A hippocampus polypeptide and use thereof

Abstract

The application belongs to the technical field of peptide preparation, and relates to a hippocampus polypeptide and application thereof. The amino acid sequence of the hippocampus polypeptide is shown in the sequence table SEQ ID NO. 1. The hippocampus polypeptide provided in the application has excellent hypoxia tolerance activity, and by using the characteristic, the hippocampus polypeptide can be added as an active ingredient into food, beverage or dietary supplement to prepare functional food or health products for daily use by specific groups of people to enhance the tolerance to hypoxic environment.

Description

Technical Field

[0001] This invention belongs to the field of peptide preparation technology, and relates to a hippocampal polypeptide and its uses. Background Technology

[0002] Seahorse ( Hippocampus As a traditional and precious Chinese medicinal herb, seahorse is recorded in classic texts such as the *Compendium of Materia Medica* as having effects such as warming the kidneys and strengthening yang, dispersing nodules and reducing swelling. Modern pharmacological studies have shown that it is rich in proteins and polypeptides, exhibiting great potential in anti-aging, anti-oxidation, and hypoxia tolerance. However, the development and research of active ingredients in seahorse are still significantly limited, restricting the research and application of its high-value-added products.

[0003] Currently, the development of seahorse resources faces three major bottlenecks: 1) Traditional extraction techniques (such as high-temperature water extraction and organic solvent extraction) are inefficient and easily damage peptide activity; 2) Existing research lacks a precise understanding of the structure and efficacy mechanism of seahorse peptides; 3) Mainstream chemical anti-fatigue / antioxidant agents on the market have safety and bioavailability issues. Therefore, there is an urgent need to use modern biotechnology to directionally prepare seahorse peptides with clear antioxidant and hypoxia-resistance activities to overcome technical barriers and achieve high-value utilization of seahorse resources. Summary of the Invention

[0004] The purpose of this invention is to provide a polypeptide with hypoxia-resistant activity prepared from seahorse, thereby improving the high-value utilization of seahorse resources.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a hippocampal polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing.

[0006] Furthermore, the present invention provides the use of the said hippocampal polypeptide as an active ingredient added to food, beverage or diet to prepare supplements, functional foods or health products.

[0007] Furthermore, the present invention provides a food, beverage, or dietary supplement containing the aforementioned hippocampal polypeptide.

[0008] Furthermore, the present invention provides a functional food or health product comprising the aforementioned hippocampal polypeptide or supplement.

[0009] The hippocampal polypeptide provided by this invention has excellent hypoxia tolerance activity. Utilizing this property, the hippocampal polypeptide can be added as an active ingredient to food, beverage or dietary supplement to prepare functional food or health products for daily use by specific groups to enhance their tolerance to hypoxic environments. Attached Figure Description

[0010] Figure 1This is a comparison diagram of the hypoxia tolerance activities of different components of hippocampal polypeptide in the embodiments of the present invention; wherein, 1. Control; 2. Hippocampal polypeptide stock solution; 3. Component F1; 4. Component F2; 5. Component F3; 6. Component F4;

[0011] Figure 2 This is a graph showing the relationship between the hypoxia resistance activity of the hippocampal polypeptide shown in SEQ ID NO.1 and the dosage in an embodiment of the present invention; wherein, 1. control group; 2. low-dose group; 3. medium-dose group; 4. high-dose group. Detailed Implementation

[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] Example 1: Preparation of hippocampal polypeptides with hypoxia tolerance activity

[0014] After drying and pulverizing the hippocampus, distilled water was added at a material-to-liquid ratio of 1:50 using water as the extraction solvent. Protease preparation was added at a ratio of 3% (by weight of the dried hippocampus powder). Enzymatic hydrolysis was carried out in a constant-temperature water bath at 50°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated at 90°C for 15 minutes. The hydrolysate was centrifuged at 10,000 rpm for 20 minutes, and the supernatant was dried to obtain hippocampal polypeptide. 10.00 g of hippocampal polypeptide powder was accurately weighed and diluted to 100 mL with ultrapure water to prepare a 100 mg / mL stock solution. Separation and purification were performed using a high-performance liquid chromatography (HPLC) system equipped with a Dubhe C18 preparative column (250 × 20 mm, 10 μm) at a flow rate of 8 mL / min, monitored at room temperature with a detection wavelength of 220 nm. Different polarity eluents were collected using a gradient elution program: the 5% methanol eluent was designated as fraction F1, the 20% methanol eluent as fraction F2, the 40% methanol eluent as fraction F3, and the 80% methanol eluent as fraction F4.

[0015] The activity of different hippocampal peptide components was screened through a mouse hypoxia tolerance experiment. The results showed that component F3 exhibited a significant hypoxia tolerance effect, with an average survival time of 23.23 minutes, significantly better than the control group and other experimental groups. This result indicates that component F3 is rich in hippocampal peptides with hypoxia tolerance activity. Specific experimental data are as follows: Figure 1 As shown.

[0016] The structure of hippocampal peptide F3 was identified using an LCMS-9030 Q-TOF liquid chromatography-mass spectrometry (LC-MS) system. Chromatographic separation was performed using an Inertsil HILIC C18 reversed-phase column with gradient elution at 0.3 mL / min at a column temperature of 30°C. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The elution program was: 0–5 min hold at 5% B, 5–10 min increase to 10% B, 10–15 min increase to 20% B, and 15–20 min increase to 40% B. Mass spectrometry detection was performed using an electrospray ionization source in positive ion mode. The ion source interface voltage was 4.0 kV, and the rates of the drying gas, heating gas, and collision gas were all 10.0 L / min. The interface temperature was 300°C, the heating module temperature was 400°C, and the scan range was m / z 50–2000. The obtained mass spectrometry data were identified using proteomics techniques, yielding 38 valid peptides.

[0017] AMPK is a key energy regulatory target in the body's response to hypoxic stress. Under hypoxic conditions, cellular ATP synthesis is inhibited, leading to an increase in the AMP / ATP ratio, which in turn activates AMPK. Activated AMPK helps cells maintain energy homeostasis in low-oxygen environments through mechanisms such as promoting glucose transport, enhancing glycolysis efficiency, and regulating mitophagy, ultimately improving the tissue and body's tolerance to hypoxia.

[0018] To verify the interaction mechanism between the active ingredient and AMPK, this invention employs a molecular docking strategy combining AutoDockFR and RosettaFlexPepDock. This method first examines the flexible conformational changes of the receptor protein using AutoDockFR, then refines the binding conformation and accurately assesses the binding free energy using RosettaFlexPepDock. Finally, based on a comprehensive score, the binding mode and interaction strength between the hippocampal peptide and AMPK are predicted, providing a molecular-level mechanistic basis for its hypoxia tolerance efficacy. The prediction results are shown in the table. Five peptides have high comprehensive scores, suggesting hypoxia tolerance activity, with the peptide shown in SEQ ID NO.1 having the highest comprehensive score, suggesting the best hypoxia tolerance activity.

[0019] Table 1. Overall Score of Hippocampal Peptide Molecular Docking

[0020] .

[0021] Example 2: Determination of the hypoxia tolerance activity of hippocampal polypeptide (SEQ ID NO.1)

[0022] (1) Synthesis of target polypeptide sequence

[0023] The target polypeptide sequence (SEQ ID NO.1) was successfully prepared using solid-phase synthesis technology.

[0024] (2) Laboratory animals and materials

[0025] Experimental materials: physiological saline, electronic balance, stopwatch, gavage syringe, 250mL ground glass bottle, petroleum jelly, soda lime (or equal amounts of sodium hydroxide and calcium carbonate).

[0026] Animals: Healthy male ICR or Kunming mice, 6-8 weeks old, weighing 18-22g.

[0027] Rearing environment: Standard laboratory conditions (temperature 22±2°C, humidity 50±10%, 12 / 12 hour light / dark cycle), free access to food and water.

[0028] Experimental sample: Hippocampal polypeptide was obtained at a material-to-liquid ratio of 1:60, a temperature of 55℃, an enzymatic hydrolysis time of 5 hours, and an enzyme content of 5%.

[0029] (3) Grouping and administration

[0030] Blank control group: administered an equal volume of physiological saline by gavage;

[0031] Low-dose hippocampal polypeptide group: hippocampal polypeptide (300 mg / kg) administered by gavage.

[0032] Medium-dose hippocampal polypeptide group: oral administration of hippocampal polypeptide (600 mg / kg).

[0033] High-dose hippocampal polypeptide group: hippocampal polypeptide (900 mg / kg) administered by gavage.

[0034] Administration cycle: 30 consecutive days via gavage.

[0035] One hour after mice in each dose group were orally administered different concentrations of the test sample and the control group was given an equal amount of solvent, each group of mice was placed individually in a 250mL ground glass bottle containing 5g of soda lime. The bottle mouth was sealed with Vaseline to ensure airtightness, and the survival time of the mice in the closed hypoxic environment was immediately recorded, with respiratory arrest as the observation endpoint.

[0036] Evaluation of hypoxia tolerance in mouse models showed that the peptide significantly prolonged the survival time of experimental animals, exhibiting a clear dose-response relationship. Figure 2 As shown, compared with the control group, the survival time of mice in each dose group was significantly increased, with the survival time of the high dose group reaching 22.93 min, which fully demonstrates that the synthetic polypeptide has significant hypoxia resistance biological activity.

Claims

1. A hippocampal polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing.

2. The application of the hippocampal polypeptide according to claim 1, characterized in that: This hippocampal polypeptide can be added as an active ingredient to food or beverages to prepare supplements, functional foods, or health products.

3. A food or beverage supplement, characterized in that: The supplement contains the hippocampal polypeptide of claim 1.

4. A functional food or health product comprising the hippocampal polypeptide of claim 1 or the supplement of claim 3.

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