Application of Chinese soft-shelled turtle egg protein peptide extract in preparation of anti-aging drugs, health products or functional foods
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-16
- Publication Date
- 2026-08-11
AI Technical Summary
目前，国内外对中华草龟的研究大部分聚焦于草龟蛋的营养价值、龟蛋孵化条件和胚胎发育的影响因素研究，而对草龟蛋蛋白肽的研究与高值化利用较少
[0028]与现有技术相比，本申请的有益效果是：在实验动物给予100～400 mg/kg/天的剂量范围内，中华草龟蛋蛋白肽提取物可显著改善D-半乳糖诱导衰老小鼠带来的体重下降；显著提高小鼠血清中抗氧化酶活力，同时降低血清中脂质过氧化产物MDA的含量；可缓解D-半乳糖对衰老小鼠的肝脏和肾脏带来的氧化应激损伤和功能损伤，可显著降低D-半乳糖诱导衰老小鼠肝脏中的衰老标志物p62、p53、p-p53、p16蛋白表达水平。以上均证实了中华草龟蛋蛋白肽提取物具有抗衰老作用，能够用于制备衰老的延缓、预防和/或治疗药品、保健品和/或功能食品。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of medicine, specifically relating to the application of a Chinese pond turtle egg protein peptide extract in the preparation of anti-aging drugs, health products or functional foods. Background Technology
[0002] Aging is a complex biological process characterized by a decline in bodily functions and a corresponding decrease in the body's ability to respond to and adapt to environmental stresses as we age. In recent years, with the accelerating pace of population aging, the incidence of age-related chronic diseases such as diabetes and cancer has been rising, posing serious threats to society and individuals. Therefore, research on the mechanisms of aging and anti-aging measures has become a focus of social attention.
[0003] Several hypotheses exist regarding the mechanisms of aging. Among them, the oxygen free radical theory posits that aging is the result of harmful attacks on cellular components by oxygen free radicals produced by metabolism, and is a key intrinsic mechanism of aging. In biological systems, the production of reactive oxygen species (ROS) is unavoidable, and the human body partially neutralizes their harmful effects through antioxidant defense mechanisms. The most important components of the antioxidant enzyme defense system include superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT). Besides ROS, oxidative stress also produces large amounts of malondialdehyde (MDA). SOD, GSH-Px, and CAT are primarily responsible for balancing free radicals or repairing antioxidant molecules; their activity or expression levels can indirectly reflect the oxidative stress state of cells or tissues. MDA is a highly reactive compound produced by lipid peroxidation under oxidative stress-related conditions. It can irreversibly modify protein residues, such as lysine, arginine, and histidine, and can attach to autologous biomolecules, thereby generating new epitopes that can induce potentially adverse biological responses. Therefore, scavenging excess free radicals in the body is a potentially effective strategy for delaying aging.
[0004] Another important characteristic of cellular senescence is cell cycle arrest, primarily due to the regulation of the p16 / RB and p53 / p21 pathways. The regulation and alteration of these two pathways may represent two theoretical signaling mechanisms leading to cellular senescence. p21 can universally bind to various cellular complexes, thus inhibiting their formation, reducing the phosphorylation level of RB, preventing the release of E2F, and hindering DNA production. Consequently, cells cannot enter the S phase and remain arrested in the G phase, ultimately inducing cellular senescence. The p53 gene participates in cell growth, apoptosis, cell cycle arrest, senescence, and DNA repair; reducing the expression level of the p53 gene can alleviate skin aging to some extent.
[0005] The liver is the body's main metabolic and detoxification organ, while the kidneys are excretory and humoral regulation organs; both are extremely sensitive to changes in oxidative stress levels. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) are normally found in hepatocytes, and their levels in the blood can be used to assess the extent of liver damage. Furthermore, with age, functional renal masses, renal blood flow, and glomerular filtration rate decrease, often accompanied by glomerulosclerosis, tubular atrophy, and interstitial fibrosis; renal function can be assessed through serum urea (UREA) and creatinine (CREA) levels.
[0006] Natural products contain abundant antioxidants, and utilizing these natural antioxidants to prevent aging and related diseases has become a research hotspot in modern biomedicine and nutrition. The Chinese pond turtle (Chinemysreevesii), also known as the grass turtle, mud turtle, or common pond turtle, lives in freshwater and is distributed throughout China. It is a nutritious and delicious aquatic product, and also a medicinal ingredient with significant medicinal value. Since 2017, my country's freshwater turtle production has consistently remained above 45,000 tons, with aquaculture production projected to reach approximately 55,000 tons in 2024. As a major edible turtle species in my country, the scale of Chinese pond turtle farming is expanding, and the production of its eggs, as a byproduct, is also increasing, making it a significant area for development and utilization. Currently, most domestic and international research on the Chinese pond turtle focuses on the nutritional value of its eggs, incubation conditions, and factors influencing embryonic development, while research and high-value utilization of turtle egg protein peptides are relatively limited. Therefore, developing new applications for turtle egg protein peptides in the anti-aging field has significant practical implications. Summary of the Invention
[0007] To address the above problems, this invention provides an application of Chinese pond turtle egg protein peptide extract in the preparation of anti-aging drugs, health products, or functional foods.
[0008] The primary objective of this invention is to provide the application of Chinese pond turtle egg protein peptide extract in the preparation of a drug that inhibits the expression of liver aging-related biomarkers.
[0009] As a preferred embodiment of this application, the Chinese pond turtle egg protein peptide extract contains peptide segments with amino acid sequences as shown in SEQ ID NO. 1 to 6.
[0010] The second objective of this invention is to provide an application of Chinese pond turtle egg protein peptide extract in the preparation of anti-aging drugs.
[0011] As a preferred embodiment of this application, the anti-aging effect specifically refers to improving D-galactose-induced aging symptoms.
[0012] As a preferred embodiment of this application, the Chinese pond turtle egg protein peptide extract exerts its anti-aging effect through at least one of the following mechanisms:
[0013] (a) Improves symptoms of age-related weight loss induced by D-galactose;
[0014] (b) Increase the activity of antioxidant enzymes (CAT), superoxide dismutase (SOD) and / or glutathione peroxidase (GSH-Px) in the serum of test subjects, while reducing the content of malondialdehyde (MDA), a lipid peroxidation product, in the serum of test subjects.
[0015] (c) Alleviate age-mediated oxidative stress damage and functional impairment of the liver and / or kidneys;
[0016] (d) Down-regulate the expression levels of aging-related biomarkers in the liver of the subjects.
[0017] As a preferred embodiment of this application, the aging biomarkers include p62 protein, p53 protein, p-p53 protein, and p16 protein.
[0018] As a preferred embodiment of this application, the inhibition of the expression of liver aging-related biomarkers is achieved by downregulating the expression of p62, p53, p-p53, and p16 proteins in the liver.
[0019] A third objective of the present invention is to provide an anti-aging pharmaceutical composition comprising a therapeutically effective amount of Chinese pond turtle egg protein peptide extract and a pharmaceutically acceptable carrier; said pharmaceutical composition is used to inhibit the expression of liver aging-related markers or to improve D-galactose-induced aging.
[0020] As a preferred embodiment of this application, the Chinese pond turtle egg protein peptide extract is prepared by the following steps: freeze-dried powder of Chinese pond turtle egg white is dissolved in pure water, the pH is adjusted to 5-6, papain is added at an addition rate of 8000-16000 U / g, and enzymatic hydrolysis is performed for 100-200 min under ultrasonic power of 200-300 W and temperature of 45-50 ℃; after enzymatic hydrolysis, inactivation, centrifugation, and filtration are performed, the supernatant is purified, and freeze-dried to obtain the Chinese pond turtle egg protein peptide extract.
[0021] As a preferred embodiment of this application, the Chinese pond turtle eggs are from captive-bred Chinese pond turtles (Chinemysreevesii).
[0022] As a preferred embodiment of this application, the centrifugation conditions are 5000 r / min, 4 ℃, and 10 min.
[0023] As a preferred embodiment of this application, in the purification step, the supernatant (i.e., crude peptide hydrolysate) is filtered through a 0.45 μm aqueous membrane; the membrane-filtered protein peptide solution is passed through a Sephadex G-25 dextran gel column using an AKTA fully automated protein purification instrument, and then eluted with deionized water.
[0024] As a preferred embodiment of this application, the dosage form of the pharmaceutical composition is a pharmaceutically permissible oral dosage form, topical patch, or injectable dosage form.
[0025] As a further preferred embodiment of this application, the dosage form of the pharmaceutical composition is a pharmaceutically permissible injectable dosage form.
[0026] As a preferred embodiment of this application, the dosage of the pharmaceutical composition is 100–400 mg / kg / day.
[0027] The fourth objective of this invention is to provide an anti-aging health product or functional food, comprising Chinese pond turtle egg protein peptide extract and a food-grade acceptable carrier; the health product or functional food is used to inhibit the expression of liver aging-related markers or improve D-galactose-induced aging.
[0028] Compared with existing technologies, the beneficial effects of this application are as follows: Within a dosage range of 100–400 mg / kg / day administered to experimental animals, the extract of *Trionyx sinensis* egg protein peptides significantly improved the weight loss induced by D-galactose in aging mice; significantly increased the activity of antioxidant enzymes in mouse serum while reducing the content of MDA, a lipid peroxidation product, in serum; alleviated oxidative stress damage and functional impairment of the liver and kidneys caused by D-galactose in aging mice; and significantly reduced the expression levels of aging markers p62, p53, p-p53, and p16 proteins in the liver of D-galactose-induced aging mice. All of the above confirms that the extract of *Trionyx sinensis* egg protein peptides has anti-aging effects and can be used to prepare drugs, health products, and / or functional foods for delaying, preventing, and / or treating aging. Attached Figure Description
[0029] Figure 1 The effects of different samples on the body weight of aging model mice: (A) Body weight gain rate of mice in each group; (B) Body weight change curves of mice in each group. (N is the normal group, M is the model group, Vc is the positive control drug L-ascorbic acid group, CREP-1 L is the low-dose group of Chinese pond turtle egg protein peptide extract (100 mg / kg), CREP-1 M is the medium-dose group of Chinese pond turtle egg protein peptide extract (200 mg / kg); CREP-1 H is the high-dose group of Chinese pond turtle egg protein peptide extract (400 mg / kg). Different letters indicate significant differences (P < 0.05).
[0030] Figure 2The effects of different samples on the activity of antioxidant enzymes and the content of MDA in the serum of aging mice. (A) CAT; (B) SOD; (C) GSH-Px; (D) MDA. (N represents the normal group, M represents the model group, Vc represents the positive control drug L-ascorbic acid group, CREP-1 L represents the low-dose group of Chinese pond turtle egg protein peptide extract (100 mg / kg), CREP-1 M represents the medium-dose group of Chinese pond turtle egg protein peptide extract (200 mg / kg), and CREP-1 H represents the high-dose group of Chinese pond turtle egg protein peptide extract (400 mg / kg). Different letters indicate significant differences (P < 0.05).
[0031] Figure 3 The images show the pathological results of liver and kidney tissue sections from mice. (N represents the normal group, M represents the model group, Vc represents the positive control drug L-ascorbic acid group, CREP-1 L represents the low-dose group (100 mg / kg) of Chinese pond turtle egg protein peptide extract, CREP-1 M represents the medium-dose group (200 mg / kg) of Chinese pond turtle egg protein peptide extract, and CREP-1 H represents the high-dose group (400 mg / kg) of Chinese pond turtle egg protein peptide extract.)
[0032] Figure 4 The effects of different samples on liver and kidney function in aging mice. (A) ALT; (B) AST; (C) ALB; (D) ALP; (E) UREA; (F) CREA. (N represents the normal group, M the model group, Vc the positive control drug L-ascorbic acid group, CREP-1 L the low-dose group of Chinese pond turtle egg protein peptide extract (100 mg / kg), CREP-1 M the medium-dose group of Chinese pond turtle egg protein peptide extract (200 mg / kg); CREP-1 H the high-dose group of Chinese pond turtle egg protein peptide extract (400 mg / kg). Different letters indicate significant differences (P < 0.05).
[0033] Figure 5 The effect of *Trionyx sinensis* egg protein peptide extract on the expression levels of p62, p53, p-p53, and p16 proteins in mouse liver. (A) Expression bands of p62, p53, p-p53, and p16 proteins; (B) Quantitative analysis of p62 protein; (C) Quantitative analysis of p-p53 / p53 protein phosphorylation level; (D) Quantitative analysis of p16 protein. (N represents the normal group, M represents the model group, Vc represents the positive control drug L-ascorbic acid group, CREP-1 L represents the low-dose group of *Trionyx sinensis* egg protein peptide extract (100 mg / kg), CREP-1 M represents the medium-dose group of *Trionyx sinensis* egg protein peptide extract (200 mg / kg), and CREP-1 H represents the high-dose group of *Trionyx sinensis* egg protein peptide extract (400 mg / kg). Different letters indicate significant differences (P < 0.05).
[0034] Figure 6 Basepeak plot for LC-MS / MS identification of purified Chinese pond turtle egg protein peptides.
[0035] Figure 7 A graph illustrating the anti-aging effect of combining Vitamin C with Chinese pond turtle egg protein peptides. Detailed Implementation
[0036] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
[0037] The Chinese pond turtle eggs used in this invention are all from artificially bred Chinese pond turtles (Chinemys reevesii).
[0038] Example 1
[0039] This embodiment provides a method for preparing protein peptides from Chinese pond turtle eggs, which are obtained through the following method:
[0040] The egg white and yolk of Chinese pond turtle eggs were separated. The egg white was first frozen at -80℃ for 3-4 hours, and then freeze-dried for 2-3 days to obtain Chinese pond turtle egg white powder. 0.2 g of the Chinese pond turtle egg white powder was dissolved in 50 mL of pure water, and the pH was adjusted to approximately 5.0 with 0.1 mol / L HCl solution. Papain was added at a concentration of 8000 U / g, and the mixture was then enzymatically hydrolyzed at 45℃ for 100 min using ultrasonic power of 200 W. The enzyme activity was then inactivated by boiling water for 15 min, cooled to room temperature, and centrifuged (5000 r / min, 4℃, 10 min). The supernatant was collected to obtain crude polypeptide hydrolysate. The crude polypeptide hydrolysate was filtered through a 0.45 μm aqueous membrane. The protein peptide solution was passed through a Sephadex G-25 dextran gel column using an AKTA fully automated protein purification system, followed by elution with deionized water at a rate of 5.0 mL / min. The elution was collected in 15 mL centrifuge tubes and freeze-dried to obtain Chinese pond turtle egg protein peptide powder. Analysis showed that the purity of the Chinese pond turtle egg protein peptide obtained in this embodiment reached over 98.5%.
[0041] The peptide composition and structure of Chinese pond turtle egg protein peptides were identified using LC-MS / MS technology. The mass spectrometry information was compared with PDB and NCBI databases, revealing six peptides with high content, as shown in Table 1.
[0042] Table 1. Analysis of protein peptide composition in Chinese pond turtle eggs.
[0043]
[0044] Example 2
[0045] This embodiment provides a method for preparing protein peptides from Chinese pond turtle eggs, which are obtained through the following method:
[0046] The egg white and yolk of Chinese pond turtle eggs were separated. The egg white was frozen at -80℃ for 3-4 hours, followed by freeze-drying for 2-3 days to obtain Chinese pond turtle egg white powder. 0.2 g of the Chinese pond turtle egg white powder was dissolved in 50 mL of water, and the pH was adjusted to 6 with 0.1 mol / L HCl solution. Papain was added at a concentration of 16000 U / g, followed by enzymatic hydrolysis at 50℃ for 200 min using ultrasonic power of 300 W. The enzyme activity was then inactivated by boiling water for 15 min, cooled to room temperature, and centrifuged. The supernatant was collected to obtain crude polypeptide hydrolysate. The crude polypeptide hydrolysate was filtered through a 0.45 μm aqueous membrane. The filtered protein peptide solution was passed through a Sephadex G-25 dextran gel column using an AKTA automated protein purification system, followed by elution with deionized water at a rate of 5.0 mL / min. The elution was collected in 15 mL centrifuge tubes and freeze-dried to obtain Chinese pond turtle egg white peptide powder. Analysis and testing showed that the purity of the Chinese pond turtle egg protein peptide obtained in this embodiment reached over 97%.
[0047] Example 3
[0048] This embodiment provides a method for preparing protein peptides from Chinese pond turtle eggs, which are obtained through the following method:
[0049] The egg white and yolk of Chinese pond turtle eggs were separated. The egg white was frozen at -80℃ for 3-4 hours, followed by freeze-drying for 2-3 days to obtain Chinese pond turtle egg white powder. 0.2 g of the Chinese pond turtle egg white powder was dissolved in 50 mL of water, and the pH was adjusted to 5.5 with 0.1 mol / L HCl solution. Papain was added at a concentration of 12000 U / g, and the mixture was then enzymatically hydrolyzed at 48℃ for 150 min using ultrasonic power of 250 W. The enzyme activity was then inactivated by boiling water for 15 min, cooled to room temperature, and centrifuged. The supernatant was collected to obtain crude polypeptide hydrolysate. The crude polypeptide hydrolysate was filtered through a 0.45 μm aqueous membrane. The filtered protein peptide solution was passed through a Sephadex G-25 dextran gel column using an AKTA automated protein purification system, followed by elution with deionized water at a rate of 5.0 mL / min. The elution was collected in 15 mL centrifuge tubes and freeze-dried to obtain Chinese pond turtle egg white peptide powder. Analysis and testing showed that the purity of the Chinese pond turtle egg protein peptide obtained in this embodiment reached over 95.5%.
[0050] Example 4
[0051] Take 0.1 g of the protein peptide powder of Chinemys reevesii eggs prepared in Example 1 and dissolve it in 10 mL of pure water to prepare a low-dose oral liquid (CREP-1 L). Each milliliter of the obtained preparation contains 10 mg of the protein peptide extract of Chinemys reevesii eggs, that is, the concentration of the protein peptide extract of Chinemys reevesii eggs is 10 mg / mL.
[0052] Example 5
[0053] Take 0.2 g of the protein peptide powder of Chinemys reevesii eggs prepared in Example 1 and dissolve it in 10 mL of pure water to prepare a medium-dose oral liquid (CREP-1 M). Each milliliter of the obtained preparation contains 20 mg of the protein peptide extract of Chinemys reevesii eggs, that is, the concentration of the protein peptide extract of Chinemys reevesii eggs is 20 mg / mL.
[0054] Example 6
[0055] Take 0.4 g of the protein peptide powder of Chinemys reevesii eggs prepared in Example 1 and dissolve it in 10 mL of pure water to prepare a high-dose oral liquid (CREP-1 H). Each milliliter of the obtained preparation contains 40 mg of the protein peptide extract of Chinemys reevesii eggs, that is, the concentration of the protein peptide extract of Chinemys reevesii eggs is 40 mg / mL.
[0056] Example 7 Verification of Anti-aging Effect
[0057] I. Animal Experiment
[0058] A senescence-induced mouse model was established using D-galactose. After intragastric administration of the oral liquids prepared in Examples 4-6, the anti-aging effect of the protein peptide extract of Chinemys reevesii eggs was evaluated by the changes in body weight and growth rate of mice, the activities of CAT, SOD, GSH-Px and the content of lipid peroxidation product MDA in serum, histopathological examination of liver and kidney tissues, determination of liver and kidney functions of mice, and the expression levels of p62, p53, p-p53, p16 proteins in the liver.
[0059] II. Experimental Procedure
[0060] 1. Experimental Animals
[0061] The experimental animals were 8-week-old SPF-grade male C57BL / 6 mice (body weight 20±2 g), purchased from the Experimental Animal Center of Hangzhou Medical College, with the experimental animal production license number SCXK(Zhe)2024-0002. The experimental mice were housed in a pathogen-free barrier environment at a constant temperature of 22±2 °C, relative humidity of 65±5%, with 12 h of light / dark alternation. The feed provided during the period was the standard mouse feed (AIN-93) and sterile water. The SPF-grade animal use guidelines were strictly followed to ensure the health and safety of the experimental animals.
[0062] 2. Experimental Methods
[0063] After 7 days of acclimatization, mice were randomly divided into 6 groups: blank control group (N), model group (M), positive control group (Vc), low-dose group (CREP-1 L, 100 mg / kg), medium-dose group (CREP-1 M, 200 mg / kg), and high-dose group (CREP-1 H, 400 mg / kg), with 6 mice in each group. Detailed grouping information is as follows:
[0064] Blank control group (N): Normal drinking water and feed were provided for 30 days.
[0065] Model group (M): Normal drinking water and feed were provided, and each animal was injected intraperitoneally with 0.2 mL of D-galactose (400 mg / kg) daily for 30 days.
[0066] Positive control group (Vc): Normal drinking water and feed were provided. Each animal was injected intraperitoneally with 0.2 mL of D-galactose (400 mg / kg) and administered 0.2 mL of L-ascorbic acid (400 mg / kg) by gavage daily for 30 days.
[0067] Low-dose group (CREP-1 L): Normal drinking water and feed were provided, and each animal was given 0.2 mL of D-galactose (400 mg / kg) intraperitoneally and 0.2 mL of the oral liquid preparation of Example 4 (Chinese grass turtle egg protein peptide extract 100 mg / kg) by gavage for 30 days.
[0068] Medium-dose group (CREP-1 M): Normal drinking water and feed were provided, and each animal was given 0.2 mL of D-galactose (400 mg / kg) intraperitoneally and 0.2 mL of the oral liquid preparation of Example 5 (Chinese grass turtle egg protein peptide extract 200 mg / kg) by gavage for 30 days.
[0069] High-dose group (CREP-1 H): Normal drinking water and feed were provided, and each animal was given 0.2 mL of D-galactose (400 mg / kg) intraperitoneally and 0.2 mL of the oral liquid preparation of Example 6 (400 mg / kg of Chinese pond turtle egg protein peptide extract) by gavage for 30 days.
[0070] 3. Weight measurement
[0071] Each mouse was weighed before gavage on the first day, and then the weight of each mouse was measured every three days for 30 days. The last weighing was before the mice were sacrificed and dissected.
[0072] 4. Determination of serum antioxidant enzyme (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA), a lipid peroxidation product.
[0073] Blood was collected in EP tubes and left for about 1 hour. Then, it was centrifuged at 5000 r / min for 10 min at 4 ℃. The supernatant was collected as serum. The MDA content, SOD, GSH-Px and CAT activities in mouse serum were measured according to the instructions provided by the manufacturer of the SOD, GSH-Px, CAT and MDA kits (Nanjing Jiancheng Biotechnology Co., Ltd.).
[0074] 5. Histopathological examination of mouse liver and kidney tissues
[0075] The mice were dissected, and the collected kidney and liver tissues were fixed in centrifuge tubes containing 4% paraformaldehyde. After dehydration, the tissues were embedded in paraffin, and then 4 µm sections were cut and stained with hematoxylin and eosin (H&E) for histological observation.
[0076] 6. Liver and kidney function tests in mice
[0077] Blood was collected in EP tubes and left to stand for about 1 hour. Then, the tubes were centrifuged at 5000 r / min for 20 minutes at 4 ℃, and the supernatant was collected as serum. The levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), albumin (ALB), and creatinine (CREA) in mouse serum were measured using a fully automated biochemical analyzer.
[0078] 7. Detection of p62, p53, p-p53, and p16 protein expression in mouse liver
[0079] An appropriate amount of liver tissue was placed in a grinding tube, and RIPA lysis buffer containing protease and phosphatase inhibitors and grinding beads were added. The tissue was then lysed and proteins extracted using a tissue homogenizer. After lysis, the tissue was centrifuged at 8000 r / min for 15 min, and the supernatant was collected. Protein levels were measured and quantified using the BCA method. Protein expression was then detected by immunoblotting. Specifically, proteins were separated from the sample on a 10% SDS-PAGE gel and then transferred to a 0.45 μm PVDF membrane for blocking. After blocking, the PVDF membrane was incubated overnight with primary antibody (Beyotime Biotechnology Co., Ltd.) at 4 ℃. After incubation, the primary antibody was recovered, and the membrane was washed three times with TSBT for 5 min each time. After washing, the PVDF membrane was transferred to secondary antibody (Beyotime Biotechnology Co., Ltd.) and incubated at room temperature with shaking for 1 h. After incubation, the secondary antibody was recovered, and the PVDF membrane was washed three times with TSBT. The bands were visualized, and the grayscale of the bands was analyzed using ImageJ.
[0080] 8. Data Processing
[0081] The data processing in this invention uses SPSS, ImageJ, and Graphpad Prism for data analysis and statistics. ImageJ is used to analyze protein bands, and the expression of the target protein in each group is represented by the ratio of the gray value of the target protein band to the gray value of the internal reference protein band.
[0082] All experiments were repeated three times (n=3), and results are expressed as mean ± standard deviation (N±SD). One-way ANOVA was used to compare differences among multiple groups, and independent samples t-test was used to compare differences between two groups. P < 0.05 was considered statistically significant.
[0083] III. Experimental Results
[0084] This application aims to verify the anti-aging effects of the Chinese pond turtle egg protein peptide extract (CREP-1), and a systematic evaluation was conducted from three levels: animal phenotype, organ function and pathology, and molecular mechanism.
[0085] 1. Animal phenotypic level
[0086] (1) Effect on mouse body weight
[0087] The effect of Chinese pond turtle egg protein peptide extract on mouse body weight is shown in the following results. Figure 1 As shown.
[0088] The weight of each mouse was measured every three days for a total of 30 days. The weight gain and weight growth rate curves for each group of mice were plotted, as shown below. Figure 1(A) and Figure 1 As shown in (B), Figure 1 (A) It can be seen that the weight gain rate of the model group (M group) was significantly lower than that of the other groups. Under the intervention of Chinese pond turtle egg protein peptide extract, the weight gain rate of mice increased, especially in the medium and high dose groups. Figure 1 (B) The weight change curves show that all groups of mice achieved weight gain within 30 days, with group N showing the most significant natural growth, group CREP-1 H being similar to group N, and group M showing slower weight gain. This indicates that the extract of Chinese pond turtle egg protein peptides can significantly improve the weight loss induced by D-galactose-induced aging in mice.
[0089] (2) Effects on antioxidant enzyme activity and malondialdehyde content in mouse serum
[0090] The effects of Chinese pond turtle egg protein peptide extract on the activity of antioxidant enzymes and malondialdehyde content in mouse serum are shown in the following results. Figure 2 (A)- Figure 2 As shown in (D).
[0091] Figure 2 (A) shows that group M had the lowest CAT activity among all groups. After intervention with Chinese pond turtle egg protein peptide extract, CAT activity was significantly improved. Figure 2 (B) shows that there was no significant difference in SOD activity between the low-dose group and the M group, but SOD activity was significantly enhanced under medium and high doses of intervention. Figure 2 (C) Comparison of GSH-PX activity in mice of different groups. Group N had the highest content. High doses of Chinese scorpion turtle egg protein peptide extract can significantly enhance GSH-PX in mice. Figure 2 The results of MDA content determination in mouse serum in (D) showed that the MDA content in group M mice was significantly higher than that in the other groups, and the high-dose group of *Typha orientalis* egg protein peptide extract significantly reduced the MDA content in mouse serum. In summary, the high-dose group of *Typha orientalis* egg protein peptide extract significantly increased the activities of CAT, SOD, and GSH-PX in mouse serum and reduced the MDA content. This indicates that *Typha orientalis* egg protein peptide extract can significantly increase the activities of antioxidant enzymes CAT, SOD, and GSH-Px in the serum of D-galactose-induced aging mice, while simultaneously reducing the content of lipid peroxidation product MDA in the serum of D-galactose-induced aging mice.
[0092] 2. Organ function and pathology
[0093] (1) Histopathological results of liver and kidney tissue sections from mice
[0094] Histopathological results of mouse liver and kidney tissue sections are shown in the figure. Figure 3 As shown.
[0095] Figure 3 It was found that group M showed the most severe liver tissue damage, with near-complete loss of liver lobular structure, disordered and broken hepatic cords, widespread vacuolar degeneration of hepatocytes, and pyknosis, fragmentation, and even dissolution of some cell nuclei. The central vein wall structure was destroyed, with significant erythrocyte exudation and inflammatory cell infiltration around it. In the high-dose group, the liver tissue morphology was closest to that of group N, with clear liver lobular structure, tightly and orderly arranged hepatocytes, and near-complete disappearance of vacuolar degeneration and inflammatory infiltration, with only occasional mild swelling of a very small number of hepatocytes. Group M showed the most severe kidney tissue damage, with disordered glomerular structure, mesangial matrix proliferation, and atrophy of some glomeruli; widespread vacuolar degeneration and detachment of renal tubular epithelial cells, and dilation and deformation of the lumen; significant inflammatory cell infiltration and erythrocyte exudation were observed in the renal interstitium. The renal tissue morphology of the high-dose group of *Trionyx sinensis* egg protein peptide extract was closest to that of the normal control group, with clear glomerular structure and no obvious proliferation in the mesangial area; the renal tubular epithelial cells were tightly and orderly arranged, with vacuolar degeneration and inflammatory infiltration basically disappearing, and only a very small number of renal tubular epithelial cells showing mild swelling. *Trionyx sinensis* egg protein peptide extract can, to some extent, alleviate the oxidative stress damage to the liver and kidneys caused by D-galactose. This indicates that *Trionyx sinensis* egg protein peptide extract can alleviate the oxidative stress damage to the liver and kidneys caused by D-galactose in aging mice.
[0096] (2) Effects on liver and kidney function in aging mice
[0097] The effects of Chinese pond turtle egg protein peptide extract on liver and kidney function in aging mice are shown in the following results. Figure 4 As shown.
[0098] Figure 4 (A)- Figure 4 (F) shows the effects on liver and kidney function in aging mice. The results showed that under the influence of excessive D-galactose, the levels of ALT, AST, and ALP in the liver of group N were significantly increased, all higher than in other groups; the levels of ALT, AST, and ALP in the high-dose group decreased to lower levels; and the ALT level in the high-dose group decreased to the same level as in group N. Regarding ALB, group N had the highest content, while the Chinese pond turtle egg protein peptide extract intervention groups showed a trend of gradually increasing ALB levels with increasing concentration, and group M had the lowest ALB content. Figure 4 In (E), the UREA content in group M differed significantly from other groups; under high-dose intervention with *Sinapis alba* egg protein peptide extract, the UREA content decreased to the same level as group N. CREA content determination results showed that group N had the lowest CREA content; under intervention with *Sinapis alba* egg protein peptide extract, the CREA content gradually decreased with increasing concentration. These findings indicate that *Sinapis alba* egg protein peptide extract can reduce the damage to liver and kidney function caused by D-galactose in aging mice.
[0099] 3. The effects on the expression levels of p62, p53, p-p53, and p16 proteins in mouse liver were investigated at the molecular mechanism level.
[0100] The effects of Chinese pond turtle egg protein peptide extract on the expression levels of p62, p53, p-p53, and p16 proteins in mouse liver are shown in the following results. Figure 5 (A)- Figure 5 As shown in (D).
[0101] Figure 5 (B)- Figure 5 (D) illustrates the effect of CREP-1 on the expression levels of p62, p53, p-p53, and p16 proteins in mouse liver. Compared with the normal control group (N group), the expression levels of p62, p53, p-p53, and p16 proteins in the liver of mice in the aging model group (M group) were significantly increased, indicating that the D-galactose-induced aging model was successfully established and that these proteins are key biomarkers of liver aging.
[0102] Compared to group M, the high-dose intervention group of *Trionyx sinensis* egg protein peptide extract significantly downregulated the expression levels of p62, p53, p-p53, and p16 proteins, with p16 protein expression even approaching the level of the normal control group. Its mechanism of action may manifest in two aspects: First, blocking upstream factors of aging. In group M, the expression levels of proteins such as p53 and p-p53, caused by oxidative stress damage, increased significantly. High-dose *Trionyx sinensis* egg protein peptide extract alleviated oxidative stress damage and significantly reduced p53 expression and its phosphorylation activation level, resulting in a significant decrease after intervention, indicating that it can inhibit upstream aging signals by reducing oxidative stress. Second, directly regulating core aging pathways. Notably, p16, a key effector protein of cell cycle arrest, was significantly overexpressed in group M. Although the positive control group (Vc) could increase antioxidant enzyme activity and downregulate p16 to some extent, the downregulation was far less than that in the high-dose CREP-1 group; there is a fundamental difference in the intensity of their anti-aging effects. Vitamin C's antioxidant effect is fundamental, and its intervention in the aging process remains at the level of "slowing down damage." In contrast, CREP-1's anti-aging effect is proactive and targeted, more effectively blocking the expression of aging-related proteins and restoring or even lowering levels of indicators such as p16 to those in the normal control group. It is noteworthy that CREP-1's anti-aging effect is multi-dimensional and synergistic: on the one hand, it reduces oxidative stress damage through antioxidation; on the other hand, it directly regulates core aging pathways. These two mechanisms work together, resulting in a more significant downregulation effect on aging biomarkers. Vitamin C, relying solely on its single antioxidant mechanism, cannot achieve this synergistic intervention, thus its downregulation effect is relatively much weaker.
[0103] In summary, within the dose range of 100 - 400 mg / kg administered to experimental animals, the protein peptide extract from Chinese soft-shelled turtle eggs can significantly improve the weight loss in D-galactose-induced senescent mice; significantly increase the activities of antioxidant enzymes CAT, SOD, and GSH-Px in the serum of mice, while reducing the content of lipid peroxidation product MDA in the serum; relieve the oxidative stress damage and functional damage to the liver and kidneys of senescent mice caused by D-galactose, and significantly reduce the protein expression levels of senescence markers p62, p53, p-p53, and p16 in the liver of D-galactose-induced senescent mice. All of the above confirm that the protein peptide extract from Chinese soft-shelled turtle eggs has anti-aging effects and can be used to prepare drugs, health products, and / or functional foods for delaying, preventing, and / or treating senescence.
[0104] Example ⑧ Verification of the combined anti-aging effect
[0105] I. Animal experiment
[0106] Using a D-galactose-induced senescent mouse model, after intragastric administration of a high-dose protein peptide + Vc combined liquid, the anti-aging effect of the combination was evaluated by the protein expression level of p16 in the liver of mice.
[0107] II. Experimental procedure
[0108] 1. Experimental animals
[0109] The experimental animals were 8-week-old SPF-grade male C57BL / 6 mice (weighing 20 ± 2 g), purchased from the Experimental Animal Center of Hangzhou Medical College, and the experimental animal production license number was SCXK(Zhe) 2024-XXX. The experimental mice were raised in a pathogen-free barrier environment with a constant temperature of 22 ± 2 °C, a relative humidity of 65 ± 5%, and a 12 h light / dark cycle. During this period, the provided feed was the standard mouse feed (AIN-93) and sterile water. Strictly abide by the SPF-grade animal use guidelines to ensure the health and safety of the experimental animals.
[0110] 2. Experimental method
[0111] After 7 days of adaptive feeding, the mice were randomly divided into 5 groups, namely the blank control group (N), the model group (M), the positive control group (Vc), the high-dose protein peptide group (CREP-1, 400 mg / kg), and the high-dose protein peptide and Vc combined group (CREP-1, 400 mg / kg; Vc, 400 mg / kg), with 6 mice in each group. The detailed grouping is as follows:
[0112] Blank control group (N): Normal drinking water and feed were given for 30 days.
[0113] Model group (M): Normal drinking water and feed were provided, and each animal was injected intraperitoneally with 0.2 mL of D-galactose (400 mg / kg) daily for 30 days.
[0114] Positive control group (Vc): Normal drinking water and feed were provided. Each animal was injected intraperitoneally with 0.2 mL of D-galactose (400 mg / kg) and administered 0.2 mL of L-ascorbic acid (400 mg / kg) by gavage daily for 30 days.
[0115] High-dose protein peptide group (CREP-1): Normal drinking water and feed were provided, and each animal was given 0.2 mL of D-galactose (400 mg / kg) intraperitoneally and 0.2 mL of the oral liquid preparation of Example 6 (400 mg / kg of Chinese pond turtle egg protein peptide extract) by gavage for 30 days.
[0116] High-dose protein peptide and vitamin C combined group (Vc+CREP-1): Normal drinking water and feed were provided. Each animal was given 0.2 mL of D-galactose (400 mg / kg) intraperitoneally, 0.1 mL of Chinese pond turtle egg protein peptide extract (400 mg / kg) and 0.1 mL of L-ascorbic acid (400 mg / kg) by gavage daily for 30 days.
[0117] 3. Detection of p16 protein expression in mouse liver
[0118] An appropriate amount of liver tissue was placed in a grinding tube, and RIPA lysis buffer containing protease and phosphatase inhibitors and grinding beads were added. The tissue was then lysed and proteins extracted using a tissue homogenizer. After lysis, the tissue was centrifuged at 8000 r / min for 15 min, and the supernatant was collected. Protein levels were measured and quantified using the BCA method. Protein expression was then detected by immunoblotting. Specifically, proteins were separated from the sample on a 10% SDS-PAGE gel and then transferred to a 0.45 μm PVDF membrane for blocking. After blocking, the PVDF membrane was incubated overnight with primary antibody (Beyotime Biotechnology Co., Ltd.) at 4 ℃. After incubation, the primary antibody was recovered, and the membrane was washed three times with TSBT for 5 min each time. After washing, the PVDF membrane was transferred to secondary antibody (Beyotime Biotechnology Co., Ltd.) and incubated at room temperature with shaking for 1 h. After incubation, the secondary antibody was recovered, and the PVDF membrane was washed three times with TSBT. The bands were visualized, and the grayscale of the bands was analyzed using ImageJ.
[0119] 4. Data Processing
[0120] The data processing in this invention uses SPSS, ImageJ, and Graphpad Prism for data analysis and statistics. ImageJ is used to analyze protein bands, and the expression of the target protein in each group is represented by the ratio of the gray value of the target protein band to the gray value of the internal reference protein band.
[0121] All experiments were repeated three times (n=3), and results are expressed as mean ± standard deviation (N±SD). One-way ANOVA was used to compare differences among multiple groups, and independent samples t-test was used to compare differences between two groups. P < 0.05 was considered statistically significant.
[0122] III. Experimental Results
[0123] 1. Effects on p16 protein expression levels in mouse liver
[0124] The effect of high-dose protein peptides and vitamin C combined on the expression level of p16 protein in mouse liver is shown in the following results. Figure 7 As shown.
[0125] The p16 protein expression level in group M was significantly higher than that in the other groups. With intervention from vitamin C (400 mg / kg), the p16 protein level was significantly downregulated, decreasing to 0.928 compared to group M, and showing no significant difference from group N. With intervention from the protein peptide CREP-1 alone (400 mg / kg), the p16 protein expression level further decreased to 0.641, significantly lower than groups M, Vc, and N (P < 0.05). With the combined intervention of the protein peptide and vitamin C, the p16 protein expression level decreased to 0.195, showing a significant difference compared to the other four groups (P < 0.05), fully demonstrating that the combined use of CREP-1 and vitamin C has a better anti-aging effect.
[0126] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. Application of Chinese pond turtle egg protein peptide extract in the preparation of drugs that inhibit the expression of liver aging-related biomarkers.
2. Application of Chinese Turtle Egg Protein Peptide Extract in the Preparation of Anti-aging Drugs.
3. Use according to claim 2, characterized in that, The anti-aging effect specifically refers to improving D-galactose-induced aging symptoms.
4. Use according to claim 2 or 3, characterized in that, The Chinese pond turtle egg protein peptide extract exerts its anti-aging effect through at least one of the following mechanisms: (a) Improves symptoms of age-related weight loss induced by D-galactose; (b) Increase the activity of antioxidant enzymes, superoxide dismutase and / or glutathione peroxidase in the serum of the test subjects, while reducing the content of malondialdehyde, a lipid peroxidation product, in the serum of the test subjects; (c) Alleviate age-mediated oxidative stress damage and functional impairment of the liver and / or kidneys; (d) Down-regulate the expression levels of aging-related biomarkers in the liver of the subjects.
5. Use according to claim 4, characterized in that, The aging biomarkers include p62 protein, p53 protein, p-p53 protein and / or p16 protein.
6. Use according to claim 1, characterized in that, The inhibition of liver aging-related biomarkers is achieved by downregulating the expression of p62, p53, p-p53 and / or p16 proteins in the liver.
7. An anti-aging pharmaceutical composition, characterized by, The pharmaceutical composition contains a therapeutically effective amount of Chinese pond turtle egg protein peptide extract and a pharmaceutically acceptable carrier; the pharmaceutical composition is used to inhibit the expression of liver aging-related markers to improve D-galactose-induced aging.
8. The pharmaceutical composition of claim 7, wherein, The Chinese pond turtle egg protein peptide extract was prepared through the following steps: The egg white and yolk of a Chinese pond turtle egg were separated. The egg white was frozen at -80℃ for 3-4 hours, followed by freeze-drying for 2-3 days to obtain Chinese pond turtle egg protein powder. The freeze-dried egg white powder was then dissolved in pure water, and the pH was adjusted to 5-6. Papain was added at a dosage of 8000-16000 U / g, and the mixture was enzymatically hydrolyzed for 100-200 minutes under ultrasonic power of 200-300 W and temperature of 45-50℃. After enzymatic hydrolysis, the extract was inactivated, centrifuged, filtered, and the supernatant was purified and freeze-dried to obtain the Chinese pond turtle egg protein peptide extract.
9. The pharmaceutical composition of claim 8, wherein, The eggs of the Chinese pond turtle came from artificially bred Chinese pond turtles.
10. An anti-aging health food or functional food, characterized by, It contains Chinese pond turtle egg protein peptide extract and a food-grade acceptable carrier; the health product or functional food is used to inhibit the expression of liver aging-related markers or improve D-galactose-induced aging.