Eggshell membrane peptide with antioxidant and improving exercise capacity of aging body and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUIZHOU MILAIMI FOOD CO LTD
- Filing Date
- 2026-04-29
- Publication Date
- 2026-07-10
Smart Images
Figure CN122356221A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and mainly to an eggshell membrane peptide with antioxidant properties and the ability to improve the movement of aging organisms, and its applications. Background Technology
[0002] As we age, the functions of various organs and tissues gradually decline, with a particularly significant decrease in motor function. Maintaining motor function depends on the supporting function of the skeletal system and the contractile power of skeletal muscles; these two work synergistically to ensure the body's activity level. Skeletal muscle decline manifests as muscle atrophy, weakened strength, and reduced exercise endurance; skeletal aging is characterized by bone loss, destruction of bone microstructure, and decreased osteoblast function. This synergistic decline in skeletal and skeletal muscle function leads to decreased motor function in the elderly, limiting the body's range of motion and metabolic levels, further exacerbating muscle loss, bone loss, and systemic functional decline, creating a vicious cycle that ultimately shortens lifespan. Therefore, delaying the decline in motor function has become one of the key strategies for intervening in aging and extending healthy lifespan.
[0003] Oxidative stress is a core mechanism driving the decline in athletic performance. During aging, the body's endogenous antioxidant defense system weakens, leading to the continuous accumulation of reactive oxygen species in skeletal muscle and bone tissues, causing lipid peroxidation, protein oxidative damage, and mitochondrial dysfunction. In skeletal muscle, oxidative stress directly damages the membrane structure and contractile proteins of muscle cells, weakening muscle contraction efficiency. In the skeletal system, oxidative stress impairs the proliferation and differentiation capacity of osteoblasts, inhibiting bone formation and disrupting bone metabolic balance. The combined damage from both ultimately leads to decreased exercise endurance, increased fatigue susceptibility, and a comprehensive decline in athletic ability. Therefore, enhancing the ability of skeletal muscle cells and osteoblasts to resist oxidative stress damage is an important way to delay the decline in athletic function and improve the athletic performance of aging bodies.
[0004] In recent years, research on bioactive compounds from food sources has gradually become a hot topic in the field of anti-aging. Bioactive peptides released through enzymatic hydrolysis of natural food sources possess various biological functions, including antioxidant, immunomodulatory, and hypoglycemic effects. Eggshell membranes, as a major byproduct of poultry egg processing, are produced in large quantities and are rich in protein. However, the insolubility of eggshell membranes limits their development. Enzymatic hydrolysis to release broadly bioactive peptides from insoluble eggshell membranes is a common method to increase their bioavailability. Studies have shown that eggshell membrane peptides have excellent antioxidant activity. However, research on specific peptides that protect muscle and skeletal muscle under oxidative stress conditions is still insufficient. More importantly, there is a lack of eggshell membrane peptides that can simultaneously improve the motor function of aging organisms. Therefore, developing an eggshell membrane peptide that can delay the decline in motor function caused by aging has significant practical implications. Summary of the Invention
[0005] The purpose of this invention is to provide an eggshell membrane peptide with antioxidant properties and the ability to improve the motor function of aging organisms, and its applications, to solve the problems existing in the prior art. This invention identifies an antioxidant peptide from eggshell membrane hydrolysate. This eggshell membrane peptide can improve the motor function and antioxidant stress resistance of aging organisms. It can be used not only to prepare antioxidants, but also to prepare functional foods, health products, and pharmaceutical compositions that improve the motor function of aging organisms, showing broad application prospects.
[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an eggshell membrane peptide with antioxidant properties and the ability to improve the physical function of aging organisms. The amino acid sequence of the eggshell membrane peptide is shown in SEQ ID NO.1, specifically Glu-Gln-Pro-Trp-Phe-Pro-Ala-Gly (EQPWFPAG).
[0007] Furthermore, the eggshell membrane peptides possess antioxidant activity. This antioxidant activity includes, but is not limited to, scavenging reactive oxygen species in the body and enhancing the body's resistance to oxidative stress.
[0008] Furthermore, the eggshell membrane peptide has the ability to improve the declining motor function of aging organisms. This ability to improve the declining motor function of aging organisms includes, but is not limited to, increasing the frequency of movement, exercise endurance, or exercise coordination of aging organisms.
[0009] Furthermore, the activity of improving the motor function of aging organisms includes the fact that the eggshell membrane peptide also has the activity of protecting bone cells against oxidative stress damage.
[0010] The present invention also provides the application of the eggshell membrane peptide described in any of the above claims in the preparation of products that improve the motor function of aging organisms.
[0011] The present invention also provides a product for improving the motor function of aging organisms, comprising the above-mentioned eggshell membrane peptides, and pharmaceutically acceptable carriers or excipients.
[0012] Optionally, the dosage form of the product for improving the motor function of aging bodies is selected from any one of tablets, capsules, granules, powders, oral liquids, or emulsions.
[0013] The present invention has the following technical effects: This invention identifies a novel eggshell membrane peptide, EQPWFPAG, from eggshell membrane hydrolysate for the first time, and evaluates its function using a Caenorhabditis elegans aging model and a skeletal cell model. The results show that this eggshell membrane peptide significantly reduces reactive oxygen species levels and lipofuscin accumulation in aging nematodes, enhances their resistance to H2O2 stress, and ultimately effectively improves the motility of aging nematodes and prolongs their healthy lifespan. Simultaneously, cell experiments confirmed that this peptide can directly protect skeletal cells against oxidative stress damage, further supporting its efficacy in improving aging motor function. This eggshell membrane has significant application value in functional foods, health products, and pharmaceuticals. Attached Figure Description
[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0015] Figure 1 This is a secondary mass spectrometry pattern for identifying eggshell membrane peptide EQPWFPAG; Figure 2 This is a diagram showing the interaction between the eggshell membrane peptide EQPWFPAG and the Keap1 protein; Figure 3 The effect of eggshell membrane peptide EQPWFPAG on ROS content in Caenorhabditis elegans; Figure 4 The effect of eggshell membrane peptide EQPWFPAG on the H2O2 stress resistance of Caenorhabditis elegans; Figure 5 The effect of eggshell membrane peptide EQPWFPAG on the locomotion ability of Caenorhabditis elegans; Figure 6 The effect of eggshell membrane peptide EQPWFPAG on lipofuscin accumulation in Caenorhabditis elegans; Figure 7 The effect of eggshell membrane peptide EQPWFPAG on the lifespan of Caenorhabditis elegans; Figure 8 The effect of eggshell membrane peptide EQPWFPAG on the viability of MC3T3-E1 cells under oxidative stress. Detailed Implementation
[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0021] Example 1: Preparation and screening of eggshell membrane peptides.
[0022] Preparation: Eggshell membrane was used as raw material. After ultrafine pulverization, double-distilled water was added at a powder-to-water volume ratio of 1:20. The mixture was then treated with an ultrasonic homogenizer at 200 W for 10 minutes. The pH of the solution was adjusted to 9.0, and alkaline protease (10000 U / g) was added. After mixing, the mixture was enzymatically hydrolyzed in a 55℃ constant temperature shaking water bath for 6 hours, maintaining the pH at 9.0. After enzymatic hydrolysis, the pH was adjusted to 7.0, and the enzyme was inactivated at 90℃ for 15 minutes. After cooling to room temperature, the mixture was centrifuged (4000×g, 14 minutes). The supernatant was desalted using a 200 Da dialysis bag for 46 hours. Finally, the eggshell membrane peptide powder was obtained by vacuum freeze-drying.
[0023] Peptide Identification and Screening: The eggshell membrane peptide powder obtained in step 1 was identified by HPLC-MS / MS. Subsequently, the peptides were evaluated and ranked using the online prediction tools Peptide Ranker and ToxinPred, and their toxicity was predicted. The novelty of the peptides was verified using the BIOPEP-UWM database. The antioxidant activity of the peptides was further predicted using the AnOxPP tool. The peptides were molecularly docked with Keap1, a core regulatory protein of the antioxidant pathway, using the molecular docking software AutoDock Vina. The EQPWFPAG peptide showed the best docking performance, with a binding energy of -9.8 kcal / mol. The molecular docking results indicated that the peptides mainly bind to Keap1 protein through hydrogen bonds, hydrophobic interactions, and van der Waals forces. The secondary mass spectrum of the eggshell membrane peptide is shown below. Figure 1 As shown in the diagram, the molecular docking schematic is as follows: Figure 2 As shown.
[0024] Table 1. Activity prediction, toxicity, and molecular docking results of eggshell membrane peptides.
[0025] Example 2: In vivo activity evaluation of eggshell membrane peptides.
[0026] The selected eggshell membrane peptide EQPWFPAG was synthesized in a solid-phase manner. The decline in lifespan and motility of *C. elegans* is closely related to the degeneration of muscle function; therefore, *C. elegans* was used as an aging model to evaluate its in vivo antioxidant and motility activities. The specific process is as follows: Culture and grouping of Caenorhabditis elegans: Uracil synthesis-deficient *E. coli* OP50 was cultured under aseptic conditions and then concentrated 10-fold for later use. In sterile NGM culture dishes, 200 μL of concentrated OP50 was added to the blank control group and spread evenly; 200 μL of concentrated OP50 and 200 μL of 1.0 mg / mL eggshell membrane peptide EQPWFPAG were added to the eggshell membrane peptide group, mixed, and spread evenly. Wild-type *C. elegans* N2 was cultured on fresh NGM plates for 3 days and then synchronized, incubated overnight at 20°C. The obtained L1 stage larvae were transferred to the blank control group and the eggshell membrane peptide group culture medium, respectively, for subsequent experiments.
[0027] ROS content experiment: Using *C. elegans* on its 8th day of adulthood as an aging model, the nematodes were divided into a blank control group and an eggshell membrane peptide group. After washing with M9 buffer, the nematodes were stained with dichlorofluorescein (final concentration 1 μmol / L) and incubated at 37°C in the dark for 2 hours. After staining to remove residual dye, the nematodes were transferred to a 2% agarose gel and photographed using a laser confocal microscope under the FITC channel. Results are as follows: Figure 3As shown, compared with the blank control group, the ROS fluorescence intensity of the eggshell membrane peptide group in nematodes was significantly reduced. This indicates that the eggshell membrane peptide EQPWFPAG has strong antioxidant activity.
[0028] H2O2 stress resistance experiment: Nematodes were placed in 96-well plates containing 10 mmol / L H₂O₂ solution. The number of surviving nematodes was observed and recorded under a microscope every 10 minutes until all nematodes died. The results are as follows: Figure 4 As shown, the survival time of nematodes in the eggshell membrane group under oxidative stress was increased to 30 hours, and compared with the blank control group, the average survival time of nematodes in the eggshell membrane peptide group was significantly prolonged by 16.24%. This indicates that the eggshell membrane peptide EQPWFPAG can resist oxidative damage by enhancing the antioxidant defense system of nematodes.
[0029] Motor ability experiment: Head waving was measured on day 1 (adulthood) and day 8 (senescence) of the nematodes. The nematodes in each group were washed with M9 buffer and transferred to blank NGM medium. The number of head wavings within 30 seconds was recorded under a stereomicroscope. Results are as follows: Figure 5 As shown, during the aging period, the head-shaking frequency of nematodes in the eggshell membrane peptide-treated group was significantly increased by 16.78% compared to the blank control group. The results indicate that the eggshell membrane peptide can effectively improve age-related motor function decline.
[0030] Lipofuscin accumulation experiment: Lipofuscin is a cellular waste product that accumulates in lysosomes with age and oxidative damage, and can serve as an indicator of aging. Lipofuscin content was measured in nematodes on day 8 of adulthood. After washing with M9 buffer, the nematodes were fixed in 4% paraformaldehyde, transferred to 2% agarose gel pads, and observed and photographed under an inverted microscope. Results are as follows: Figure 6 As shown, compared with the blank control group, the autofluorescence intensity of lipofuscin in the nematodes treated with eggshell membrane peptide was significantly reduced, indicating that the eggshell membrane peptide can reduce the accumulation of lipofuscin during aging and has the effect of delaying aging.
[0031] Lifetime experiment: Nematodes synchronized to stage L4 were transferred to blank culture medium containing the sterilizing agent FUDR and culture medium containing eggshell membrane peptides, 100 nematodes per group, and cultured overnight at 20°C, recorded as day 0 of survival. The number of surviving nematodes was then observed and recorded every two days under a stereomicroscope. Simultaneously, surviving nematodes were transferred to freshly prepared culture medium with the same concentration of the drug and continued culturing until all nematodes died. Results are as follows: Figure 7As shown, the maximum lifespan of nematodes treated with eggshell membrane peptides increased from 23 days to 26 days. Compared with the blank control group, the average lifespan of nematodes in the eggshell membrane peptide group was significantly extended by 8.77%. This indicates that eggshell membrane peptide EQPWFPAG improves the motility of aging nematodes while also prolonging their lifespan.
[0032] Example 3: Determination of the viability of MC3T3-E1 cells under oxidative stress by eggshell membrane peptides.
[0033] MC3T3-E1 cells were cultured in α-MEM complete medium containing 10% fetal bovine serum and penicillin antibiotics, and incubated at 37 °C. When the cells reached 80%-90% confluence, they were passaged using 0.25% trypsin. After two passages, cells were incubated at 1×10⁻⁶ cells / year. 6 Cells were seeded in wells. Cell differentiation was induced using differentiation medium containing 10 mmol / L sodium β-glycerophosphate and 50 µg / mL ascorbic acid, with simultaneous treatment with different concentrations of eggshell membrane peptides and 400 µmol / L H2O2. Cell viability was measured using a CCK-8 assay kit (purchased from Beyotime Biotechnology Co., Ltd.). Results are as follows: Figure 8 As shown, compared with the blank control group, the cell viability of the H2O2 damage model group was only 60%, while the cell viability of the eggshell membrane peptide group was significantly increased. The cell viability of the 10 μg / mL, 30 μg / mL and 50 μg / mL eggshell membrane peptide groups were 85%, 91% and 104%, respectively, indicating that the eggshell membrane peptide EQPWFPAG can effectively protect MC3T3-E1 cells against oxidative stress damage.
[0034] In summary, the eggshell membrane peptide EQPWFPAG of this invention can effectively improve the motor function of aging bodies and extend their healthy lifespan. Therefore, it is suitable for producing products that improve the motor function of aging bodies, such as functional foods, dietary supplements, sports nutrition products, health products, or pharmaceutical compositions. These products can be manufactured by containing the eggshell membrane peptide EQPWFPAG and pharmaceutically acceptable carriers or excipients, and the dosage form can be selected from any one of tablets, capsules, granules, powders, oral liquids, or emulsions.
[0035] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An eggshell membrane peptide with antioxidant properties and the ability to improve the motor function of aging organisms, characterized in that: The amino acid sequence of the eggshell membrane peptide is shown in SEQ ID NO:
1.
2. The eggshell membrane peptide according to claim 1, which has antioxidant properties and improves the body's ability to move in an aging organism, is characterized in that: The eggshell membrane peptide has the ability to improve the decline in motor function in aging organisms.
3. The eggshell membrane peptide according to claim 2, which has antioxidant properties and improves the body's ability to move in an aging organism, is characterized in that... The ability to improve the declining motor function of aging organisms includes the ability of the eggshell membrane peptides to protect bone cells against oxidative stress damage.
4. The use of the eggshell membrane peptide with antioxidant properties and the ability to improve the motor function of aging organisms as described in any one of claims 1-3 in the preparation of products that improve the motor function of aging organisms.
5. A product for improving the motor function of an aging body, characterized in that, It comprises the eggshell membrane peptide according to any one of claims 1-3, and a pharmaceutically acceptable carrier or excipient.
6. The product for improving the motor function of an aging body according to claim 5, characterized in that, The dosage form of the product is selected from any one of tablets, capsules, granules, powders, oral liquids, or emulsions.