Use of a linear peptide in the preparation of a medicine or functional food for improving cold resistance of the body

Abstract

This invention discloses the application of a linear peptide or a pharmaceutically acceptable salt thereof in the preparation of drugs or functional foods for enhancing the body's cold resistance. Animal experiments showed that oral administration of the linear peptide significantly prolonged the survival time of mice in a 4°C low-temperature environment and significantly enhanced their ability to maintain rectal temperature; simultaneously, serum levels of thyroid hormones (T3, T4) and adrenaline were significantly increased (P < 0.05).

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and functional food technology, specifically relating to the application of a linear heptapeptide in the preparation of cold-resistant drugs or functional foods. Background Technology

[0002] Cold exposure triggers a series of compensatory physiological responses to maintain core body temperature homeostasis, primarily including peripheral vasoconstriction to reduce heat loss, shivering-induced thermogenesis, and enhanced metabolic thermogenesis through activation of the hypothalamic-pituitary-thyroid axis and the sympathetic-adrenal medullary system. Thyroid hormones (T3 and T4) play a central role in regulating basal metabolic rate and adaptive thermogenesis. Studies have shown that local T3 production in brown adipose tissue (BAT), catalyzed by type II T4 5′-deiodinase, is a key step in the thyroid hormone-dependent cold-adaptive thermogenesis response, and cold exposure itself can activate this deiodinase through the sympathetic nervous system, thereby enhancing the thermogenesis effect. Furthermore, T3 can promote mitochondrial thermogenesis by activating thyroid hormone receptor and uncoupling protein 1 (UCP1) gene expression.

[0003] In the field of cold-resistant drug development, current technologies mainly focus on the research of antifreeze peptides (AFPs). Natural antifreeze proteins are known to exist in cold-resistant organisms such as fish, insects, plants, and bacteria, and they function through an adsorption-inhibition mechanism by adsorbing onto the surface of ice crystals and inhibiting ice crystal growth. In recent years, research on antifreeze peptides has further expanded into the field of artificial design. For example, in 2025, Zhang Lei's team at Tianjin University reported a method for artificially designing antifreeze peptides based on the "site-to-distance" design principle in the *Journal of the American Chemical Society* (JACS), achieving the rational design of antifreeze peptides. Furthermore, in 2025, Pan et al. synthesized antifreeze peptide oligomers through protease catalysis and found that sequence order is the key factor determining antifreeze activity, rather than solely depending on amino acid composition. However, these antifreeze peptides mainly function through physical means (inhibiting ice crystal growth and protecting cells from freeze-thaw damage), and their mechanism of action is fundamentally different from enhancing the body's systemic cold resistance.

[0004] On the other hand, existing studies have reported the effects of cold exposure on the human endocrine system. A review by Leppäluoto et al. (2005) indicated that long-term cold exposure accelerates thyroid hormone metabolism, increases T3 clearance and production, and may lead to a decrease in serum free T3 levels. However, this physiological phenomenon has not yet been translated into effective pharmacological intervention strategies. Off-label use of low-dose thyroxine (T4) or triiodothyronine (T3) is occasionally seen clinically to enhance metabolic thermogenesis, but such applications lack standardized clinical trial data and pose potential cardiovascular and metabolic risks.

[0005] The inventors of this application previously isolated and identified a linear heptapeptide, TYVPKAF, from deer antler and found that it can be used to treat osteoarthritis and promote bone defect healing (CN119080880A). However, there are currently no reports on this peptide or any variant thereof in enhancing the body's cold resistance. Existing antifreeze peptides mainly exert their effects by physically inhibiting ice crystal growth, which is fundamentally different from strategies that systematically enhance cold resistance by regulating endocrine hormone levels, and cannot meet the needs of the art for safe and effective cold-resistant drugs. Therefore, there is an urgent need in the art for a novel drug or functional food that can enhance the body's cold resistance by regulating thyroid hormone and adrenaline levels. Summary of the Invention

[0006] This invention provides a novel use for linear peptides, specifically involving the application of a linear heptapeptide with the sequence TYVPKAF (SEQ ID NO:1) in the preparation of drugs or functional foods for enhancing the body's cold resistance, in order to solve the technical problem of the lack of safe and effective drugs or functional foods that can systematically enhance the body's cold resistance in the prior art.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides the use of a linear peptide or a pharmaceutically acceptable salt thereof in the preparation of a drug or functional food for enhancing the body's cold resistance, wherein the linear peptide comprises the amino acid sequence shown in SEQ ID NO:1 (TYVPKAF).

[0009] In one embodiment of the present invention, the application is the use of a linear peptide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving the body's ability to maintain body temperature and / or prolonging survival time under low-temperature conditions.

[0010] In one embodiment of the present invention, the linear peptide is a linear heptapeptide with the amino acid sequence TYVPKAF.

[0011] In one embodiment of the invention, the pharmaceutically acceptable salt is selected from acetate, hydrochloride, phosphate, sulfate, citrate, or trifluoroacetate.

[0012] In one embodiment of the present invention, the drug is administered orally.

[0013] In one embodiment of the present invention, the drug is an oral preparation, which includes at least one of oral liquid, tablet, capsule, and granule.

[0014] In one embodiment of the present invention, the functional food includes at least one of beverages, solid beverages, compressed candies, and gel candies.

[0015] In one embodiment of the present invention, the drug or functional food enhances cold resistance by increasing the body's thyroid hormone and / or adrenaline levels.

[0016] In one embodiment of the invention, the thyroid hormones include triiodothyronine (T3) and / or thyroxine (T4).

[0017] In one embodiment of the present invention, the drug or functional food is used to prevent or alleviate bodily damage caused by low-temperature exposure.

[0018] In one embodiment of the present invention, the temperature of the low-temperature exposure is ≤4°C.

[0019] In one embodiment of the invention, the linear peptide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 0.5-200 mg / kg body weight in a pharmaceutical or functional food.

[0020] In one embodiment of the invention, the daily dose of the linear peptide or a pharmaceutically acceptable salt thereof in a drug or functional food is 50-150 mg / kg body weight.

[0021] The present invention also provides a pharmaceutical composition for enhancing the body's cold resistance, the pharmaceutical composition comprising a therapeutically effective amount of a linear peptide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, the linear peptide comprising the amino acid sequence shown in SEQ ID NO:1: TYVPKAF.

[0022] In one embodiment of the present invention, the dosage form of the pharmaceutical composition is an oral preparation, which is selected from at least one of oral liquid, tablet, capsule or granule.

[0023] In one embodiment of the invention, the pharmaceutically acceptable carrier is selected from one or more of diluents, binders, lubricants, and disintegrants.

[0024] The present invention also provides a method for enhancing the body's cold resistance for non-therapeutic purposes, comprising the step of orally administering an effective amount of a linear peptide or a pharmaceutically acceptable salt thereof to a subject in need, said linear peptide comprising the amino acid sequence shown in SEQ ID NO:1: TYVPKAF.

[0025] Compared with the prior art, the present invention has the following beneficial effects and technical advantages:

[0026] (1) First discovery of a novel cold-resistance use for the linear heptapeptide TYVPKAF. This invention first discovers and confirms the novel use of the linear heptapeptide TYVPKAF and its pharmaceutically acceptable salt to enhance the body's cold resistance. Animal experiments show that after oral administration of this linear heptapeptide, the survival time of mice in a 4°C low-temperature environment was significantly prolonged (all mice in the normal water group died after 6 hours of 4°C cold exposure, while 40% of the mice in the oral peptide group survived after 6 hours and 10% survived after 8 hours), and the body temperature maintenance ability was significantly enhanced (the rectal temperature of the mice in the oral peptide group remained at around 25°C after 6 hours of 4°C cold exposure, while all mice in the normal water group died), indicating that this peptide can systematically enhance the body's cold resistance, providing a novel active substance for the development of cold-resistance drugs and functional foods.

[0027] (2) The mechanism of action is clear: it exerts a systemic cold-resistance effect by regulating endocrine hormone levels. This invention confirms that the linear heptapeptide TYVPKAF, after oral administration, can significantly increase the levels of thyroid hormones (T3, T4) and adrenaline in the body (P < 0.05). Thyroid hormones and adrenaline are core hormones for regulating basal metabolic rate and adaptive thermogenesis in the body, and their increased levels can directly enhance the body's heat production capacity and cold resistance. This mechanism of action is completely different from the existing antifreeze peptides' mechanism of action of inhibiting ice crystal growth through physical means (existing antifreeze peptides mainly inhibit ice crystal growth by adsorbing onto the surface of ice crystals, which is fundamentally different from the mechanism of action of this invention, which systematically enhances cold resistance by regulating endocrine hormone levels), providing a new strategy and direction for the development of cold-resistant products.

[0028] (3) Oral administration, high safety and convenient use. The linear heptapeptide TYVPKAF described in this invention can be administered orally. Animal experiments show that it has good tolerability, with no abnormalities observed during 32 weeks of administration, and a wide safety window. Compared with invasive methods such as injection, oral formulations are more convenient to use, have higher patient compliance, and are suitable for promotion and application among people who need to improve their cold resistance, such as those working in cold environments, polar exploration, and military training. Attached Figure Description

[0029] Figure 1 A schematic diagram of a peptide administered orally to mice;

[0030] Figure 2 Survival curves of mice in the normal drinking water group and the oral peptide group under 4°C cold exposure conditions;

[0031] Figure 3 The graph shows the changes in rectal temperature of mice in the normal drinking water group and the oral peptide group at different time points under 4℃ cold exposure conditions.

[0032] Figure 4The image shows the ELISA results of serum triiodothyronine (T3) levels in mice in the normal drinking water group and the oral peptide group.

[0033] Figure 5 The image shows the ELISA results of serum thyroxine (T4) levels in mice in the normal drinking water group and the oral peptide group.

[0034] Figure 6 The image shows the ELISA results of serum adrenaline (EPI) levels in mice in the normal drinking water group and the oral peptide group. Detailed Implementation

[0035] The technical solution of the present invention will be described in more detail with reference to specific embodiments and accompanying drawings. In implementing the present invention, except for the experimental steps, conditions, and methods described in detail below, all other techniques and common knowledge in the art are employed, and no additional special limitations are imposed.

[0036] It should be noted that the embodiments described herein are only some typical examples of the present invention, and not all implementation methods. The following description of exemplary embodiments is for illustrative purposes only and does not constitute any limitation on the present invention or its application scenarios.

[0037] In the demonstration and discussion of the various embodiments, the specific numerical values ​​and parameters involved are for illustrative purposes only and are not intended to be limiting. Therefore, other embodiments based on the concept of this invention can have their relevant numerical values ​​adjusted according to actual needs.

[0038] In addition, unless otherwise specified, the experimental materials used in the examples are all commercially available biochemical reagents, and the experimental operations also follow the standard procedures in this field.

[0039] Example 1: Effect of linear heptapeptide TYVPKAF on cold tolerance in mice

[0040] 1.1 Laboratory Animals and Grouping

[0041] Forty SPF-grade male C57BL / 6 mice (purchased from Shanghai BK Company, catalog number: BK-C57-56W), aged 56 weeks, were randomly divided into two groups after one week of acclimatization: a normal water intake group (Control group, n=20) and an oral peptide group (PP group, n=20). All mice were housed in a constant temperature (22±2℃), constant humidity (50±10%) environment with 12-hour light-dark cycles, and had free access to water and food. Experimental procedures were conducted in accordance with relevant ethical guidelines for laboratory animal welfare.

[0042] 1.2 Administration Method

[0043] Mice in the oral peptide group were given a daily dose of 100 mg / kg body weight of the linear heptapeptide TYVPKAF (abbreviated as PP, sequence Thr-Tyr-Val-Pro-Lys-Ala-Phe, SEQ ID NO:1, synthesized by Genscript Biotech Co., Ltd., purity ≥95%) dissolved in drinking water, and allowed free access to the water. Mice in the normal drinking water group were given only drinking water without the peptide. Both groups of mice were administered the drug for 32 weeks.

[0044] 1.3 Cold Exposure Experiment

[0045] After 32 weeks of drug administration, 10 mice from each of the two groups were randomly selected for a cold tolerance test. Mice were transferred to a pre-cooled 4°C incubator, placed individually in cages (cages for each group were randomly arranged within the incubator), with normal ventilation maintained. No food was provided throughout the test, but pre-cooled drinking water was available. Mice survival was recorded every hour, and rectal temperature was measured every 2 hours. The cold exposure duration was 6 hours; if mice survived, it could be extended to 8-10 hours.

[0046] (1) Survival rate observation

[0047] like Figure 2 As shown, in the normal drinking water group, more than half of the mice died after 4 hours of cold exposure at 4°C (survival rate 40%), and all died after 6 hours (survival rate 0%). In contrast, in the oral peptide group, more than half of the mice died after 6 hours of cold exposure at 4°C (survival rate 40%), and some still survived after 8 hours (survival rate 20%). Kaplan-Meier survival curve analysis showed a statistically significant difference in survival time between the two groups (P < 0.01, Log-rank test). The results indicate that the cold tolerance of the mice in the oral peptide group was significantly better than that in the normal drinking water group.

[0048] (2) Changes in rectal temperature

[0049] like Figure 3 As shown, after 2 hours of cold exposure at 4℃, the rectal temperature of mice in the normal drinking water group (28.5±1.2℃) was significantly lower than that in the oral peptide group (32.1±0.9℃), with a statistically significant difference (P < 0.05, Student's t-test). After 4 hours of cold exposure, the rectal temperature of mice in the normal drinking water group was below 20℃ (18.7±1.5℃), and the difference from that in the oral peptide group (26.4±1.1℃) was even more significant (P < 0.01). After 6 hours of cold exposure, all mice in the normal drinking water group had died, while the rectal temperature of surviving mice in the oral peptide group remained at around 25℃ (25.3±0.8℃). These results further indicate that oral administration of the linear heptapeptide TYVPKAF can significantly improve the body temperature maintenance ability of mice under low temperature conditions.

[0050] 1.4 Hormone Level Detection

[0051] (1) Sample collection

[0052] Another group of mice (n=10 per group) were anesthetized, and whole blood was collected in EDTA anticoagulant tubes (BD Company, catalog number: 367525). Within 30 minutes after collection, the blood was centrifuged at 3000×g for 30 minutes at 2-8℃. The supernatant was collected, aliquoted, and stored at -80℃ for later use.

[0053] (2) ELISA detection

[0054] Serum thyroid hormone (T3, T4) and epinephrine (EPI) levels were detected using enzyme-linked immunosorbent assay (ELISA). The kits used are as follows:

[0055] Mouse triiodothyronine (T3) ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number: ml300909);

[0056] Mouse thyroxine (T4) ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number: ml001955);

[0057] Mouse adrenaline (EPI) ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number: ml002049).

[0058] The specific steps are as follows:

[0059] (a) Preparation of Standards and Samples: Remove the required strips from the aluminum foil bag. Seal the remaining strips in a resealable bag and store them at 4°C for drying. Set up wells for standards, samples, and blanks. Add 50 μL of standards at concentrations of 0, 0.75, 1.5, 3, 6, and 12 pmol / L to wells T3 sequentially; add 50 μL of standards at concentrations of 0, 25, 50, 100, 200, and 400 ng / mL to wells T4 sequentially; add 50 μL of standards at concentrations of 0, 1, 2, 4, 8, and 16 ng / mL to wells epinephrine sequentially. Add 10 μL of the sample to be tested to the wells, followed by 40 μL of sample diluent; do not add any reagents to the blank wells.

[0060] (b) Antibody addition and incubation: Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well, seal with sealing film and incubate at 37°C for 60 minutes (incubator: Shanghai Yiheng Scientific Instruments Co., Ltd., model: DHP-9054).

[0061] (c) Washing: Discard the liquid in the well, pat dry, fill each well with washing solution, let stand for 1 minute and then shake dry. Repeat the washing process 5 times, and finally pat dry thoroughly on absorbent paper.

[0062] (d) Color development and termination: Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well and measure the optical density (OD value) of each well at 450 nm within 15 minutes (ELISA reader: BioTek, model: ELx808).

[0063] (e) Calculation of results: Plot a linear regression curve with the standard concentration as the x-axis and the corresponding OD value as the y-axis. The sample concentration calculated by the curve equation is multiplied by the dilution factor (5 times) to obtain the measured concentrations of T3, T4 and adrenaline in the sample.

[0064] (3) Statistical analysis

[0065] All experimental data are expressed as mean ± standard deviation (Mean ± SD). Comparisons between two groups were performed using Student's t-test (for normally distributed data) or Mann-Whitney U test (for non-normally distributed data). Survival analysis was performed using the Kaplan-Meier method to plot survival curves, and differences between groups were compared using the Log-rank test. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly statistically significant. All statistical analyses were performed using GraphPad Prism 9.0 software.

[0066] (4) Test results

[0067] like Figure 4 As shown, the serum T3 level in mice in the oral peptide group (5.82±0.61 pmol / L) was significantly higher than that in the normal drinking water group (3.15±0.43 pmol / L), and the difference was highly statistically significant (P < 0.01).

[0068] like Figure 5 As shown, the serum T4 level in mice in the oral peptide group (112.5±10.8 ng / mL) was significantly higher than that in the normal drinking water group (68.3±8.5 ng / mL), and the difference was highly statistically significant (P < 0.01).

[0069] like Figure 6 As shown, the serum adrenaline level in mice in the oral peptide group (6.74±0.82 ng / mL) was significantly higher than that in the normal drinking water group (3.42±0.56 ng / mL), and the difference was highly statistically significant (P < 0.01).

[0070] The above results indicate that, after oral administration, the linear heptapeptide TYVPKAF can systematically enhance the body's cold tolerance by increasing the levels of thyroid hormones (T3, T4) and adrenaline.

[0071] The embodiments described in this specification are only intended to help those skilled in the art better understand and implement the present invention. For those skilled in the art, appropriate adjustments, substitutions, or extensions can be made to these embodiments based on the content disclosed in this invention without requiring creative effort, and the basic principles of the invention can be applied to other similar scenarios. Therefore, the disclosed embodiments should not be considered as limiting the scope of protection of this invention. Any equivalent modifications, improvements, or alternatives made based on this invention without departing from its essential spirit should fall within the scope of protection of this invention.

Claims

1. The use of a linear peptide or a pharmaceutically acceptable salt thereof in the preparation of a medicine or functional food for enhancing the body's cold resistance, said linear peptide comprising the amino acid sequence shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt is selected from acetate, hydrochloride, phosphate, sulfate, citrate, or trifluoroacetate.

3. The application according to claim 1 or 2, characterized in that, The drug is administered orally; preferably, the drug is an oral preparation selected from at least one of oral liquid, tablets, capsules or granules.

4. The application according to any one of claims 1 to 3, characterized in that, The functional food is selected from at least one of beverages, solid beverages, compressed candies, or gel candies.

5. The application according to any one of claims 1 to 4, characterized in that, The drug or functional food enhances cold resistance by increasing the body's thyroid hormone and / or adrenaline levels; preferably, the thyroid hormones include triiodothyronine (T3) and / or thyroxine (T4).

6. The application according to any one of claims 1 to 5, characterized in that, The drug or functional food is used to prevent or alleviate bodily damage caused by low-temperature exposure; preferably, the low-temperature exposure temperature is ≤4℃.

7. The application according to any one of claims 1 to 6, characterized in that, The daily dose of the linear peptide or its pharmaceutically acceptable salt in a drug or functional food is 0.5-200 mg / kg body weight; more preferably, the daily dose is 50-150 mg / kg body weight.

8. The application according to any one of claims 1 to 7, characterized in that, The application refers to the use of linear peptides or pharmaceutically acceptable salts thereof in the preparation of drugs for improving the body's ability to maintain body temperature and / or prolonging survival time under low-temperature conditions.

9. A pharmaceutical composition for enhancing the body's cold resistance, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of a linear peptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein the linear peptide comprises the amino acid sequence shown in SEQ ID NO: 1; the dosage form of the pharmaceutical composition is preferably an oral formulation selected from at least one of oral liquids, tablets, capsules or granules; the pharmaceutically acceptable carrier is preferably selected from one or more of diluents, binders, lubricants or disintegrants.

10. A method for enhancing the body's cold resistance for non-therapeutic purposes, characterized in that, The method includes the step of orally administering an effective amount of a linear peptide or a pharmaceutically acceptable salt thereof to a subject in need, said linear peptide comprising the amino acid sequence shown in SEQ ID NO:1.

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