Anti-aging polypeptide and application thereof

Abstract

The invention relates to the technical field of polypeptide application, and particularly discloses an anti-aging polypeptide and application thereof.The structural general formula of the anti-aging polypeptide is H2N-(AA) m-Phe-Lys-Val-Pro-Asn-Met-(AA) n-COOH, AA represents any amino acid, and m and n are integers ranging from 0 to 4. Experiments prove that the anti-aging polypeptide provided by the invention can effectively inhibit oxidative stress to resist inflammation, can also improve the memory level, and has a remarkable anti-aging effect. The invention provides powerful technical support for further exploration of clinical application of peptide molecules and development of health food and medicines.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptide application, and particularly relates to an anti-aging polypeptide and application thereof. BACKGROUND

[0002] Aging is a complex biological process of systemic decline in physiological function of the body, and the cognitive dysfunction caused by it, especially the loss of episodic and spatial memory, has become a core problem affecting human health. This memory decline is closely related to structural damage to neurons and reduced synaptic plasticity in the hippocampus, and oxidative stress has been confirmed as a key mechanism driving this pathological process. Under physiological conditions, the body maintains the dynamic balance of reactive oxygen species (ROS) through endogenous antioxidant systems such as superoxide dismutase (SOD) and glutathione peroxidase (GPx); however, as age increases, mitochondrial dysfunction leads to excessive accumulation of ROS, while antioxidant defense capacity decreases, triggering persistent oxidative stress. Excessive ROS attacks biological macromolecules, inducing lipid peroxidation to damage the integrity of neuronal cell membranes and mitochondria, causing oxidative modification of proteins to inactivate synapse-related proteins (such as PSD-95 and Synaptophysin), and causing DNA oxidative damage to activate the apoptosis pathway. In the hippocampus, a key area for memory formation, oxidative stress damages neural function through multiple pathways - it inhibits the phosphorylation process of the CREB transcription factor, reduces the expression of brain-derived neurotrophic factor (BDNF), weakens synaptic strengthening and neuronal survival; at the same time, it activates microglia to release TNF-alpha, IL-1 beta and other pro-inflammatory factors, induces neuroinflammation and inhibits hippocampal neurogenesis; in addition, it also disrupts calcium homeostasis, inducing mitochondrial calcium overload and excitotoxicity.

[0003] Although existing intervention strategies such as exogenous antioxidants such as vitamin C / E, artificially synthesized anti-aging target drugs, etc. attempt to alleviate oxidative stress, regulate inflammation, and repair damage, their low bioavailability, poor blood-brain barrier penetration, insufficient neuronal targeting, and significant toxic side effects severely limit their efficacy. Therefore, finding new anti-aging active substances and developing new anti-aging products have urgent clinical value for improving memory, reducing oxidative stress levels, and delaying aging.

[0004] In recent years, peptide substances have been widely studied as a class of functional substances with high safety and good processing characteristics. They have been scientifically verified to have multiple biological activities, including but not limited to anti-aging, anti-viral, anti-tumor, antioxidant, and metabolic regulation, etc., due to their excellent bioavailability. However, most existing polypeptides are mixtures obtained from animals and plants, and the components need to be separated to enrich monomers with excellent activity, further improving their anti-aging efficacy.

[0005] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0006] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide an anti-aging polypeptide and its application, aiming to solve the problems of less types of existing anti-aging polypeptides and poor anti-aging effect.

[0007] The technical scheme of the present application is as follows:

[0008] An anti-aging polypeptide, wherein the structure general formula of the anti-aging polypeptide is: H2N-(AA) m -Phe-Lys-Val-Pro-Asn-Met-(AA) n -COOH, wherein AA represents any amino acid, and m and n are both integers between 0 and 4.

[0009] The anti-aging polypeptide, wherein the anti-aging polypeptide is one of Ala-Phe-Lys-Val-Pro-Asn-Met-Lys, Leu-Ala-Phe-Lys-Val-Pro-Asn-Met-Phe-Gln, Leu-Asn-Ala-Phe-Lys-Val-Pro-Asn-Met-Phe-Gln-Ile, Asn-Asn-Ala-Gln-Phe-Lys-Val-Pro-Asn-Met-Leu-Leu-Phe, and Phe-Lys-Val-Pro-Asn-Met.

[0010] Use of the anti-aging polypeptide as described in the present application in the preparation of an anti-aging drug or anti-aging health product.

[0011] The use, wherein the anti-aging drug or anti-aging health product further comprises one or more pharmaceutically acceptable excipients, and the excipients include carriers, diluents, excipients, adjuvants, buffers, pH regulators, preservatives, antioxidants, bacteriostatic agents, stabilizers, suspending agents, solubilizers, surfactants, coloring agents, and isotonicity solutes.

[0012] The use, wherein the anti-aging drug or anti-aging health product is in the form of an oral dosage form.

[0013] The use, wherein the oral dosage form includes sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, syrup, buccal preparations, granules, drop pills, powders, ointments, suspensions, and powders.

[0014] Beneficial effects: The present application provides an anti-aging polypeptide, which has been verified by experiments to effectively inhibit oxidative stress against inflammation and also improve memory level, and has a significant anti-aging effect. The present application provides strong technical support for further exploring the clinical application of peptide molecules and developing health food and drugs. Attached Figure Description

[0015] Figure 1 The graph shows the in vitro DPPH clearance rate of the anti-aging peptide in Example 1.

[0016] Figure 2 This is a graph showing the in vitro cell viability test results of the anti-aging peptides in Example 2.

[0017] Figure 3 This is a graph showing the results of the mouse escape latency test in Example 4.

[0018] Figure 4 The image shows the results of the test on the time the mouse spent on the platform in Example 4.

[0019] Figure 5 The image shows the results of the test on the number of times the mouse crossed the platform in Example 4. Detailed Implementation

[0020] This invention provides an anti-aging polypeptide and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0021] Peptides are intrinsic signaling molecules for many physiological functions, offering a therapeutic intervention opportunity that closely mimics natural pathways, and have thus attracted widespread attention as a therapeutic approach. Compared to small-molecule chemical drugs, peptides possess unique therapeutic properties and higher efficacy due to their high bioactivity and specificity. They also exhibit high safety, good tolerability, and are less prone to accumulation in the body, resulting in higher bioavailability and easier absorption and utilization by the human body. Compared to targeted drugs, long-term use of peptides is less likely to induce tolerance, providing a stable and long-term anti-aging effect.

[0022] Based on this, the present invention provides an anti-aging polypeptide, the amino acid sequence of which is H2N-(AA). m -SEQ ID No.5-(AA) n As shown in the diagram, -COOH is specifically H2N-(AA). m -Phe-Lys-Val-Pro-Asn-Met-(AA) n -COOH, where AA represents any amino acid, and m and n are both integers between 0 and 4. The anti-aging polypeptides provided by this invention can be obtained through natural extraction or chemical synthesis, preferably through conventional chemical synthesis (solid-phase / liquid-phase synthesis) or enzymatic synthesis.

[0023] In some embodiments, the amino acid sequence of the anti-aging polypeptide is as shown in SEQ ID No. 1, specifically: Ala-Phe-Lys-Val-Pro-Asn-Met-Lys, denoted as Seq1, and the corresponding structural formula is:

[0024] In some embodiments, the amino acid sequence of the anti-aging polypeptide is as shown in SEQ ID No. 2, specifically: Leu-Ala-Phe-Lys-Val-Pro-Asn-Met-Phe-Gln, denoted as Seq2, and the corresponding structural formula is:

[0025] In some embodiments, the amino acid sequence of the anti-aging polypeptide is as shown in SEQ ID No. 3, specifically: Leu-Asn-Ala-Phe-Lys-Val-Pro-Asn-Met-Phe-Gln-Ile, denoted as Seq3, and the corresponding structural formula is:

[0026] In some embodiments, the amino acid sequence of the anti-aging polypeptide is as shown in SEQ ID No. 4, specifically: Asn-Asn-Ala-Gln-Phe-Lys-Val-Pro-Asn-Met-Leu-Leu-Phe, denoted as Seq4, and the corresponding structural formula is:

[0027]

[0028] In some embodiments, the amino acid sequence of the anti-aging polypeptide is as shown in SEQ ID No. 5, specifically: Phe-Lys-Val-Pro-Asn-Met, and the corresponding structural formula is:

[0029]

[0030] In some embodiments, the application further provides a use of the anti-aging polypeptide as described in the application in the preparation of an anti-aging drug or an anti-aging health product.

[0031] In particular, the anti-aging drug or anti-aging nutraceutical is further added with one or more pharmaceutically acceptable excipients, including carriers, diluents, excipients, adjuvants, buffers, pH adjusting agents, preservatives, antioxidants, bacteriostatic agents, stabilizers, suspending agents, solubilizers, surfactants, coloring agents, and isotonicity solutes. Suitable carriers, diluents, excipients, and the like can be found in standard pharmaceutical books, see, for example, Handbook of Pharmaceutical Additives, 2ndEdition (Editors M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA); Remington's Pharmaceutical Science, 18thEdition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 2ndEdition, 1994.

[0032] In some embodiments, the anti-aging drug or anti-aging nutraceutical is in an oral dosage form, including, but not limited to, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, syrups, buccal agents, granules, drop pills, powders, pastes, suspensions, and powders.

[0033] To verify that the anti-aging polypeptide provided by the present application can effectively inhibit oxidative stress against inflammation, and also improve memory level, has a significant anti-aging effect, the present application is specifically described below in combination with specific examples. It is necessary to point out here that the following examples are only used to further illustrate the present application, and cannot be understood as limiting the protection scope of the present application. Some non-essential improvements and adjustments of the present application made by those skilled in the art according to the content of the present application still fall within the protection scope of the present application.

[0034] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximations. The endpoints of the ranges and any values are understood to be approximate, and in actual practice, individual values can vary from the stated values. The ranges and individual values are understood to encompass values approximating the stated values within the precision of the measurement, the precision of the measurement being understood to vary from instrument to instrument. For example, the ranges and individual values are understood to encompass values that are ± 10% of the stated value, unless otherwise explicitly stated.

[0035] Example 1

[0036] DPPH radical scavenging capacity is considered a key indicator for evaluating the antioxidant efficacy of peptides. This study evaluated the antioxidant activity of anti-aging peptides using DPPH radical scavenging rate. Seq1, Seq2, Seq3, Seq4, and Seq5 were used as the research subjects, with vitamin C as a positive control (both experimental groups and vitamin C concentration were 0.2 mg / mL). The experimental setup included: Group A1 (anti-aging peptides or vitamin C in DPPH solution), Group A2 (ethanol in anti-aging peptides or vitamin C solution), and Group A3 (ethanol in DPPH solution), with four replicates for each group. A 96-well plate was used, with 100 µL of the corresponding reagent added to each well. After the reaction was conducted in the dark, the absorbance at 517 nm was measured. Based on the measured data, the DPPH radical scavenging rate Y was calculated using the formula. D (As shown in Equation I), the result is as follows Figure 1 As shown, In the formula, A1 represents the absorbance of group A1 at 517 nm; A2 represents the absorbance of group A2 at 517 nm; and A3 represents the absorbance of group A3 at 517 nm.

[0037] from Figure 1 The results show that, compared with the same mass concentration of vitamin C, the anti-aging peptides described in this application have comparable antioxidant capacity to vitamin C, with Seq5 exhibiting the best antioxidant capacity. Therefore, Seq5 was used as the test subject in subsequent experiments.

[0038] Example 2

[0039] This embodiment provides a method for studying in vitro cell viability.

[0040] PC-12 cells were seeded at a density of 3 × 10 cells / mL in 6-well plates (1 mL / well) or 96-well plates (100 μL / well) and cultured at 37°C in a 5% CO2 incubator for 24 hours until the cells were stable. Then, a senescence model was constructed: the medium was replaced with one containing 20 mg / mL D-gal, and the cells were cultured for another 24 hours. After modeling, the cells were divided into groups: (1) Experimental group: different concentrations of Seq5 (0.01, 0.1, 1, 10 mg / mL) were added; (2) Blank control group: no D-gal or Seq5 was added, and the cells were cultured normally; (3) Positive control group: 50 μg / mL Vitamin C was added. After 24 hours of further treatment in each group, cell viability was detected using a CCK-8 assay kit. The results are as follows: Figure 2 As shown.

[0041] from Figure 2 The results show that the anti-aging peptide Seq5 of this application has a significant effect on improving the viability of senescent cells at concentrations of 0.01-10 mg / mL, and its effect on improving in vitro cell viability reaches its optimal level when the concentration reaches 1 mg / mL.

[0042] Example 3

[0043] This embodiment provides a research method for evaluating in vivo antioxidant activity.

[0044] I. Animal experiment design: Seq5 was used as the test substance, and SPF female KM mice (body weight 20-25 g) were selected as experimental animals. The mice were randomly divided into 4 groups (n=5): (1) blank control group; (2) model group; (3) Seq5 low-dose group; (4) Seq5 high-dose group.

[0045] II. Modeling and dosing regimen: Except for the blank control group, the other three groups of mice received daily neck subcutaneous injection of 100 mg / kg D-galactose (dissolved in normal saline) to induce aging model. On this basis: model group and blank control group: given equal volume of normal saline by gavage daily. Seq5 low-dose group and high-dose group: respectively given 50 mg / kg and 200 mg / kg of Seq5 solution by gavage daily.

[0046] III. Sample collection and processing: After 4 weeks of continuous intervention, whole blood samples were collected by eyeball blood collection method, and then the mice were euthanized. The whole blood samples were centrifuged at 3500 r / min for 20 min at 4°C, and the serum was separated and stored at -80°C for testing.

[0047] IV. Serum index detection: The activities of creatine kinase (CK), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in serum were determined using biochemical reagent kits, and the results are shown in Table 1.

[0048] Table 1 Effect of anti-aging polypeptide on antioxidant enzyme system in mouse serum

[0049]

[0050] Note: Different lowercase letters in the same column indicate significant differences (P<0.05)

[0051] The activity level of antioxidant enzymes (such as CAT, SOD, GSH-Px) in serum is an important indicator for evaluating the systemic oxidative stress state of the body, and has high physiological representativeness. Creatine kinase (CK): mainly distributed in skeletal muscle and myocardial tissue. When myocardial injury occurs, CK can be released into the blood in large quantities, causing a significant increase in its serum level, and is one of the sensitive biomarkers of myocardial injury. Catalase (CAT): its main function is to catalyze the decomposition of hydrogen peroxide (H2O2) into water and oxygen. Under oxidative stress, H2O2 is one of the reactive oxygen species (ROS), and its generation will abnormally increase. Therefore, maintaining a high level of CAT activity is crucial for removing excess H2O2 and protecting biological macromolecules and cell structures from oxidative damage. Superoxide dismutase (SOD): as a key antioxidant enzyme containing metal ions, SOD is responsible for catalyzing the dismutation of superoxide anion radicals (O2⁻•) into H2O2 and oxygen, constituting the first line of defense against oxidation in the body. The maintenance of SOD activity is potentially associated with various physiological and pathological processes such as cardiovascular disease, tumor development, and aging process (by reducing cellular lipid peroxidation), and is believed to play an important protective role. Glutathione peroxidase (GSH-Px): this enzyme belongs to the peroxidase family, and its function is to use reduced glutathione (GSH) as a substrate to catalyze the reduction of various lipid peroxides (ROOH) and H2O2, generating the corresponding harmless alcohols (ROH) or water. This process plays a core role in removing harmful peroxides, maintaining cellular redox homeostasis, and protecting cell membranes and organelles from oxidative damage.

[0052] Table 1 shows that compared with the model control group, the activity of serum antioxidant enzymes (CAT, SOD, GSH-Px) of the experimental animals intervened by Seq5 was significantly increased, and the CK level was significantly decreased.

[0053] Five, brain tissue index detection: take the mouse brain tissue, add appropriate amount of pre-cooled phosphate buffer solution (PBS) for homogenate. The homogenate is centrifuged at 3500 r / min for 20 min at 4°C, and the supernatant is collected. The SOD activity, malondialdehyde (MDA) content, interleukin-6 (IL-6) content and interleukin-1β (IL-1β) content in the brain tissue are detected by using the corresponding kit, and the results are shown in Table 2.

[0054] Table 2 Activity and content of oxidative stress factors and inflammatory factors in brain tissue of anti-aging polypeptide-treated mice

[0055]

[0056] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0057] SOD and MDA (malondialdehyde) content reflects the level of oxidative stress in the brain, IL-6 and IL-1β can reflect the level of inflammation in the aging brain tissue. The data in Table 2 show that Seq5 can act on the brain, delay the aging of the brain, and the activity is dose-dependent.

[0058] The experimental results of this example show that the anti-aging polypeptide of the application has a significant inhibitory effect on D-galactose-induced oxidative stress damage.

[0059] Example 4

[0060] This example provides an in vivo behavioral evaluation method.

[0061] Using D-gal-induced aging mice as a model, the experimental animals were divided into six groups, namely, a blank group, a model group, a Vc treatment group (100 mg / kg, Vc), a low-dose Seq5 treatment group (100 mg / kg, SL), a medium-dose Seq5 treatment group (200 mg / kg, SM), and a high-dose Seq5 treatment group (400 mg / kg, SH). Except for the blank group, each group of mice was subcutaneously injected with a D-gal solution (500 mg / kg) every day for 8 weeks, and during the 4th to 8th weeks, the corresponding test drugs were administered by gavage. The blank group and the model group were replaced with an equal volume of distilled water. After the administration was completed, the behavioral water maze experiment was performed.

[0062] Morris water maze experiment design: This experiment includes two stages of spatial learning training (days 1-5) and spatial memory detection (days 7, 9).

[0063] (I) Spatial learning training stage (days 1-5)

[0064] The circular pool is divided into four quadrants, and the pool wall is marked with different visual cues. The hidden platform is placed 2 cm below the water surface in the target quadrant, and non-toxic black dye is added to the pool water to ensure visual shielding. Each mouse is allowed to freely explore and locate the hidden platform from a random quadrant each day, with a single training time limit of 60 seconds. If the mouse successfully reaches the platform and stays on it for ≥3 seconds, the training is terminated; if it fails to locate the platform within the time limit, it is guided to the platform with a guide rod. The escape latency (i.e., the time required to locate the platform) is recorded during all training processes by a video tracking system, and the results are shown in Figure 3 .

[0065] As can be seen from Figure 3 , the time for the model group mice to find the escape platform did not significantly decrease, however, the time for the medium-dose and high-dose Seq5 treatment group mice to find the escape platform gradually decreased as the training progressed, indicating that the learning ability of the mice treated with medium-dose and high-dose Seq5 was significantly improved compared to the model group.

[0066] (ii) Spatial memory probe phase (day 9)

[0067] Remove the hidden platform. All mice were put into water from a fixed starting quadrant (randomly selected) and freely explore for 60 seconds. The following parameters were recorded:

[0068] (1) Goal quadrant residence time (the quadrant where the original platform was located), the results are shown in Figure 4 .

[0069] (2) Platform region crossing times (a virtual area centered on the original platform location), the results are shown in Figure 5 .

[0070] Figure 4 and Figure 5 indicate that after Seq5 treatment, the number of times mice cross the escape platform and the residence time significantly increase, which shows that Seq5 can significantly improve the memory level of mice, especially after medium and high dose Seq5 treatment, the memory level of mice is improved more significantly.

[0071] In summary, the anti-aging polypeptide provided by the present application can effectively inhibit oxidative stress and anti-inflammation, and also can improve the memory level, and has a significant anti-aging effect. The present application provides strong technical support for further exploring the clinical application of peptide molecules, developing health care food and drugs.

[0072] It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. An anti-aging polypeptide, characterized in that, The structural general formula of the anti-aging polypeptide is: H2N-(AA) m -Phe-Lys-Val-Pro-Asn-Met-(AA) n -COOH, wherein AA represents any amino acid, and m and n are both integers between 0 and 4.

2. Anti-aging polypeptide according to claim 1, characterized in that, The anti-aging polypeptide is one of Ala-Phe-Lys-Val-Pro-Asn-Met-Lys, Leu-Ala-Phe-Lys-Val-Pro-Asn-Met-Phe-Gln, Leu-Asn-Ala-Phe-Lys-Val-Pro-Asn-Met-Phe-Gln-Ile, Asn-Asn-Ala-Gln-Phe-Lys-Val-Pro-Asn-Met-Leu-Leu-Phe, Phe-Lys-Val-Pro-Asn-Met.

3. Use of the anti-aging polypeptide of claim 1 or 2 in the preparation of an anti-aging drug or an anti-aging health product.

4. Use according to claim 3, characterized in that, The anti-aging drug or anti-aging health product further comprises one or more pharmaceutically acceptable excipients, including carriers, diluents, excipients, adjuvants, buffers, pH regulators, preservatives, antioxidants, bacteriostatic agents, stabilizers, suspending agents, solubilizers, surfactants, coloring agents, and isotonicity solutes.

5. Use according to claim 3, characterized in that, The anti-aging drug or anti-aging health product is in an oral dosage form.

6. Use according to claim 5, characterized in that, The oral dosage form includes sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, syrups, buccal agents, granules, drop pills, powders, pastes, suspensions, and powders.

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