Application of tuna blood peptide component in preparation of anti-fatigue product and composition of tuna blood peptide component

Abstract

The invention belongs to the technical field of functional food and health care products, and particularly relates to application of a tuna blood peptide component in preparation of an anti-fatigue product and a composition of the tuna blood peptide component. The tuna blood peptide component with the molecular weight smaller than 1000 Da is applied to preparation of a product for preventing or relieving exercise-induced fatigue. The invention reveals and verifies that the specific substance, namely the tuna blood peptide component with the molecular weight of less than 1000 Da, has a remarkable anti-fatigue effect for the first time. The method is a brand new discovery that a commonly abandoned by-product is converted into a high-value functional raw material, and waste is turned into wealth.

Description

Technical Field

[0001] This invention belongs to the field of functional food and health product technology, and specifically relates to the application of tuna blood peptide component in the preparation of anti-fatigue products and its composition. Background Technology

[0002] Exercise-induced fatigue refers to a complex physiological phenomenon characterized by a temporary decline in work capacity after physical activity, caused by excessive energy consumption, accumulation of metabolic waste products, and disruption of the internal environment. Its mechanisms primarily involve the "energy depletion theory" and the "free radical theory." The former posits that fatigue results from the depletion of energy substances (such as liver glycogen) and the accumulation of metabolic waste products (such as blood lactate and blood urea nitrogen). The latter suggests that high-intensity exercise leads to a rate at which the body produces reactive oxygen species (ROS) far exceeding the clearance capacity of the endogenous antioxidant system. Excessive free radicals trigger oxidative stress, damaging cell membranes, proteins, and DNA, and are a significant cause of muscle fatigue and injury.

[0003] Therefore, developing natural and safe active substances that can simultaneously regulate energy metabolism and enhance the body's antioxidant capacity is of great significance for preventing and alleviating exercise-induced fatigue. Bioactive peptides, due to their small molecular weight, easy absorption, and strong functionality, have become a hot topic in anti-fatigue research. Existing studies have isolated peptides with anti-fatigue activity from various biological resources such as oysters, sea cucumbers, and deer antler blood.

[0004] Tuna blood, a major byproduct of seafood processing, is rich in protein, but its in-depth development and high-value utilization are still in their early stages. While some studies have investigated its enzymatic hydrolysis to prepare peptides, these studies have largely focused on preparation processes or broad in vitro antioxidant activity. The existence of specific components in tuna blood peptides with clearly defined, in vivo-proven anti-fatigue effects, and their specific mechanisms of action, remains undisclosed in current technology. Therefore, discovering and confirming a specific peptide component with significant anti-fatigue activity from this neglected resource of tuna blood would be a work of significant innovative and applied value. Summary of the Invention

[0005] This invention provides the application of tuna blood peptide components in the preparation of products for preventing or alleviating exercise fatigue.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] Application of a tuna hemopeptide component with a molecular weight of less than 1000 Da in the preparation of products for preventing or relieving exercise fatigue.

[0008] Preferably, the tuna blood peptide component with a molecular weight of less than 1000 Da is obtained by ultrafiltration of the enzymatic hydrolysis product of tuna blood.

[0009] Preferably, the enzymatic hydrolysis product of the tuna blood is prepared by enzymatic hydrolysis of the tuna blood using an acidic protease.

[0010] As a preferred embodiment, the preparation method of the tuna blood peptide component with a molecular weight of less than 1000 Da includes the following steps: (1) Enzymatic hydrolysis: Take freshly collected tuna blood, filter, freeze dry, and pulverize to obtain tuna blood powder; take tuna blood powder, add distilled water at a material-to-liquid ratio of 1:80-100 g / mL, and adjust the pH to 4.0-4.5; add acidic protease and enzymatically hydrolyze at 49°C for 7 hours; inactivate the enzyme, cool, centrifuge and take the supernatant to obtain crude enzymatic hydrolysate; (2) Ultrafiltration separation: pass the above crude enzymatic hydrolysate through ultrafiltration membranes with molecular weight cutoff of 10000 Da and 5000 Da in sequence, and discard the cutoff liquid; pass the permeate through an ultrafiltration membrane with molecular weight cutoff of 1000 Da again, collect and freeze dry the permeate component with a molecular weight of less than 1000 Da, and obtain the tuna blood peptide component with a molecular weight of less than 1000 Da as described in this invention.

[0011] Preferably, the prevention or relief of exercise-induced fatigue is achieved by regulating energy metabolism. Preferably, regulating energy metabolism includes: reducing the concentration of lactic acid and / or blood urea nitrogen in the blood after exercise, and / or increasing liver glycogen reserves.

[0012] Preferably, the prevention or relief of exercise-induced fatigue is achieved by enhancing the body's antioxidant capacity. Preferably, enhancing the body's antioxidant capacity includes: increasing the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and / or catalase (CAT) in the liver, and / or reducing the content of malondialdehyde (MDA).

[0013] Preferably, the prevention or relief of exercise fatigue also includes protecting muscle tissue by inhibiting the extravasation of creatine kinase.

[0014] Preferably, the product is a functional food, health product, or dietary supplement.

[0015] A composition for preventing or relieving exercise-induced fatigue, the composition comprising a therapeutically effective amount of a tuna hemopeptide component with a molecular weight of less than 1000 Da, and a food- or pharmaceutically acceptable carrier.

[0016] The beneficial effects of this invention are:

[0017] This invention is the first to reveal and confirm that a specific substance, "tuna blood peptide component with a molecular weight of less than 1000 Da," possesses significant anti-fatigue effects. This is a novel discovery that transforms a typically discarded byproduct into a high-value functional raw material, achieving the goal of turning waste into treasure.

[0018] This invention systematically demonstrates, through in vitro chemical experiments (DPPH, ABTS, etc.) and cell-level experiments (H2O2-induced RAW 264.7 cell oxidative damage model), that tuna hemopeptide components with a molecular weight <1000 Da (TFBP) possess a strong ability to directly scavenge free radicals and protect cells from oxidative damage, thus confirming its in vitro antioxidant properties. TFBP remains stable over a wide pH and temperature range, and importantly, it retains high antioxidant activity even after digestion with simulated pepsin and trypsin.

[0019] This invention demonstrates, through anti-fatigue experiments using a zebrafish model, that TFBP can effectively inhibit the increase of ROS levels, exhibiting significant in vivo anti-fatigue capabilities. Using a mouse weight-bearing swimming model, it was shown that TFBP can significantly prolong the time of exhaustion in swimming (P<0.01), providing the most direct and compelling evidence of its anti-fatigue efficacy and indicating its excellent effect in improving exercise endurance.

[0020] This invention further elucidates that the anti-fatigue effect of tuna hemopeptide components with a molecular weight less than 1000 Da is achieved through a dual mechanism of "regulating energy metabolism" and "enhancing antioxidant defense." Regarding the regulation of energy metabolism, it effectively reduces the concentration of lactic acid and blood urea nitrogen in the blood after exercise, while significantly increasing liver glycogen reserves. This indicates that the substance can accelerate the clearance of metabolic waste and store more energy for the body, fundamentally delaying the onset of fatigue. Regarding the enhancement of antioxidant defense, it can significantly increase the activity of key antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver, and significantly reduce the content of malondialdehyde (MDA), a lipid peroxidation product. This demonstrates that it can effectively combat exercise-induced oxidative stress and protect the body's cells from free radical damage.

[0021] This invention also demonstrates that the tuna hemopeptide component can inhibit the extravasation of creatine kinase (CK) from muscle cells. CK is a marker enzyme for muscle damage, and this effect indicates that the active substance of this invention can effectively protect the integrity of the myofibril membrane and reduce muscle damage during high-intensity exercise.

[0022] The tuna blood peptide component with a molecular weight of less than 1000 Da described in this invention has stable product performance, is non-toxic, and can be efficiently enriched and separated using mature ultrafiltration technology, which facilitates industrial production and product quality control. Attached Figure Description

[0023] Figure 1 The effect of pH on the antioxidant activity of tuna hemopeptides;

[0024] Figure 2 The effect of metal ions on the DPPH free radical scavenging ability of enzymatic hydrolysis products;

[0025] Figure 3 The effect of metal ions on the scavenging ability of ABTS free radicals in the enzymatic hydrolysis product;

[0026] Figure 4 The effect of temperature on the antioxidant activity of tuna hemopeptides;

[0027] Figure 5 The effect of in vitro digestion simulation on the antioxidant activity of tuna blood peptides;

[0028] Figure 6 The chromatogram shows the molecular weight distribution of the enzymatic hydrolysate of tuna hemopeptides (A) and the percentage of each peptide segment with different molecular weights in tuna hemopeptides (B). In (B), there are four components: > 10000 Da (F1), 5000 Da ~ 10000 Da (F2), 1000 Da ~ 5000 Da (F3), and < 1000 Da (F4).

[0029] Figure 7 This study measured the DPPH free radical scavenging rate of each component in ultrafiltration at different concentrations. Note: Different uppercase letters indicate statistically significant differences between concentrations (P < 0.05), while lowercase letters indicate significant differences between groups (P < 0.05). The same applies below.

[0030] Figure 8 It is the determination of the hydroxyl radical scavenging rate of each component of ultrafiltration at different concentrations;

[0031] Figure 9 It is a determination of the ABTS free radical scavenging rate of each component of ultrafiltration at different concentrations;

[0032] Figure 10 It is a determination of the FRAP capacity of each component in ultrafiltration at different concentrations;

[0033] Figure 11 This is a gel chromatography separation diagram;

[0034] Figure 12 It is the determination of the DPPH free radical scavenging rate of each component;

[0035] Figure 13 The cytotoxicity of different concentrations of TFBP on RAW 264.7 cells;

[0036] Figure 14The effect of different concentrations of H2O2 on the viability of RAW 264.7 cells;

[0037] Figure 15 This study demonstrates the protective effect of TFBP against H2O2-induced damage in RAW 264.7 cells. Note: * or ** indicates a significant difference (P<0.05) or highly significant difference (P<0.01) compared to the control group; # or ## indicates a significant difference (P<0.05) or highly significant difference (P<0.01) compared to the H2O2-damaged group, and the same applies below.

[0038] Figure 16 The effect of TFBP on intracellular ROS content in H2O2-induced damaged RAW 264.7 cells;

[0039] Figure 17 These are fluorescence images of RAW 264.7 cells treated with TFBP for H2O2; Note: A: Control group; B: 500 μM H2O2 damage group; CG: H2O2 damage groups pretreated with 12.5, 25, 50, 100, and 200 μg / mL TFBP.

[0040] Figure 18 The effect of TFBP on LDH content in H2O2-induced damaged RAW 264.7 cells;

[0041] Figure 19 The effect of TFBP on SOD activity in H2O2-induced damaged RAW 264.7 cells;

[0042] Figure 20 The effect of TFBP on CAT activity in H2O2-induced damaged RAW 264.7 cells;

[0043] Figure 21 The effect of TFBP on GSH content in H2O2-induced damaged RAW 264.7 cells;

[0044] Figure 22 The effect of TFBP on MDA content in H2O2-induced damaged RAW 264.7 cells;

[0045] Figure 23 The values ​​represent the ROS fluorescence intensity of different experimental groups. Note: Different letters indicate significant differences, P<0.05.

[0046] Figure 24 These are the ROS staining results in zebrafish from different experimental groups. Note: A: Control group; B: Model group; C: L-TFBP group (100 μg / mL); D: M-TFBP group (200 μg / mL); E: H-TFBP group (500 μg / mL).

[0047] Figure 25 This refers to the change in mouse body weight over different number of days;

[0048] Figure 26 This is a graph showing the observation of liver toxicity in mice;

[0049] Figure 27 The effect of TFBP on the motor ability of mice;

[0050] Figure 28 The effect of TFBP on lactate levels in mice;

[0051] Figure 29 The effect of TFBP on blood urea nitrogen levels in mice;

[0052] Figure 30 The effect of TFBP on liver glycogen content in mice;

[0053] Figure 31 The effect of TFBP on serum creatine kinase levels in mice;

[0054] Figure 32 The effect of TFBP on serum LDH activity in mice;

[0055] Figure 33 The effect of TFBP on SOD activity in mice;

[0056] Figure 34 The effect of TFBP on GSH-Px activity in mice;

[0057] Figure 35 The effect of TFBP on CAT activity in mice;

[0058] Figure 36 The effect of TFBP on MDA activity in mice;

[0059] Figure 37 This is a Pearson correlation analysis of antioxidant and anti-fatigue effects. Note: * indicates a significant correlation at the 0.05 level (two-tailed); ** indicates a significant correlation at the 0.01 level (two-tailed). Detailed Implementation

[0060] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0061] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. The reagents used in the following embodiments, unless otherwise specified, can be purchased from conventional biochemical reagent stores.

[0062] CCK-8 kit, DCFH-DA fluorescent probe kit for reactive oxygen species (ROS), and BCA protein detection kit were purchased from Beyotime Biotechnology Co., Ltd.; ABTS kit (visible spectrophotometry) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; RAW 264.7 cell line was purchased from Shanghai Cell Bank, Chinese Academy of Sciences; RAW 264.7 complete culture medium was purchased from Wuhan Pronosei Biotechnology Co., Ltd.; and weighted solder bars were purchased from Delixi Electric Co., Ltd.

[0063] The following kits were purchased from Nanjing Jiancheng Bioengineering Institute: DPPH free radical scavenging ability kit, hydroxyl radical assay kit, total antioxidant capacity (T-AOC) assay kit (FRAP method), total superoxide dismutase (T-SOD) assay kit, superoxide dismutase (SOD) assay kit, malondialdehyde (MDA) assay kit, reduced glutathione (GSH) assay kit, glutathione peroxidase (GSH-Px) assay kit, catalase (CAT) assay kit, creatine kinase (CK) assay kit (colorimetric method), lactate dehydrogenase (LDH) kit (microplate method), lactate dehydrogenase (LDH) kit (colorimetric method), and glycogen assay kit.

[0064] Experimental data are expressed as mean ± standard deviation. Data analysis was performed using SPSS, and graphs were generated using Origin 8.0 and Graphpad Prism 8.0 software.

[0065] Example: Stability, isolation, purification and antioxidant capacity evaluation of tuna blood peptides

[0066] I. Experimental Treatment and Methods

[0067] 1 Sample Preparation

[0068] Freshly collected tuna blood was filtered through gauze, freeze-dried, and then pulverized into a uniform powder to obtain tuna blood powder. The tuna blood powder was then mixed with distilled water at a ratio of 1:100 (g / mL), and the pH was adjusted to 4.5 with 1 mol / L hydrochloric acid solution. Acidic protease (enzyme activity approximately 50,000 U / g) was added, and the mixture was enzymatically hydrolyzed at 49°C for 7 hours. After the reaction was complete, the enzyme was inactivated by heating at 100°C for 15 minutes. After cooling, the supernatant was collected by centrifugation to obtain the crude tuna blood peptide hydrolysate.

[0069] The enzymatic hydrolysate was centrifuged at 4000 r / min for 20 min to remove impurities and then freeze-dried to prepare tuna blood peptides.

[0070] 2. Stability Study of Tuna Blood Peptides

[0071] A tuna hemopeptide solution with a mass concentration of 5 mg / mL was prepared. The effects of various variables, including metal ions (50, 100, 150, 200 and 250 μg / mL of CuCl2, MgCl2 and KCl), temperature (4, 25, 37, 65 and 100℃), and pH (3, 5, 7, 9 and 11), were investigated. After shaking the reaction solution for 2 h at room temperature, the supernatant was collected, and the scavenging abilities of DPPH radicals and ABTS radicals were determined.

[0072] Gastrointestinal simulation: A 5 mg / mL tuna hemopeptide solution was pH-adjusted to an acidic environment of 2.0 using 1 mol / L hydrochloric acid solution. Pepsin was added at a mass-to-volume ratio of 4%. The reaction system was placed in a 37°C constant-temperature shaker to initiate the enzymatic hydrolysis process. Samples were taken at 60 min and 120 min of the reaction. Each stage of the sample was inactivated by boiling in a 100°C water bath for 15 min. After natural cooling to room temperature, the sample was centrifuged at 10000 r / min for 10 min. The supernatant was collected after centrifugation; this was the pepsin-digested sample. The supernatant was then adjusted to pH 7.5 using 1 mol / L NaOH, and 4% trypsin was added. The reaction continued in a 37°C water bath. The treated solution was then placed in a 100°C constant-temperature water bath for 15 min to inactivate the reaction. After the reaction was terminated, the mixture was transferred to a centrifuge after natural cooling, and the supernatant was collected to obtain the degraded sample after trypsin digestion. Finally, the DPPH and ABTS free radical scavenging abilities of untreated, pepsin-treated, and trypsin-treated samples were determined.

[0073] 3. Molecular weight distribution of tuna hemopeptides

[0074] According to the standard QB / T 2879—2007, the chromatographic conditions for this experiment were as follows: A TSKgel 2500 PWXL column with dimensions of 30 cm × 7.8 mm and a packing particle size of 7 µM was used. The mobile phase consisted of acetonitrile, water, and trifluoroacetic acid in a volume ratio of 45:55:0.1. The sample injection volume was 10 µL, the concentration was set at 5 mg / mL, and the flow rate was controlled at 0.5 mL / min. Detection was performed using a UV detector at a wavelength of 220 nm, the column temperature was controlled at 25℃, and the elution time was 25 min. Furthermore, by establishing a standard curve, the molecular weight distribution of the sample was calculated using the elution volumes of aprotinin (6511 Da), bacitracin (1422 Da), acetaminophen-acetaminophen-tyrosine-arginine (451 Da), and acetaminophen-acetaminophen-acetaminophen (189 Da).

[0075] 4. Ultrafiltration separation of tuna blood peptides

[0076] Tuna hemopeptides were separated and fractionated using different ultrafiltration membranes, yielding four fractions based on molecular weight: > 10000 Da (F1), 5000 Da ~ 10000 Da (F2), 1000 Da ~ 5000 Da (F3), and < 1000 Da (F4). Each fraction was freeze-dried and then sealed and stored in a desiccator for later use.

[0077] 5. Purification of tuna hemopeptides by gel chromatography

[0078] The ultrafiltration fractions were further purified using an AKTA pure protein separation and purification system, employing a Superdex Peptide 10 / 300 GL gel column. Before operation, the column and pump were cleaned with an acetate-sodium acetate buffer solution at pH 4.6 for 1 hour. Lyophilized powders with a molecular weight < 1000 Da were prepared into a 50 mg / mL solution using the mobile phase, filtered through a 0.22 μm aqueous membrane, and then fed into the system.

[0079] The separation conditions were set as follows: column inlet pressure was maintained at 1 MPa, injection volume was 0.5 mL, flow rate was 0.2 mL / min, and the measurement wavelength was set to 280 nm. During elution, each 1 mL of eluent was collected as one tube, and the collected elution peaks were concentrated and lyophilized for subsequent use.

[0080] 6. Analysis of the antioxidant activity of tuna blood peptides based on free radical scavenging ability

[0081] DPPH radical scavenging capacity was determined using a DPPH radical scavenging kit, with a vitamin C standard solution as a positive control. Hydroxyl radical scavenging capacity was determined using a vitamin C standard solution as a positive control. ABTS radical scavenging capacity was determined using a vitamin C standard solution as a positive control. The ultrafiltration components with different molecular weights were prepared into solutions with concentrations of 2, 4, 6, 8, and 10 mg / mL. 3+ The reducing power was determined using a Vc standard solution as a positive control, and the final result was expressed as the antioxidant capacity equivalent to Trolox per gram of lyophilized sample (μmol / g).

[0082] 7. Antioxidant Activity Analysis of Tuna Blood Peptides Based on Cell Biology

[0083] 7.1 RAW 264.7 Cell Culture and Treatment RAW 264.7 cells were cultured in complete culture medium at 37°C in a 5% CO2 cell culture incubator. The passage ratio was 1:2, and cells were passaged every other day until the cells covered approximately 80% of the T25 culture flask, at which point subsequent experiments could be performed.

[0084] 7.2 Cytotoxicity assay: RAW 264.7 cell suspension was prepared at a concentration of 1 × 10⁻⁶ cells per well. 4 Cells were evenly seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. Once the cells reached a stable adherent growth state, the culture medium was discarded, and 100 μL of tuna hemolysin solution at gradient concentrations (0, 5, 10, 25, 50, 100, 200, 400, and 500 μg / mL) was sequentially injected into each well of the experimental group. The control group maintained the basal culture medium environment, thus establishing a dose-response curve study system. Incubation was then continued for another 24 h. Cell viability was measured using the CCK-8 assay: 100 μL of CCK-8 assay reagent diluted to a 10% concentration from the basal culture medium was added to each well, followed by incubation at 37°C for 20–40 min. Immediately after incubation, the absorbance of each well was measured at 450 nm.

[0085] 7.3 Cellular Oxidative Damage Model: RAW 264.7 cells grown to 80%–90% confluence in culture flasks were seeded into 96-well plates, with the seeding density adjusted to 1 × 10⁻⁶ cells / well. 4100 µL of cell suspension was added to each well of a 96-well plate used in the experiment, and PBS was added to the outermost wells. The plates were then incubated for 24 h. Cells were then incubated with H2O2 at concentrations ranging from 100 to 800 μmol / L for 4 h. The H2O2 was discarded, and the plate was replaced with the drug-treated medium. The plates were then returned to the incubator for another 24 h of cell incubation. Cell viability was then assessed, and the appropriate damage concentration was determined based on the cell survival rate under different treatment concentrations for subsequent experiments.

[0086] 7.4 Protective effect against cell damage: RAW 264.7 cells grown to 80%–90% confluence in culture flasks were seeded into 96-well plates, with the seeding density adjusted to 1 × 10⁻⁶ cells / well. 4 100 µL of cell suspension was added to each well of a 96-well plate used in the experiment, and PBS was added to the outermost wells. The plates were then incubated for 24 h. Cells were incubated with 500 μmol / L H2O2 for 4 h, the H2O2 was discarded, and different concentrations of drug-treated culture medium (5-200 µg / mL) were added. The plates were returned to the incubator for another 24 h of cell incubation, after which cell viability was assessed. Based on the cell survival rate under different treatment concentrations, the appropriate drug concentration was determined for subsequent experiments.

[0087] 7.5 ROS content determination: RAW 264.7 cells grown to 80%–90% confluence in the culture flasks were seeded into 6-well plates, and the cell seeding density was adjusted to 1 × 10⁻⁶ cells / well. 5 Add 2 mL of culture medium to each well of the plate and incubate for 24 h. Set up blank, damage (500 μmol / L H2O2), and protection groups (12.5, 25, 50, 100, 200 µg / mL TFBP + 500 μmol / L H2O2) and continue incubation for 24 h, followed by 4 h of stimulation with 500 μmol / L H2O2. Remove the plate and discard all culture medium. Add fluorescent probe solution to the plate according to the kit instructions, ensuring the added dye completely covers the cells. Place the plate back in the incubator and incubate for 20 min. Remove the plate and discard the solution. Add serum-free culture medium or PBS to the plate. Wash the cells three times, digest and collect them, and label them. Detect intracellular fluorescence signals using a fluorescence spectrophotometer at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Stronger fluorescence signals indicate higher intracellular ROS content. Fluorescence intensity was observed using a fluorescence microscope, and the reactive oxygen species content was positively correlated with the fluorescence intensity.

[0088] 7.6 LDH content determination: 1×10 5Cells were seeded into each well of a 6-well plate, with control, damage, and protection groups established. The protection groups were supplemented with different concentrations of TFBP (12.5, 25, 50, 100, and 200 μg / mL), and cultured for 24 h, followed by stimulation with 500 μmol / L H₂O₂ for 4 h. After culture, the culture medium in the wells was collected, and the cell pellet was removed by centrifugation at 1000 rpm for 4 min at 4°C. The extracellular LDH content was then measured according to the kit's instructions, and the results were recorded in U / L.

[0089] 7.7 SOD, CAT, GSH, and MDA were measured by seeding 1×10⁻⁶ cells into each well of a 6-well plate. 5 Cells were collected and divided into a control group, a damaged group, and a protection group. The protection group was supplemented with different concentrations of TFBP (12.5, 25, 50, 100, and 200 μg / mL) and cultured for 24 h, followed by stimulation with 500 μmol / L H2O2 for 4 h. After culture, cell homogenate was prepared using the following steps: cells were gently collected by pipetting and centrifuged at 4°C and 1000 rpm for 4 min to obtain a pellet; cell lysis buffer was then added, and the cells were lysed on ice for 15 min; subsequently, the pellet was removed by centrifugation at 3000 rpm for 10 min, and the resulting supernatant was the cell homogenate. Following the kit instructions, the protein concentration of the homogenate was determined using the BCA method, and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) were simultaneously measured.

[0090] II. Results Analysis

[0091] 1. Results of antioxidant stability of tuna blood peptides

[0092] The effect of different pH values ​​on the antioxidant activity of tuna hemopeptides, such as Figure 1 As shown, within the pH range of 3-11, the scavenging rates of tuna hemopeptides showed a trend of first increasing and then decreasing with increasing pH, exhibiting better antioxidant activity under both weakly acidic and neutral conditions. The highest DPPH radical scavenging rate (84.25%) was observed at pH 5; the ABTS radical scavenging rate also reached its peak at this pH value (29.07%).

[0093] The effect of metal ions on the activity of tuna hemopeptides, such as Figure 2 and Figure 3 As shown. The degree to which the DPPH scavenging activity of tuna hemopeptides is affected by metal ions is Mg. 2+ >Cu 2+ >K + The degree to which ABTS scavenging activity is affected by metal ions is Cu 2+ >Mg 2 >K+ As the concentration of different metal ions increased, the ABTS and DPPH scavenging rates of tuna hemopeptides decreased to some extent. 2+ At a mass concentration of 250 μg / mL, the ABTS clearance rate of tuna hemopeptide was only 27.80%.

[0094] The effect of temperature on the antioxidant activity of tuna hemopeptides, such as Figure 4 As shown in the figure, the study found that the antioxidant capacity of this peptide significantly decreased in both DPPH and ABTS free radical systems when the temperature increased from 20℃ to 100℃. The scavenging capacity decreased by 2.22% in the DPPH free radical system, while the decrease was more pronounced in the ABTS free radical system, reaching 11.52%. These changes in thermal stability confirm the temperature sensitivity of bioactive peptides. Although high temperature inhibited free radical scavenging ability, tuna blood peptides still maintained high antioxidant activity. The results of this study indicate that tuna blood peptides possess good stability within a certain temperature range.

[0095] Figure 5 The effects of pepsin and trypsin on the stability of tuna blood peptides were demonstrated. After 1 h of pepsin digestion, the DPPH free radical scavenging rate of tuna blood peptides decreased significantly (P<0.05), but increased significantly with increasing digestion time and trypsin digestion (P<0.05). This may be because in the early stages of digestion, changes in protein structure and the production of some intermediate products reduced the DPPH scavenging rate, but as digestion continued and trypsin digestion progressed, more peptides with antioxidant activity were released, thus increasing the DPPH scavenging rate. In the determination of ABTS free radical scavenging capacity, the antioxidant capacity after gastric-trypsin digestion was significantly lower than that in the undigested stage, and the ABTS free radical scavenging rate gradually decreased with increasing digestion time.

[0096] 2. Molecular weight distribution of tuna hemopeptides

[0097] The molecular weight distribution range of tuna hemopeptides was determined using HPLC. The molecular weight distribution characteristics of tuna hemopeptides are as follows: Figure 6 As shown in (A). By Figure 6 (B) Data shows that 81.3% of the peptides have a molecular weight below 5000 Da, with 55.4% below 1000 Da and 25.9% between 1000 and 5000 Da, indicating that the enzymatic hydrolysis products are mainly oligopeptides. Studies have confirmed that low molecular weight oligopeptides exhibit stronger antioxidant activity. Among them, peptides with a molecular weight of 500–1800 Da show particularly significant antioxidant efficacy, while components below 1000 Da exhibit even better activity.

[0098] 3. Study on the chemical antioxidant activity of ultrafiltration components of tuna blood peptides

[0099] 3.1 Determination of DPPH free radical scavenging ability

[0100] The DPPH free radical scavenging performance of each component of ultrafiltration is as follows: Figure 7 As shown in the data, the clearance rate of each component increased with increasing sample concentration. At a concentration of 10 mg / mL, component F4 performed best, with a clearance rate of 73.03%, slightly lower than the ascorbic acid (Vc) control. In contrast, the clearance capacity of components F1, F2, and F3 decreased sequentially.

[0101] 3.2 Hydroxyl radical scavenging capacity determination

[0102] like Figure 8 As shown, the hydroxyl radical scavenging capacity of each ultrafiltration component increased with increasing sample concentration. Among them, the scavenging efficiency of components F4, F3, and F2 showed no significant difference, while the scavenging rate of group F1 was significantly lower than the other groups. When the concentration reached 10 mg / mL, component F4 exhibited the highest scavenging rate (72.13%), but it was still significantly lower than the positive control Vc.

[0103] 3.3 Determination of ABTS free radical scavenging ability

[0104] from Figure 9 Data analysis revealed a significant positive correlation between ABTS radical scavenging rate and sample concentration gradient (P<0.05). When the treatment concentration was increased to 10 mg / mL, fraction F3 showed the highest ABTS radical scavenging rate, reaching 69.97%. F1 had the second highest ABTS radical scavenging rate at this concentration, followed by fraction F4, while F2 had the lowest ABTS radical scavenging rate.

[0105] 3.4 Determination of the total reducing power of FRAP

[0106] from Figure 10 It can be seen that increasing the sample concentration led to an increase in the total reducing power of all components. Among them, the increase in component F1 was particularly significant. Within the concentration range of 2-10 mg / mL, component F1 exhibited a higher total reducing power than other components.

[0107] 4. Results of Tuna Blood Peptide Isolation and Purification

[0108] Based on the preceding ultrafiltration experiments, it was found that fraction F4 exhibited the best DPPH and ABTS radical scavenging rates, and also showed good performance in hydroxyl radical scavenging and total reducing power. Based on these results, fraction F4 was selected for subsequent gel chromatography separation. Gel chromatography technology achieves separation based on differences in molecular size. Figure 11As shown, the tuna hemopeptide F4 fraction, after ultrafiltration, was further separated by gel chromatography to obtain three main elution fractions. The fractions are labeled F4-1, F4-2, and F4-3 from left to right according to their elution order.

[0109] 5. Determination of DPPH free radical scavenging rate of each component of tuna hemopeptide

[0110] The F4 fraction (molecular weight less than 1 kDa) separated by ultrafiltration and the F4 1-3 fractions treated by gel permeation chromatography were dissolved separately to prepare solutions with a concentration of 1 mg / mL. Subsequently, their antioxidant properties were evaluated using a DPPH free radical scavenging assay. Figure 12 As shown, the results indicated that fraction F4 exhibited a clearance rate of 44.3%, significantly higher than that of F4-1 (40.6%), F4-2 (38.1%), and F4-3 (37.1%), with a statistically significant difference (P<0.01). This significant difference may be attributed to the fact that ultrafiltration retains a richer variety of compounds compared to the fraction obtained from gel filtration, resulting in a synergistic effect of multiple components. Therefore, fraction F4, with its stronger antioxidant capacity, was selected for further experiments.

[0111] 6. Study on the antioxidant activity of tuna blood peptides in cells

[0112] 6.1 Effects of different concentrations of tuna hemopeptide on the viability of RAW 264.7 cells

[0113] Different concentrations of TFBP (ultrafiltration component F4) were applied to RAW 264.7 cells for 24 h to observe its effect on cell viability. Figure 13 It can be seen that TFBP can promote the growth of RAW 264.7 cells when the concentration is in the range of 5-400 μg / mL, indicating that it has no obvious toxicity to RAW 264.7 cells.

[0114] 6.2 Establishment of a cellular oxidative stress model

[0115] Hydrogen peroxide (H₂O₂), a typical reactive oxygen species (ROS) inducer, can generate hydroxyl radicals and superoxide anions intracellularly via the Fenton reaction, triggering lipid peroxidation, protein carbonylation, and DNA oxidative damage, ultimately leading to cell cycle arrest and programmed cell death. This study used H₂O₂ concentration gradients from 100 to 800 μM to treat RAW 264.7 macrophage cells in an in vitro culture system to establish an in vitro oxidative stress model. Cell viability was assessed using the CCK-8 colorimetric assay. Figure 14As shown, cell viability decreased in a dose-dependent manner. At a concentration of 500 μM, the survival rate dropped to (49.72±2.59)%, which met the critical concentration threshold (40-60%) for constructing the oxidative damage model. Under this concentration condition, cells maintained basal metabolic activity while exhibiting typical oxidative stress characteristics, and therefore it was selected as the parameter for subsequent experiments.

[0116] 6.3 Protective effect of tuna hemopeptides on oxidative stress cells

[0117] To investigate the protective effect of tuna hemolysin (TFBP) against hydrogen peroxide (H2O2)-induced damage in RAW 264.7 cells, the experiment employed a procedure of drug intervention followed by modeling: cells were first pretreated with TFBP, and then oxidative damage was induced with H2O2. Figure 15 As shown, cell viability in the H2O2-damaged group was significantly reduced, only (49.7±3.6)% of that in the control group. Furthermore, within the concentration range of 12.5-50 μg / mL, the effect of TFBP on cell viability was concentration-dependent. The cell viability in the TFBP-treated group increased from (68.1±2.1)% to (91.7±6.5)%, both showing highly significant differences compared to the damage group (P<0.01). At a TFBP concentration of 50 μg / mL, cell viability was already close to that of the control group. However, when the TFBP concentration was in the range of 100-200 μg / mL, cell viability decreased, showing highly significant differences compared to both the damage group and the control group (P<0.01). Therefore, the addition of TFBP in this experiment can mitigate the damage caused by H2O2 to cells, and the optimal concentration of TFBP is 25-100 μg / mL.

[0118] 6.4 Effects of tuna hemopeptide on ROS levels in oxidative stress cells

[0119] ROS (Reactive Oxygen Species) free radicals include various components such as hydroxyl radicals, superoxide anions, and peroxides. Their dynamic homeostasis in vivo is crucial for physiological regulation, and intracellular ROS levels are a key indicator reflecting the degree of oxidative damage. Through analysis... Figure 16 and Figure 17 The data showed that RAW 264.7 cells treated with 500 μM H2O2 exhibited a significant increase in intracellular ROS levels, reaching 1.56 times that of the control group, confirming that H2O2 successfully induced oxidative stress and triggered ROS accumulation. Further research indicated that pretreatment with different concentrations of tuna hemolysin (TFBP) significantly reduced ROS levels in damaged cells (P<0.01), accompanied by a simultaneous decrease in fluorescence intensity (P<0.01). The 50 μg / mL TFBP treatment group showed particularly significant effects, with ROS levels decreasing to 1.14 times that of the control group. Although still slightly higher than the blank control group, this was close to the normal range, indicating that TFBP possesses the ability to inhibit ROS accumulation and resist oxidative damage.

[0120] 6.5 Effects of tuna hemopeptide on LDH content in cells under oxidative stress

[0121] LDH is a key enzyme in maintaining cell homeostasis and can be widely used in cytotoxicity detection by assessing cell membrane integrity and the degree of cellular stress damage. Results are as follows... Figure 18 As shown, H2O2 treatment significantly increased extracellular LDH levels, with the model group's LDH level reaching 1.46 times that of the blank control group, indicating that cell membrane integrity had been disrupted. Compared to the model group, TFBP significantly reduced LDH release from RAW 264.7 cells at concentrations of 12.5, 25, 50, 100, and 200 mg / mL, decreasing by 12.78%, 21.27%, 25.35%, 19.66%, and 9.67%, respectively. These results demonstrate that TFBP can alleviate oxidative damage to RAW 264.7 cells caused by LDH leakage.

[0122] 6.6 Effects of tuna hemopeptide on intracellular SOD, CAT, GSH and MDA levels in cells under oxidative stress

[0123] Depend on Figure 19 It was found that after H2O2 treatment, superoxide dismutase (SOD) activity showed a significant decreasing trend, with the measured value being only 31.90% of that in the normal control group. This is because the introduction of exogenous H2O2 can induce endogenous oxidative stress in cells, leading to impaired function of the antioxidant enzyme system and resulting in significant oxidative stress damage such as cell membrane lipid peroxidation. Compared with the model group, only cells pretreated with 50 μg / mL TFBP showed a significant increase in SOD activity (P<0.05). Although the intracellular SOD activity of cells treated with other concentrations of TFBP was higher than that of the model group, the difference was not statistically significant (P>0.05). This indicates that TFBP enhances its antioxidant effect by increasing intracellular SOD activity, thereby reducing cell damage caused by oxidative stress.

[0124] CAT is a heme-containing enzyme that catalyzes the decomposition of hydrogen peroxide into water, preventing damage to the body from endogenously produced hydrogen peroxide. According to Figure 20As shown, CAT activity decreased significantly by 69.20% after H2O2 treatment (P<0.01). However, when pretreated with 12.5 μg / mL TFBP, CAT activity increased significantly (P<0.01), recovering to 42.08% of the control group. With increasing TFBP concentration, CAT activity further increased, recovering to 88.71% at a TFBP concentration of 50 μg / mL, with no significant difference from the control group (P>0.05). This study found that the loss of CAT activity exacerbates oxidative stress-induced DNA damage, while increased CAT activity is a protective response of cells to oxidative stress, indicating a crucial role for CAT in antioxidant defense.

[0125] GSH participates in redox processes in the body, inhibits free radical damage to organs, and is a major non-protein physiological antioxidant in cells. GSH deficiency may lead to cardiovascular disease, immune disorders, and diabetes. Figure 21 It was found that after RAW 264.7 cells were treated with H2O2, their GSH enzyme activity significantly decreased to 49.58% of the control group, with a statistically significant difference (P<0.01), indicating that the oxidative damage model was successfully established. Compared with the model group, the GSH content of the 50 and 100 μg / mL drug administration groups was significantly increased (P<0.01), with the 50 μg / mL group showing the highest GSH content, recovering to 97.69%, which was not significantly different from the control group (P>0.05). There were no significant differences between the other drug administration groups and the model group, indirectly indicating that 50 μg / mL had the best cell protective effect.

[0126] Lipid peroxidation refers to a series of chain reactions between reactive oxygen species (ROS) and lipids. This process produces various byproducts that may threaten cellular physiological functions and cause cell damage. Malondialdehyde (MDA), as the final metabolite of the lipid peroxidation chain reaction, can serve as a biomarker for the degree of oxidative stress damage due to its intracellular accumulation level. Figure 22The results showed that when RAW 264.7 cells were treated with H2O2, the MDA content significantly increased to 2.86 times that of the control group (P<0.01), indicating a high level of lipid peroxidation and severe oxidative damage. However, after TFBP pretreatment, the MDA content significantly decreased (P<0.01), especially when the TFBP concentration was 50 μg / mL, the MDA content was not significantly different from the control group (P>0.05). This indicates that TFBP has a significant ameliorative effect on the excessive lipid peroxidation response caused by oxidative damage in RAW 264.7 cells. Studies have found that antioxidant peptides can significantly reduce MDA production in HepG2 cells. Compared with the control group, the MDA content in the model group was significantly increased, while the MDA level was significantly reduced after peptide pretreatment, which is consistent with the decrease in intracellular ROS levels. The results indicate that antioxidant peptides protect cells from oxidative damage by inhibiting ROS-induced lipid peroxidation.

[0127] In this embodiment, tuna hemopeptides were further separated and purified using ultrafiltration and gel column chromatography techniques to obtain components with higher antioxidant properties. The protective effects of the obtained antioxidant components on oxidatively damaged cells were investigated through in vitro antioxidant experiments and cell experiments, yielding the following conclusions: (1) Strong acid and alkaline environments significantly reduced the activity of tuna hemopeptides (P<0.05), while tuna hemopeptides maintained high antioxidant activity in pH 3-5 environments; (2) Four components were successfully separated using 1, 5, and 10 kDa ultrafiltration membranes and named F1 (>10 kDa), F2 (5~10 kDa), F3 (1~5 kDa), and F4 (<1 kDa), respectively. Considering the scavenging capacity of the three free radicals and the total reducing power of FRAP as evaluation indicators, the F4 component with a molecular weight less than 1 kDa showed the best antioxidant activity. (3) A hydrogen peroxide (H2O2)-induced oxidative stress injury model of RAW 264.7 cells was constructed, and the cytotoxicity and antioxidant activity of TFBP were evaluated using changes in cell viability as a key assessment indicator. TFBP significantly reduced (P<0.05) the levels of ROS and LDH in cells, significantly enhanced (P<0.05) the activities of endogenous antioxidant enzymes SOD, CAT, and GSH, and significantly reduced (P<0.05) the content of MDA, demonstrating that TFBP has a certain protective effect on the RAW 264.7 cell oxidative damage model.

[0128] Preparation Example 1: Preparation of Tuna Hemopeptide Components (TFBP) with a Molecular Weight Less Than 1000 Da

[0129] (1) Enzymatic hydrolysis: Freshly collected tuna blood was filtered through gauze, freeze-dried, and then pulverized into a uniform powder using a grinder to obtain tuna blood powder. The tuna blood powder was mixed with distilled water at a ratio of 1:100 (g / mL), and the pH was adjusted to 4.5 with 1 mol / L hydrochloric acid solution. Acidic protease (enzyme activity approximately 50,000 U / g) was added, and the mixture was hydrolyzed at 49℃ for 7 hours. After the reaction, the enzyme was inactivated by heating at 100℃ for 15 minutes. After cooling, the supernatant was collected by centrifugation to obtain crude tuna blood peptide hydrolysate.

[0130] In the crude enzymatic hydrolysate, components with a molecular weight less than 1000 Da accounted for approximately 55.4%. The total amino acid content of the product was measured to be 58.56 mg / g, of which essential amino acids accounted for 45.66% of the total amino acid content, and branched-chain amino acids (valine, isoleucine, and leucine) accounted for 29.30% of the total amino acid content, which was a relatively large proportion.

[0131] (2) Ultrafiltration separation: The above crude enzymatic hydrolysate is passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 10000 Da and 5000 Da, and the retentate is discarded. The permeate is then passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the permeate fraction with a molecular weight cutoff of less than 1000 Da is collected and freeze-dried to obtain the tuna hemopeptide fraction with a molecular weight of less than 1000 Da described in this invention, denoted as TFBP.

[0132] The foregoing content has confirmed the in vitro antioxidant properties of tuna blood peptides. The following further uses zebrafish fatigue model, mouse exhaustive swimming and free swimming experiments to evaluate its anti-fatigue efficacy from multiple dimensions, including zebrafish in vivo reactive oxygen species (ROS) levels, mouse exercise endurance, post-exercise metabolite accumulation, muscle tissue protection and antioxidant capacity, etc.

[0133] Example 1 of the efficacy experiment: Anti-fatigue experiment using a zebrafish model

[0134] To construct the model, 4 dpf AB strain zebrafish were placed in 6-well plates, with 30 zebrafish in each 5 mL system. A blank control group (i.e., zebrafish treated with fish farming water), a model control group (model) treated with 3 mg / mL anhydrous sodium sulfite, and experimental groups (treated with 3 mg / mL anhydrous sodium sulfite and 100, 200, and 500 μg / mL TFBP aqueous solutions), were designated as L-TFBP group, M-TFBP group, and H-TFBP group, respectively. All groups were incubated at 28℃ for 18 h.

[0135] The activity of reactive oxygen species (ROS) scavenging was assessed by staining zebrafish with a ROS staining kit. After incubation at 28.5°C in the dark for 30 min, the samples were washed three times with distilled water. Juvenile fish were then anesthetized with 0.16% tricaine, and zebrafish samples were photographed tail by tail under a fluorescence microscope. The anti-fatigue effect of the samples was evaluated by statistically analyzing the fluorescence intensity of the yolk sac.

[0136] Sodium sulfite, due to its ability to react with dissolved oxygen in water, leading to a decrease in ambient oxygen levels, is often used to construct a hypoxic fatigue model in zebrafish. Under this hypoxic stress, the physiological functions of zebrafish are impaired, resulting in fatigue. Experimental results are as follows... Figure 23 and Figure 24 As shown, compared to the Control group, the ROS content in the zebrafish of the Model group was significantly increased (P<0.01), and the fluorescence intensity was also significantly enhanced. Compared to the Model group, the fluorescence intensity of the L-TFBP, M-TFBP, and H-TFBP treatment groups was significantly decreased (P<0.05), and the fluorescence intensity showed a continuous upward trend with increasing TFBP concentration. In summary, TFBP can effectively inhibit the increase of ROS levels, demonstrating significant in vivo anti-fatigue ability.

[0137] Example 2 of efficacy experiment: Acute toxicity test

[0138] Sample preparation: TFBP powder was dissolved in physiological saline, filtered through a 0.22 μm membrane and then sterilized, and stored at 4°C.

[0139] Experimental Animals and Husbandry Conditions: Ten healthy male KM mice (18-22 g, SPF grade) aged 7 weeks were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. The research was conducted in the Barrier System Laboratory. In this laboratory, the air cleanliness level met or exceeded Class 10,000 standards, and the ventilation frequency was maintained between 10 and 20 times per hour. The temperature was maintained at 20–26℃, with a daily temperature difference ≤3℃, and the relative humidity was maintained at 40–70%. The mice were provided with a 12-hour light and 12-hour dark cycle environment. After one week of acclimatization, they were randomly divided into two groups of 10 mice each.

[0140] After the animal experiments were completed and the groups were adapted to different feeding conditions, the following experimental groups were selected for different treatments: Control group: administered the same volume of physiological saline, with fasting for 8 hours before and after gavage; TFBP dosage group: administered 3 g / (kg·bw) TFBP, followed by a second gavage administration 8 hours later, for a total dosage of 6 g / (kg·bw). Mice were continuously monitored for 7 days after administration, with detailed observation of their appearance, behavior, respiration, secretions, defecation, and mortality, and their weight changes recorded for 7 consecutive days. If death occurred, the cause of death and its correlation with the drug were analyzed; if no death occurred, the animals were further assessed for potential signs of toxicity, such as abnormal behavior or changes in physiological indicators.

[0141] In acute toxicity tests in mice, body weight change is an important indicator for assessing the toxic effects of the test substance. Mouse body weight parameters and liver toxicity results are as follows: Figure 25 , Figure 26 After a 7-day course of treatment, the mice showed no significant abnormalities in appearance, behavior, respiration, limb movement, diet, or excretion, and no deaths were observed. There was also no significant difference in body weight between the treated group and the control group.

[0142] Example 3 of efficacy experiment: In vivo verification of the anti-fatigue effect of TFBP (mouse weight-bearing swimming experiment).

[0143] I. Experimental Methods

[0144] Sample preparation: The sample was prepared by mixing TFBP powder with physiological saline, sterilized after filtration through a 0.22 μm membrane, and stored at 4°C. Positive control drug policosanol (PC) was prepared by mixing policosanol with 1% sodium carboxymethyl cellulose, sterilized after filtration through a 0.22 μm membrane, and stored at room temperature for later use.

[0145] Experimental animals and feeding conditions: Thirty healthy male KM mice (weighing 18-22 g, SPF grade) aged 7 weeks were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. Feeding conditions were the same as in Example 2 of the drug efficacy experiment. After one week of acclimatization feeding, they were randomly divided into 5 groups of 6 mice each.

[0146] After the animal groups completed their adaptive feeding, they were divided into the following experimental groups for different experimental treatments: Control group: Administered the same volume of physiological saline via gavage. Experimental groups A, B, and C were administered different doses of L-TFBP (0.5 mg / g), M-TFBP (1.5 mg / g), and H-TFBP (3 mg / g), respectively. Experimental group D was administered PC (0.01 mg / g). All groups underwent a 28-day dosing period, with administration once daily via gavage.

[0147] Mouse weight and organ index: Before administration of the drug to each batch of mice daily, the weight of the mice was measured. The mice were then administered the drug by gavage according to their weight, and the weight was recorded once a day. The average weight of each group of mice was then calculated. After the swimming experiment, the liver and spleen of the mice were removed, photographed, weighed, rinsed with cold physiological saline to remove blood, dried with filter paper, and weighed. The weight was expressed as organ mass / body weight ratio.

[0148] Weight-bearing swimming experiment: The weight-bearing exhaustion swimming experiment was conducted in week 4 (day 28). After administration, mice were allowed to rest for 30 minutes, and then placed in a swimming tank with a tin bar bearing 5% of their body weight on their tail base. The water depth was no less than 30 cm, and the water temperature was 25℃±1.0℃. The exhaustion swimming time was recorded when the mouse's nose tip was submerged in water for more than 5 seconds. If any mouse stopped swimming by arching its back or hovered to rest, the surrounding water was stirred with a glass rod to force it to keep moving.

[0149] Following the weighted swimming experiment, mice underwent a 30-minute resting period before whole blood samples were collected using the fundus venous plexus sampling method. The collected blood was transferred to coagulation incubation tubes at room temperature (120 min). The samples were then centrifuged at 4000 rpm for 15 min at 4°C, and the supernatant serum was collected. The aliquoted serum samples were immediately stored at -80°C for later analysis. The assays included exercise metabolic markers such as lactate (LA), blood urea nitrogen (BUN), and lactate dehydrogenase (LDH).

[0150] Mice were euthanized by cervical dislocation and quickly dissected. Liver tissue was separated and weighed, and pre-cooled physiological saline was added at a mass-to-volume ratio of 1:9. The mixture was then mechanically homogenized under ice bath conditions to prepare a 10% tissue homogenate. The homogenate suspension was transferred to centrifuge tubes and centrifuged at 4000 rpm for 20 min at 4°C. The supernatant was aliquoted into cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for later analysis. The levels of liver glycogen (HG), creatine kinase (CK), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) were measured according to the kit instructions.

[0151] II. Analysis of the Anti-fatigue Effect of Tuna Blood Peptides Based on a Mouse Model

[0152] 2.1 Effects of tuna hemopeptides on body weight and organ indices in mice

[0153] As shown in Table 1, during the 28-day gavage experiment, the body weight of the experimental animals in each group showed a regular increasing trend. One-way ANOVA showed that the differences in body weight gain between the positive control group (PC group) and each dose treatment group and the Control group did not reach a statistically significant level (P>0.05). This result indicates that TFBP and PC had neither promoting nor inhibiting effects on the growth and development of mice, and all mice were in a state of natural growth and normal feeding.

[0154] Table 1. Effects of TFBP on mouse body weight.

[0155]

[0156] Note: Different uppercase letters indicate significant differences in weight at different time periods (P<0.05), and different lowercase letters indicate significant differences in weight within different groups (P<0.05).

[0157] The liver is the most important metabolic organ in the body, and the spleen is the largest lymphatic organ. During acute injury, the liver is subjected to toxic effects, and hepatocytes undergo pathological changes such as degeneration and necrosis, which may lead to changes in liver indices. If liver damage is severe, cell swelling, necrosis, and inflammatory cell infiltration can relatively increase liver weight, potentially raising liver indices. As shown in Table 2, there was no significant difference in liver and spleen coefficients between the tuna blood peptide group and the control group (P>0.05), indicating that the drug had a relatively small impact on organ mass.

[0158] Table 2 Effects of TFBP on organ indices in mice

[0159]

[0160] Note: Different letters indicate significant differences between different groups (P < 0.05).

[0161] 2.2 Effects of tuna hemopeptide on the motor function of mice

[0162] The degree of fatigue in the body can be directly and objectively reflected by a decrease in exercise endurance. In studies assessing fatigue tolerance in mice, the exhaustive swimming test apparatus is considered effective in reflecting the anti-fatigue effect of a compound due to its advantages such as simplicity, ease of operation, multiple controllable factors, and high reproducibility. Specifically, the longer the exhaustive swimming time, the more significant the anti-fatigue effect of the compound. Figure 27 As shown, after 28 days of administration, compared with the Control group, the swimming time of mice in all treatment groups increased, with the high-dose group showing the most significant increase (P<0.05). There was no significant difference between the L-TFBP group and the M-TFBP group and the Control group. The PC group showed the most significant increase in swimming time (P<0.05). Furthermore, increasing the TFBP dose significantly prolonged the exhaustive swimming time, showing a clear dose-dependent relationship. This indicates that high-dose TFBP has a certain anti-fatigue effect on mice.

[0163] 2.3 Effects of tuna hemopeptides on metabolite accumulation

[0164] During strenuous exercise, muscle oxygen consumption increases dramatically, and the body's metabolic mode shifts to anaerobic pathways, leading to a significant acceleration in glycolysis and a large accumulation of lactic acid (LA). Elevated LA concentrations can cause a decrease in the pH of muscle tissue and blood, thereby interfering with cardiac circulatory function and skeletal muscle system operation, and are considered a key contributing factor to fatigue. Figure 28 It was observed that after 30 minutes of swimming, the lactate (LA) level in the H-TFBP group was significantly lower than that in the control group (P<0.05). Furthermore, LA levels showed a decreasing trend with increasing tuna hemolymph peptide dosage, indicating a clear dose-dependent relationship. This suggests that TFBP can promote LA clearance, thereby alleviating fatigue.

[0165] When engaging in prolonged, strenuous exercise without timely replenishment of glucose, the body will utilize protein for energy. In the body's physiological metabolism, urea, as a final product of protein breakdown, shows a concentration change that effectively reflects the body's metabolic state. During high-intensity physical activity, the urea content in the blood shows a significant upward trend. This phenomenon stems from the accelerated protein metabolism and altered nitrogen balance during exercise, and there is a significant negative correlation between blood urea nitrogen levels and the body's exercise adaptability. These dynamic changes in biochemical parameters provide important evidence for assessing exercise load and recovery status. Figure 29As shown, the BUN concentration in the TFBP group was higher than that in the Control group, but there was no statistically significant difference between the two groups (P>0.05). Numerically, TFBP reduced the blood urea nitrogen content in mouse serum to some extent.

[0166] 2.4 Effects of tuna hemopeptides on mouse liver glycogen

[0167] Glycogen, a core energy source during exercise, is primarily stored in the liver and muscle tissue. Liver glycogen can be broken down into glucose to maintain systemic energy metabolism. During high-intensity or prolonged aerobic exercise, the proportion of energy supplied by glycogen increases significantly; however, as exercise continues, glycogen reserves are gradually depleted, becoming a significant factor in inducing fatigue. Figure 30 As shown, the liver glycogen content in the H-TFBP group was significantly higher than that in the control group (P<0.01). Specifically, the liver glycogen levels in the L-TFBP, M-TFBP, and H-TFBP treatment groups were 1.04 times, 1.07 times, and 1.26 times that of the control group, respectively. These results indicate that tuna hemopeptide (TFBP) can significantly promote liver glycogen accumulation in mice, slow down the rate of glycogen depletion during exercise, and thus delay the onset of fatigue.

[0168] 2.5 Protective effect of tuna hemopeptides on mouse muscle

[0169] Creatine kinase (CK), a key enzyme in energy metabolism, catalyzes the phosphorylation of creatine to phosphocreatine. Phosphocreatine, through phosphate group transfer, drives the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), providing energy for cells. During exercise, the surge in ATP consumption prompts a compensatory increase in CK activity, accelerating energy synthesis to delay fatigue. Simultaneously, the altered permeability of muscle cell membranes caused by strenuous exercise leads to the release of CK into the bloodstream, resulting in elevated serum CK levels. Figure 31 As shown, compared with the Control group, the serum CK activity in both the PC group and the H-TFBP group was significantly reduced (P<0.01). This result indicates that TFBP can effectively alleviate exercise-induced muscle damage and inhibit CK extravasation.

[0170] Lactate dehydrogenase (LDH) is an enzyme that plays a crucial role in the body, primarily involved in glycolysis and gluconeogenesis under anaerobic conditions. Increased LDH activity can enhance the conversion of lactate to pyruvate in muscle tissue, thereby improving the body's resistance to fatigue. Figure 32 Compared with the Control group, the H-TFBP and PC groups showed higher LDH expression levels with significant differences (P<0.05), indicating that the anti-fatigue effect of TFBP on mice is due to its ability to increase LDH activity in vivo, reduce the accumulation of metabolites such as LA and BUN, and alleviate the burden on the body.

[0171] 2.6 Effects of tuna hemopeptides on antioxidant capacity in mice

[0172] Reactive oxygen species (ROS)-induced protein oxidation is a significant contributing factor to exercise-related skeletal muscle dysfunction. In the antioxidant defense system, superoxide dismutase (SOD) mediates the oxidation of superoxide anions (O2-). - The dismutation reaction of SOD produces hydrogen peroxide (H2O2) and oxygen (O2), while glutathione peroxidase (GSH-Px) converts H2O2 into water through a glutathione-dependent reduction reaction. Experimental data show that by regulating the enzyme activity levels of SOD and GSH-Px, the ability to protect against oxidative damage under exercise stress can be effectively improved. Figure 33 As shown, compared with the Control group, the SOD activity in mice in the PC and H-TFBP groups was significantly increased (P<0.01), and within a certain range, the dose-dependent characteristics were significantly observed. Figure 33 As shown, GSH-Px activity was increased in all TFBP groups compared to the Control group. The differences between the M-TFBP and H-TFBP groups were significant (P<0.05), with increases of 39.82% and 53.07% respectively compared to the Control group. This may be because TFBP can reduce oxidative stress damage in mice, and when oxidative stress levels decrease, the body's feedback regulation mechanism for SOD production is activated to cope with oxidative stress, thereby producing more SOD.

[0173] GSH-Px is an important component of the body's antioxidant system, catalyzing the formation of glutathione and reacting with reactive oxygen species to block their damage to the body. For example... Figure 34 As shown, PC, M-TFBP, and H-TFBP groups all significantly increased GSH-Px activity (P<0.05), and TFBP showed a dose-dependent effect. Fatigue processes generate a large number of free radicals, causing oxidative damage to cells. The active ingredients in TFBP can effectively scavenge free radicals, reduce oxidative stress levels, and stimulate GSH-Px synthesis after cells sense the reduction in oxidative stress, thus increasing GSH-Px content.

[0174] Increased CAT activity indicates that the body possesses certain antioxidant and anti-fatigue capabilities. Figure 35It was found that the CAT activity in the H-TFBP group was significantly increased compared with the Cotrol group (P<0.01). The CAT activities in the L-TFBP, M-TFBP, and H-TFBP groups were 18.90, 27.53, and 31.68 U / mgprot, respectively, showing a gradient upward trend. These results indicate that TFBP can increase CAT activity in mice and has a certain anti-fatigue effect. TFBP has the ability to scavenge free radicals, and therefore may promote the body to produce more free radical scavengers, such as CAT, thereby reducing oxidative damage to cells by free radicals and improving the anti-fatigue ability of mice.

[0175] MDA levels are an important indicator of the body's antioxidant capacity, primarily reflecting the rate of lipid peroxidation, but more importantly, the degree of cell membrane damage. Higher MDA levels indicate more severe cell damage. Therefore, MDA is a key indicator of an organism's oxidative stress level, and fatigue levels are positively correlated with MDA content. Figure 36 As shown, the control group had the highest MDA content, while all TFBP groups showed a significant decrease compared to the control group (P<0.05). Based on the above analysis, TFBP may alleviate fatigue by increasing the activity of antioxidant enzymes and inhibiting the production of lipid peroxides in cell membranes.

[0176] Correlation analysis between antioxidant and anti-fatigue

[0177] Based on the expression levels of anti-fatigue-related biochemical indicators in mice from the aforementioned pharmacodynamic experiments, the H-TFBP group showed the best anti-fatigue activity. Therefore, the H-TFBP group was selected for correlation analysis of in vivo antioxidant activity and anti-fatigue efficacy. Figure 37 As shown, Pearson correlation analysis revealed a significant positive correlation between MDA content and BUN concentration; a significant positive correlation between CAT activity and SOD activity, and a highly significant positive correlation between CAT activity and LDH activity; a significant positive correlation between SOD activity and both BUN concentration and LDH activity; and a significant positive correlation between CK activity and the energy substance HG.

[0178] Therefore, this study indicates that the in vivo antioxidant activity of TFBP is significantly positively correlated with its anti-fatigue effect. Tuna hemopeptide exerts its anti-fatigue effect by enhancing the activity of antioxidant enzymes and reducing the content of lipid peroxides, thereby effectively resisting oxidative stress damage.

[0179] This invention utilizes a zebrafish fatigue model and a mouse exhaustive swimming model to evaluate the anti-fatigue effect of tuna hemopeptide (TFBP). The results are as follows:

[0180] (1) Compared with the model group, the fluorescence intensity of zebrafish in the L-TFBP, M-TFBP and H-TFBP groups was significantly reduced (P<0.05), and the fluorescence intensity gradually increased with the increase of TFBP concentration.

[0181] (2) After gavage, the body weight of mice in the Control group, Model group, L-TFBP group, M-TFBP group and H-TFBP group all increased slightly, and there was no significant difference in body weight among the groups (P>0.05). This indicates that gavage administration of TFBP does not affect the normal growth of mice and has no obvious acute toxicity.

[0182] (3) Gavage administration of TFBP can effectively reduce the accumulation of metabolites after exercise. After swimming for 30 minutes, the swimming time of the H-TFBP group increased significantly, and TFBP showed a certain dose-dependent effect. Compared with the control group, the blood urea nitrogen of mice in the TFBP group decreased by 11.76%-14.8%, and the high-dose group had the strongest ability to clear metabolites. Compared with the control group, the HG content and LDH activity of liver tissue of mice in the TFBP group were increased to varying degrees, while the CK level decreased by 2.33%, 1.23%, and 11.85%, respectively. TFBP showed a significant ability to enhance the activity of antioxidant enzymes in mice and effectively reduce the generation of lipid peroxides. Specifically, compared with the control group, low, medium, and high doses of TFBP increased SOD activity by 21.06%, 39.47%, and 70.34%, respectively. Meanwhile, GSH-Px activity was also enhanced, with low, medium, and high doses of TFBP increasing GSH-Px activity by 22.57%, 39.82%, and 53.07%, respectively. These results indicate that different doses of TFBP have a significant effect on enhancing antioxidant enzyme activity. The MDA content in the TFBP group was significantly lower than that in the Control group (P<0.05). The CAT activity of H-TFBP was significantly increased (P<0.01).

[0183] (4) Pearson correlation analysis revealed a significant positive correlation between oxidative stress markers (SOD, MDA, etc.) and exercise endurance indicators (time to exhaustion, blood lactate, etc.).

[0184] The above-mentioned pharmacological experiments fully demonstrate that the tuna hemopeptide component (TFBP) with a molecular weight of less than 1000 Da described in this invention exerts a significant anti-fatigue effect through multiple pathways, such as regulating energy metabolism and enhancing antioxidant capacity.

[0185] Dosage form example 1: TFBP tablets

[0186] TFBP 200g, microcrystalline cellulose 100g, lactose 80g, sodium carboxymethyl starch 15g, magnesium stearate 5g. Mix TFBP with the excipients evenly, granulate, and compress to obtain 1000 tablets.

[0187] Dosage form example 2: TFBP capsules

[0188] TFBP 250g, starch 150g. Mix TFBP and starch evenly, fill into No. 0 capsule shells, to obtain 1000 capsules.

[0189] Dosage form example 3: TFBP oral solution

[0190] 100g TFBP, 200g sucrose, 5g citric acid, and 1g potassium sorbate are added to purified water to a final volume of 1000mL. All components are dissolved in water, stirred thoroughly, filled into containers, and sterilized to obtain the final product.

Claims

1. The application of a tuna hemopeptide component with a molecular weight of less than 1000 Da in the preparation of products for preventing or relieving exercise-induced fatigue.

2. The application according to claim 1, characterized in that, The tuna blood peptide component with a molecular weight of less than 1000 Da was obtained by ultrafiltration of the enzymatic hydrolysis product of tuna blood.

3. The application according to claim 2, characterized in that, The enzymatic hydrolysis product of the tuna blood is prepared by enzymatic hydrolysis of tuna blood using acidic protease.

4. The application according to claim 1, characterized in that, The prevention or relief of exercise-induced fatigue is achieved by regulating energy metabolism.

5. The application according to claim 4, characterized in that, The regulation of energy metabolism includes: reducing the concentration of lactic acid and / or blood urea nitrogen in the blood after exercise, and / or increasing liver glycogen reserves.

6. The application according to claim 1, characterized in that, The prevention or relief of exercise-induced fatigue is achieved by enhancing the body's antioxidant capacity.

7. The application according to claim 6, characterized in that, The enhancement of the body's antioxidant capacity includes: increasing the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and / or catalase (CAT) in the liver, and / or reducing the content of malondialdehyde (MDA).

8. The application according to claim 1, characterized in that, The prevention or relief of exercise-induced fatigue also includes protecting muscle tissue by inhibiting the extravasation of creatine kinase.

9. The application according to claim 1, characterized in that, The product is a functional food, health product, or dietary supplement.

10. A composition for preventing or alleviating exercise-induced fatigue, characterized in that, The composition contains a therapeutically effective amount of tuna hemopeptide components with a molecular weight of less than 1000 Da, and a food or pharmaceutically acceptable carrier.

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