shown in SEQ ID No.

1.

3. A pharmaceutical composition, characterized in that, Its active ingredient is the bioactive peptide described in claim 1 or the bioactive peptide derivative described in claim 2.

4. The pharmaceutical composition according to claim 3, characterized in that, It also contains pharmaceutically acceptable carriers.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutically acceptable carrier is any one or any combination thereof, water, isotonic saline, ethanol, phosphate buffer, sorbitol, mannitol, starch, gum, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, and cellulose.

6. The pharmaceutical composition according to any one of claims 3-5, characterized in that, Its dosage forms include oral, parenteral, inhalation, spray, topical, or transdermal formulations.

7. A raw material for daily chemical use, characterized in that, It contains the bioactive peptide of claim 1 or the bioactive peptide derivative of claim 2.

8. The use of the bioactive peptide of claim 1, the bioactive peptide derivative of claim 2, the pharmaceutical composition of any one of claims 3-6, or the daily chemical raw material of claim 7 in the preparation of skin care products or cosmetics, characterized in that, The skincare or cosmetic products described herein have anti-aging, and / or antioxidant, and / or anti-ultraviolet, and / or anti-photoaging effects.

9. The use of the bioactive peptide of claim 1, the bioactive peptide derivative of claim 2, or the pharmaceutical composition of any one of claims 3-6 in the preparation of a pharmaceutical product, characterized in that, The medicine described has anti-aging and / or antioxidant effects.

10. The application of the bioactive peptide of claim 1 or the bioactive peptide derivative of claim 2 in the preparation of health products with antioxidant effects.

11. A skincare or cosmetic product, characterized in that, It contains the bioactive peptide of claim 1, the bioactive peptide derivative of claim 2, or the daily chemical raw material of claim 7.

12. A drug, characterized in that, It contains the bioactive peptide of claim 1 or the bioactive peptide derivative of claim 2.

13. A health product with antioxidant properties, characterized in that, It contains the bioactive peptide of claim 1 or the bioactive peptide derivative of claim 2.

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