A pufferfish hippocampus antioxidant peptide, and a preparation method and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a bulging hippocampus antioxidant peptide as well as a preparation method and application thereof.The bulging hippocampus antioxidant peptide comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO.1.The present application adopts an ultrahigh pressure pretreatment and enzymatic hydrolysis technology to jointly prepare the hippocampus antioxidant peptide, and the adopted extraction process route is simple, time and production cost are effectively saved;the animal model is used to prove the antioxidant effect of the bulging hippocampus antioxidant peptide as well as the effect of improving male reproductive disorders and testicular damage;and on this basis, a new peptide segment with high activity, no toxicity and no sensitization is obtained.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antioxidant peptide from the bloated seahorse, its preparation method, and its application. Background Technology

[0002] The abdominal seahorse (Hippocampus abdominalis), a valuable marine bony fish, is considered a precious tonic in traditional East Asian medicine and is known as "Southern Ginseng." Modern research shows that seahorses are rich in various bioactive components, with a protein content as high as 60%-70%, making them an excellent raw material for the preparation of bioactive peptides.

[0003] Enzymatic hydrolysis is a relatively mild method commonly used for the preparation of bioactive peptides. However, the dense molecular structure of hippocampal proteins has hindered the hydrolysis rate and resulted in low peptide conversion rates during the screening of highly active peptides, a problem that has remained largely unresolved. Ultra-high pressure (HHP) treatment can stretch protein structures, unfolding more polypeptide chains and exposing more active sites, thereby ultimately improving hydrolysis efficiency. However, research on improving the hydrolysis efficiency of hippocampal proteins and assessing the impact of appropriate pretreatment methods on the structure and hydrolysates of hippocampal proteins is limited.

[0004] In addition, there is very little research on the development and utilization of antioxidant peptides from the bloated seahorse. Chinese patent CN119874814A discloses a neuroprotective and antioxidant stress-resistant polypeptide derived from the seahorse and its preparation method. The amino acid sequence of the polypeptide is shown in SEQ ID No. 1. The preparation steps are as follows: (1) Weigh 500 grams of fresh seahorse, clean the surface mud and sand, freeze dry at -60℃, crush the seahorse with a pulverizer, pass through a 20-mesh sieve, dialyze with distilled water for 24 hours, freeze dry, and obtain seahorse powder; (2) Weigh 200 grams of seahorse powder, add 1000 ml of 95% ethanol to extract three times to remove lipid-soluble substances, and extract the precipitate with 1 mol / L NaCl solution under 4℃ conditions, combined with ultrasound assistance, and freeze dry to obtain crude seahorse protein; (3) Take the crude seahorse protein and prepare seahorse polypeptide using ultrasound-assisted biomimetic digestion method. It has the advantages of specific molecular structure, clear mechanism of action, stability in the gastrointestinal tract, neuroprotection, and antioxidant stress. Chinese patent CN120842316A discloses a bloated hippocampal peptide and its application in the preparation of functional products for repairing damage to the male reproductive system. Utilizing green and controllable enzymatic hydrolysis technology, a protein polypeptide with nutritional, health-promoting, and medicinal value is obtained. Further research yielded a highly bioactive peptide sequence, and a high-purity bioactive peptide was obtained through chemical synthesis. The bioactivity of the peptide was verified using mouse spermatogonia. However, the preparation process of the aforementioned peptide did not effectively address the issue of the dense molecular structure of hippocampal proteins during peptide screening, nor did it effectively verify the antioxidant activity and in vivo efficacy of the peptide. Furthermore, no multifunctional hippocampal peptide with both antioxidant properties and the ability to improve male reproductive disorders and testicular damage has been found in the current technology. Summary of the Invention

[0005] The technical problems to be solved by this invention are: first, the dense molecular structure of hippocampal proteins has resulted in inhibited enzymatic hydrolysis and low peptide conversion rate, which has not been well resolved; second, no multifunctional hippocampal peptide with both antioxidant and male reproductive disorders and testicular damage has been found in the existing technology.

[0006] To address the aforementioned problems, this invention provides an antioxidant peptide from swollen seahorse, its preparation method, and its applications. The method involves pretreating swollen seahorse raw materials under ultra-high pressure to aid enzymatic hydrolysis, thereby increasing the peptide content and enhancing its activity in inhibiting free radicals such as DPPH. Furthermore, the prepared swollen seahorse antioxidant peptide has been shown to improve male reproductive disorders induced by chemical processes and in aging mice. Based on this, a novel peptide fragment with high activity, non-toxicity, and non-allergenicity was obtained.

[0007] To achieve the above objectives, the present invention is implemented through the following technical means: a bloating seahorse antioxidant peptide, comprising a polypeptide with an amino acid sequence as shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] GPAPWGF.

[0010] The preparation method of the expanded-belly seahorse antioxidant peptide powder containing the above-mentioned expanded-belly seahorse antioxidant peptides includes the following steps:

[0011] (1) Fresh bloated seahorses were freeze-dried under vacuum to remove moisture from them; (The conditions for vacuum freeze-drying were: temperature -40 ℃ ~ -50 ℃, vacuum degree 40-60 Pa, time 24 h) The dried seahorses were pulverized by a pulverizer to obtain seahorse powder.

[0012] (2) Accurately weigh the crushed seahorse powder, add distilled water, treat under high pressure of 190-210 MPa for 8.5-9.5 min, adjust the pH to 2, add pepsin at a mass ratio of 2.5-3.5%, and hydrolyze in a constant temperature water bath at 37℃ for 2.5-3.5 h.

[0013] Among the various pretreatment methods (ultra-high pressure, ultrasound, and heat treatment), ultra-high pressure exhibited the highest peptide yield and the best DPPH free radical scavenging activity. Furthermore, ultra-high pressure does not cause thermal denaturation of active components such as proteins, making it more suitable for practical applications. In the screening of various proteases (neutral protease, papain, alkaline protease, bovine collagenase, animal proteolytic enzymes, pepsin, and trypsin), pepsin showed the highest peptide yield and the best DPPH free radical scavenging activity.

[0014] (3) After enzymatic hydrolysis, the enzyme is inactivated by high temperature. After cooling to room temperature, the supernatant is collected at 4℃, 9000-10000 r / min for 14-16 min. The supernatant is obtained. The high centrifugal force of 9000×g~10000×g can effectively settle small suspended particles, unhydrolyzed macromolecular proteins and colloidal impurities in the system, and significantly improve the clarity of the supernatant. This avoids the residue from interfering with subsequent steps such as antioxidant activity detection and peptide purification.

[0015] (4) The above enzymatic hydrolysate is filtered with a microporous membrane; then it is fractionated in an ultrafiltration system with a molecular weight cutoff of 3 kDa to obtain the component with Mw < 3 kDa, which is freeze-dried to obtain the expanded seahorse antioxidant peptide powder.

[0016] Furthermore, the dried seahorse from step (1) is pulverized by a pulverizer until it can all pass through an 80-mesh sieve. The particles passing through the 80-mesh sieve can increase the specific surface area to promote full contact between the enzyme and the substrate. This allows the enzyme molecules to bind more fully to the peptide bond sites of the substrate, accelerating the hydrolysis process and thus improving the enzymatic hydrolysis efficiency.

[0017] Further, in step (2), distilled water is added at a ratio of 1:25.

[0018] Furthermore, after the enzymatic hydrolysis in step (3) is completed, the enzyme is inactivated by heating at 100°C for 15 min.

[0019] Furthermore, in step (4), the pore size of the microporous filter membrane is 0.22 μm.

[0020] A method for preparing an antioxidant peptide from a distended seahorse, which is synthesized artificially.

[0021] The application of the above-mentioned expanded hippocampal antioxidant peptides in the preparation of antioxidant products.

[0022] The above-mentioned swelling hippocampal antioxidant peptides are used in the preparation of products that improve male reproductive disorders and testicular damage.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) Compared with the prior art, the present invention uses ultra-high pressure pretreatment and enzymatic hydrolysis to prepare hippocampal antioxidant peptides. The extraction process is simple and effectively saves time and production costs. The reagents used in the experiment are readily available and low in cost, have no impact on the environment, can significantly improve the yield and antioxidant activity of peptides, and the prepared hippocampal peptides have good stability.

[0025] (2) This invention uses an animal model to explore the in vivo antioxidant mechanism of the antioxidant peptides of the bloated seahorse, which can be used as antioxidants or therapeutic drugs; it has confirmed the effect of the antioxidant peptides of the bloated seahorse on male reproductive disorders and testicular damage caused by aging, which is conducive to the high-value utilization of the bloated seahorse.

[0026] (3) The novel peptide GPAPWGF obtained in this invention has a molecular weight of 659.36 Da, high activity, and is non-toxic and non-allergenic. Attached Figure Description

[0027] Figure 1 The figure shows the DPPH radical scavenging rate of each component. Different lowercase letters (ad) in the figure indicate that there are significant differences in DPPH radical scavenging rates among the groups (p < 0.05).

[0028] Figure 2 The figure shows the DPPH radical scavenging rate of HAPH-1 under different temperature conditions. Different lowercase letters (ab) in the figure indicate significant differences in DPPH radical scavenging rates between groups. The difference between a and b is significant (p < 0.05).

[0029] Figure 3 The DPPH radical scavenging rate of HAPH-1 under different pH conditions is shown. Here, 'a' indicates no significant difference between groups (p > 0.05).

[0030] Figure 4 The figure shows the DPPH free radical scavenging rate of HAPH-1 after 4 hours of in vitro simulated gastrointestinal digestion. Different lowercase letters (ab) in the figure indicate significant differences in DPPH free radical scavenging rates between groups. The difference between a and b is significant (p < 0.05).

[0031] Figure 5 The results are from the LC-MS / MS identification of the HAPH-1 component.

[0032] Figure 6 This is the docking result between P1 and Keap1 proteins.

[0033] Figure 7 This is the secondary structure diagram of P1.

[0034] Figure 8 This is the result of the effect of GPAPWGF on sperm in a TP-induced mouse model. A shows a microscopic observation of sperm morphology; B is a bar chart of sperm motility (%); C is a bar chart of sperm abnormality rate (%); and D is a bar chart of sperm count (×10⁻⁶). 6 / ml) bar chart.

[0035] Figure 9 This study investigated the effects of GPAPWGF on the pathological morphology of testicular tissue in a mouse model of male reproductive disorders.

[0036] Figure 10 This represents the effect of GPAPWGF on TP-induced reproductive hormones in male mice. **: Significant difference between the model group and the normal control group (P < 0.01). ##: Significant difference between the peptide treatment group and the positive drug group and the model group (P < 0.01). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0039] Example 1:

[0040] A method for preparing a swelling-inducing seahorse antioxidant peptide powder includes the following steps:

[0041] 1. Preparation of antioxidant peptides from bloated seahorse:

[0042] First, fresh abdominally swollen seahorses were vacuum freeze-dried to remove excess moisture. The dried seahorses were then pulverized until they could all pass through an 80-mesh sieve. The pulverized seahorse powder was accurately weighed and added to distilled water at a ratio of 1:25 (w / v). The mixture was treated under high pressure at 200 MPa for 9 minutes, and the pH was adjusted to 2 using 1 mol / L HCl. Then, 3% (w / w) pepsin was added, and the mixture was hydrolyzed in a 37°C water bath for 3 hours. After hydrolysis, the enzymes were inactivated by heating at 100°C for 15 minutes. After cooling to room temperature, the mixture was centrifuged at 4°C, 10,000 r / min for 15 minutes, and the supernatant was collected to obtain the Hippocampus abdominalis protein hydrolysates (HAPH).

[0043] 2. Ultrafiltration separation of antioxidant peptides from distended seahorse:

[0044] The above enzymatic hydrolysate was filtered through a microporous membrane (pore size 0.22 μm). Then, it was fractionated in an ultrafiltration system using an ultrafiltration membrane with a molecular weight (Mw) cutoff of 3 kDa to obtain two fractions: Mw < 3 kDa (HAPH-1) and Mw > 3 kDa (HAPH-2). Each fraction was collected, freeze-dried, and the DPPH free radical scavenging rate was determined.

[0045] Effect verification:

[0046] 1. Determination of peptide yield:

[0047] Take 2 mL of the enzymatic hydrolysate and mix it with 10% TCA solution. Let it stand for 10 min, centrifuge at 4000 r / min for 15 min, and then take the supernatant. Dilute the solution to 50 mL with 5% TCA. Take 3 mL of this solution, add 2 mL of biuret reagent, mix well, and let it stand for 10 min. Measure the absorbance at 540 nm and plot a standard curve. The peptide yield is calculated using the following formula:

[0048] Peptide yield (%) = Peptide content in enzymatic hydrolysate / Protein content in raw material × 100.

[0049] 2. The method for determining DPPH free radical scavenging rate is as follows:

[0050] Take 1 mL of sample and add 4 mL of DPPH solution (0.04 mg / mL, dissolved in 95% ethanol). Mix the above solution well and incubate in the dark for 30 min. Measure the absorbance at 517 nm, using reduced glutathione (GSH) as a positive control. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0051] DPPH free radical scavenging rate (%) = (A1-A2) / A1×100;

[0052] Where: A1—the absorbance measured using distilled water instead of the enzyme digest;

[0053] A2—The absorbance value measured after the enzyme hydrolysate reaction.

[0054] 3. The method for determining the ABTS free radical scavenging rate is as follows:

[0055] ABTS stock solution (7 mmol / L ABTS mixed with 2.45 mmol / L potassium persulfate solution at a 1:1 (v / v) ratio) was incubated at room temperature in the dark for 12–16 h. Before use, it was diluted 40–60 times with phosphate buffer (pH 7.4) to obtain an absorbance of 0.706 ± 0.02 at 734 nm, which is the ABTS working solution. 0.2 mL of the sample was added to 5 mL of the ABTS working solution and incubated at room temperature in the dark for 10 min. Then, the absorbance at 734 nm was measured. Phosphate buffer solution was used as a control instead of the sample. The formula for calculating the ABTS free radical scavenging rate is as follows:

[0056] ABTS radical scavenging rate (%) = (A1-A2) / A1×100;

[0057] Where: A1—the absorbance measured using phosphate buffer instead of enzyme digest;

[0058] A2—The absorbance value measured after the enzyme hydrolysate reaction.

[0059] 4. The method for determining the hydroxyl radical scavenging rate is as follows:

[0060] Take 1 mL of sample, add 1 mL of 6 mmol / L ferrous sulfate and hydrogen peroxide sequentially, mix well and let stand for 10 min; then add 1 mL of 6 mmol / L salicylic acid, mix well and let stand for 30 min, then take 200 μL onto a 96-well plate and measure its absorbance at 510 nm. The formula for calculating the hydroxyl radical scavenging rate is as follows:

[0061] Hydroxyl radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100;

[0062] Where: A0—the absorbance value measured using distilled water instead of beer sample;

[0063] A1—The absorbance value measured for the sample;

[0064] A2—Absorbance measured using distilled water instead of salicylic acid.

[0065] 5. The method for measuring reducing power is as follows:

[0066] Take 1 mL of sample and add 2 mL of sodium phosphate buffer (0.2 mol / L, pH 6.6) and 2 mL of 1% potassium ferricyanide solution. Mix thoroughly and incubate at 50°C for 20 min. After cooling to room temperature, add 2 mL of 10% TCA solution and centrifuge at 3000 r / min for 10 min. Take 2 mL of the supernatant and add 2 mL of distilled water and 0.4 mL of 0.1% ferric chloride solution, respectively. React at room temperature for 10 min and then measure the absorbance at 700 nm. Use distilled water instead of the sample as a blank control. The reducing power is expressed as the difference between the absorbance of the sample and the absorbance of the blank control; the higher the absorbance, the higher the reducing power of the test sample.

[0067] 6. Stability Study:

[0068] (1) Thermal stability: HAPH-1 lyophilized powder was weighed and prepared into a 5 mg / mL solution. The solution was placed in a water bath at 20℃, 40℃, 60℃, 80℃ and 100℃ for 2 h. After cooling to room temperature, the DPPH free radical scavenging rate was measured.

[0069] (2) pH stability: Weigh HAPH-1 lyophilized powder to prepare a 5 mg / mL solution. Adjust the pH of the sample solution to 3.0, 5.0, 7.0, 9.0 and 11.0 respectively. After standing at room temperature for 2 h, adjust the pH to neutral and measure the DPPH free radical scavenging rate.

[0070] (3) Gastrointestinal digestive stability: HAPH-1 lyophilized powder was weighed and prepared into a 5 mg / mL solution, which was added to an equal volume of artificial gastric juice (1 g pepsin was added to 100 mL of distilled water, and the pH was adjusted to 2.0 with 1 mol / L HCl). The solution was incubated in a 37℃ water bath for 2 h. The enzymatic hydrolysate was taken every 30 min, and the DPPH free radical scavenging rate was measured after enzyme inactivation. After the simulated gastric digestion reaction was completed, the pH of the enzymatic hydrolysate was adjusted to 7.5 with NaOH, and trypsin (enzyme / sample = 1:25 w / w) was added and digested in a 37℃ water bath for 2 h. The enzymatic hydrolysate was taken every 30 min, and the DPPH free radical scavenging rate was measured after enzyme inactivation.

[0071] result:

[0072] 1. The peptide yield and DPPH scavenging rate of the enzymatic hydrolysate in Example 1 were measured to be 82.69% and 60.97%, respectively. This indicates that the process has extremely high enzymatic hydrolysis efficiency for expanded hippocampal protein; at the same time, the enzymatic hydrolysate has good antioxidant activity.

[0073] 2. The enzymatic hydrolysate of the distended hippocampus in Example 1 was separated by a 3 kDa ultrafiltration membrane. The DPPH free radical scavenging rates of each component were as follows: Figure 1As shown, at a peptide concentration of 5 mg / mL, HAPH-1 achieved a DPPH free radical scavenging rate of 63.47 ± 1.29%. This indicates that this step concentrates the antioxidant activity in the low molecular weight (<3 kDa) HAPH-1 fraction by retaining substances with a molecular weight >3000 Da, and confirms that the low molecular weight HAPH-1 fraction has superior antioxidant activity, laying a material basis for subsequent targeted screening of core active peptides.

[0074] The antioxidant activity results of HAPH-1 are shown in Table 1, with glutathione serving as the control group. This indicates that HAPH-1 also exhibits good scavenging activity against other free radicals, demonstrating comprehensive antioxidant activity.

[0075] Table 1: Results of HAPH-1 antioxidant activity:

[0076] .

[0077] 3. For example Figures 2-4 As shown, the DPPH clearance rate of HAPH-1 did not change significantly at temperatures of 20-60℃ and pH values ​​of 3-11. Furthermore, after 4 hours of in vitro simulated gastrointestinal digestion treatment, its DPPH clearance rate remained at a high level, indicating that HAPH-1 has high thermal stability, acid-base stability, and gastrointestinal digestibility.

[0078] Example 2:

[0079] 1. Prediction of the properties of antioxidant peptides in distended seahorse:

[0080] Potential bioactivity of the expanded hippocampal antioxidant peptides was predicted using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ). Isoelectric point (pI) and average hydrophilicity coefficient (GRAVY) were determined using the ProtParam tool in ExPASy (www.expasy.org / tools). Hydrophobicity was determined using Pepdraw (http: / / www.pepdraw.com / ). Toxicity was predicted using ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php). Sensitization was predicted using AllerTOP v2.0 (http: / / www.ddg-pharmfac.net / AllerTOP / ).

[0081] 2. Identification of the α-glucosidase inhibitory peptide sequence in the distended hippocampus by LC-MS / MS:

[0082] After desalting, the peptide sample was centrifuged and dried, then redissolved in Nano-LC mobile phase A (0.1% formic acid / water) and bottled for online LCMS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, 100 Å) at an appropriate volume, followed by a 20 μL wash for desalting. The sample was then desalted and retained on the pre-column before separation on a C18 reversed-phase column (AcclaimPepMap RSLC, 75 μm × 25 cm C18 - 2 μm 100 Å). The gradient used was a 60-minute increase in mobile phase B (80% acetonitrile, 0.1% formic acid) from 5% to 38%. Mass spectrometry was performed using a ThermoFisher Q Exactive plus system (ThermoFisher, USA) combined with a nano-spray Nano Flex ion source (ThermoFisher, USA), with a spray voltage of 1.9 kV and an ion transfer tube heating temperature of 320 °C.

[0083] 3. Molecular docking:

[0084] The peptides were molecularly docked with Keap1 (PDB ID: 4L7B). The optimal docking conformation was selected based on the binding energy. The docking results file (pdbqt) was converted to a pdb file and then imported into Pymol and the online website PLIP. https: / / plip- tool.biotec.tu-dresden.de / plip-web / plip / index The results can be visualized and analyzed.

[0085] 4. Artificially synthesized polypeptides:

[0086] The peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd. using a solid-phase synthesis method. Its secondary structure is as follows: Figure 7 As shown, the antioxidant capacity of the peptide was measured.

[0087] 5. The protective effect of bloated seahorse antioxidant peptides on the improvement of triptolide (TP)-induced male reproductive disorders.

[0088] Animal Experiment 1: Thirty-two SPF-grade male ICR mice aged 6-8 weeks were selected and, after one week of acclimatization, were randomly divided into four groups (n=8) for 21 days. The specific grouping and drug dosage are as follows.

[0089] Table 2: Specific groupings and dosages:

[0090] .

[0091] Animal Experiment 2: Thirty-two SPF-grade male ICR mice aged 6-8 weeks were selected and, after one week of acclimatization, were randomly divided into four groups (n=8) for 21 days. The specific grouping and drug dosage are as follows.

[0092] A male mouse model of reproductive disorders was established by intraperitoneal injection of TP for 21 consecutive days.

[0093] 6. Sperm parameter measurement:

[0094] (1) Sample processing: The experimental mice were fasted for 12 hours but allowed free access to water. After anesthetizing the mice, blood was collected from the orbital rim and the mice were euthanized. The left and right epididymis of the mice were accurately removed. The epididymis were immediately placed in centrifuge tubes / culture dishes containing 5 ml of physiological saline and preheated in a 37°C water bath. The epididymis was then cut into small pieces with clean ophthalmic scissors and incubated at 37°C for 5 minutes to ensure that the sperm could swim out of the epididymis. The corresponding sperm parameters were then evaluated. When collecting the sperm suspension, care should be taken to accurately aspirate it with a pipette. The sperm suspensions from different mice should be aspirated from the same location as much as possible to avoid errors.

[0095] (2) Sperm count determination: A hemocytometer containing 30 μl of sperm suspension was placed under a regular optical microscope for observation, and the sperm count was determined according to the red blood cell counting method. The number of sperm in the four square cells of the central grid was recorded under the microscope, following the principle of recording only one side (up, down, left, right) in an "S" shape. To avoid excessive error, the difference between the maximum and minimum sperm counts recorded in the selected squares had to exceed 20. The sperm counts from the four square cells were then summed.

[0096] Sperm count per milliliter of semen (10) 6 / ml) = Total sperm count in 4 square cells / 4 × N × 10 × 10 3 ;

[0097] (Note: N is the dilution factor. This experiment used a 5-fold dilution.)

[0098] (3) Measurement of sperm motility: A hemocytometer containing 30 μl of sperm suspension was placed under a regular optical microscope for observation. The number of sperm that moved rapidly forward in a straight line was randomly evaluated among 200 sperm.

[0099] Sperm motility (%) = Number of fast-moving sperm / 200 × 100%.

[0100] (4) Determination of sperm abnormality rate: 30 μL of sperm suspension was dropped onto a clean glass slide, dried, and then fixed with methanol for 10 min. Then, an appropriate amount of eosin staining solution was added, and after drying and fixing for 1 h, it was rinsed with water. The sperm morphology was observed under a 100× ordinary optical microscope, and the sperm with intact structures were counted. The number of abnormal sperm in 200 sperm cells was assessed in each mouse. Sperm abnormalities were mainly manifested in the head, followed by the tail. The main types of abnormal sperm in the head included: banana-shaped head, double head, fat head, amorphous head, and forward-curved head; the main types of abnormal sperm in the tail included: folded tail and double tail.

[0101] Sperm deformity rate (%) = number of deformed sperm / 200 × 100%.

[0102] result:

[0103] 1. The HAPH-1 fraction was identified by LC-MS / MS as containing 4033 peptides, with Mw < 3 kDa and peptide lengths ranging from 6 to 33 amino acids. Figure 5 As shown in the figure. Subsequently, the activity of bioactive peptides was predicted using the PeptideRanker website. If a peptide's score was greater than 0.5, it was considered to have potential bioactivity. To avoid false positives, 85 peptides with scores greater than 0.8 were selected. These were further screened using online analysis tools such as ExPASy, Pepdraw, ToxinPred, and AllerTOP v2.0 to identify 50 novel, unreported, non-toxic, and non-allergenic peptides with potentially high bioactivity, named P1-P50. Their chemical structures were then drawn using ChemDraw. All peptides were then docked with the Keap1 protein to obtain the required binding energy. The binding energy of the ligand and receptor was used to evaluate the degree of binding; the lower the binding energy, the easier it is for the two to bind and the stronger the interaction. The binding energy data for P1 are shown in Table 3. The binding energy of P1 with the Keap1 protein is less than -9 kcal / mol. Therefore, it is speculated that P1 has strong antioxidant activity.

[0104] Table 3: Binding energy data for P1:

[0105] .

[0106] 2. The docking results of P1 and Keap1 proteins are as follows: Figure 6 As shown in Table 4, the interactions between P1 and Keap1 are as follows. The results indicate that P1 forms hydrogen bonds with the amino acid residues Arg326, Val418, Val420, Val465, Val467, Val512, Val561, and Glu611 of the Keap1 molecule, and forms hydrophobic interactions with Ala366, Val467, and Val561. This means that the peptide can occupy key binding sites for Nrf2 through hydrogen bonding and bind to Keap1 under the influence of hydrophobic interactions, thereby activating the Keap1-Nrf2 / HO-1 signaling pathway and exerting an antioxidant effect in vivo.

[0107] Table 4: Interaction forces generated between P1 and Keap1:

[0108] .

[0109] The docking results of P1 with DPPH and ABTS radicals are shown in Table 5. The docking of P1 with DPPH and ABTS radicals also showed low binding energies, less than -3 kcal / mol. Therefore, it is speculated that P1 possesses strong antioxidant activity.

[0110] Table 5: Docking results of P1 with DPPH and ABTS free radicals:

[0111] .

[0112] 3. Through the BIOPEP-UWM database ( https: / / biochemia.uwm.edu.pl / biopep-uwm / P1 was confirmed to be a novel peptide through manual searching, therefore its antioxidant activity was verified by in vitro artificial synthesis. The purity of the synthesized peptide was >95%. The results are shown in Table 6.

[0113] Table 6: Verification of the antioxidant activity of P1 through in vitro artificial synthesis:

[0114] .

[0115] 4. Effects of GPAPWGF on sperm in a TP-induced mouse model: Figure 7 As shown in the figure, compared with the normal group, TP significantly reduced sperm motility and sperm count in mice, increased sperm morphological abnormalities, increased number of abnormal sperm, and significantly increased sperm abnormality rate (P < 0.01). GPAPWGF intervention significantly restored sperm count and sperm motility, and reduced sperm abnormality rate (P < 0.01). These results indicate that GPAPWGF has a certain ameliorative effect on TP-induced spermatogenic dysfunction in male mice.

[0116] 5. Effects of GPAPWGF on the pathological morphology of testicular tissue in a mouse model of male reproductive disorders, such as... Figure 8 As shown in the figure, the testicular tissue structure in the NC group was normal, with spermatogenic cells at different developmental stages visible, and the spermatogenic cells in the seminiferous tubules were clearly layered and neatly arranged. The M group showed severe tissue vacuolation with irregular shapes, reduced spermatogenic cell layers, disordered arrangement, and enlarged gaps; only a small number of Sertoli cells, spermatogonia, and primary spermatocytes were present. After GPAPWGF treatment, compared with the M group, the vacuolation of the testicular tissue was significantly improved, and the arrangement of spermatogenic cells was more neat. The results indicate that GPAPWGF can alleviate testicular tissue damage caused by TP.

[0117] 6. Results of the effects of GPAPWGF on TP-induced male mouse reproductive hormones are as follows: Figure 10 As shown, the results indicate that GPAPWGF can improve the damage to the reproductive axis in male mice in the TP model (P < 0.01) and maintain normal reproductive hormone levels to some extent.

[0118] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the invention. Those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. All modifications or equivalent substitutions should be included within the scope of protection of the present invention.

Claims

1. A type of anti-oxidative peptide from seahorse, characterized in that: It is a polypeptide with the amino acid sequence shown in SEQ ID NO.

1.

2. The method for preparing the anti-oxidative peptides from the bloated seahorse according to claim 1, characterized in that: It is artificially synthesized.

3. The application of the expanded hippocampal antioxidant peptide according to claim 1 in the preparation of antioxidant products.

4. The use of the expanded hippocampal antioxidant peptide according to claim 1 in the preparation of a drug for improving male reproductive disorders and testicular damage.

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