Amphippocampal peptide and application thereof in preparation of functional product for repairing male reproductive system injury

Abstract

The invention relates to the field of preparation and application of biological peptides, in particular to a hippocampal swelling peptide and application thereof in preparation of functional products for repairing male reproductive system injury. A green controllable enzymolysis technology is utilized to obtain protein polypeptide with nutritional, health-care and medicinal values, five active peptide sequences with high biological activity are obtained through further research on the basis, the high-purity active peptide is obtained through a chemical synthesis mode, and the biological activity of the peptide is verified through mouse spermatogonium.

Description

Technical Field

[0001] The present invention relates to the field of preparation and application of bio - peptides, and particularly to a belly - swollen seahorse peptide and its application in the preparation of functional products for repairing male reproductive system injuries. Background Art

[0002] Male reproductive injury refers to the dysfunction or structural damage of the reproductive system caused by various factors, which may affect sperm production, sexual function and fertility. Currently, the treatment methods mainly include antioxidants and assisted reproductive technologies. It is worth noting that nutritional support and functional foods have been proven to be able to improve antioxidant capacity and the number of spermatogenic cells, and improve reproductive injury and sexual function in mice.

[0003] Seahorses are important high - value marine fish in China's aquatic economy and have extremely high output value in the traditional Chinese medicine, marine aquarium and other markets. Research has found that seahorses contain a variety of bioactive substances and are often circulated in the traditional Chinese medicine market in the form of dried products, and are considered to have the effects of tonifying the kidney and strengthening yang, antioxidant, supplementing qi and blood, lowering blood pressure, etc. Currently, the belly - swollen seahorse (Hippocampus abdominalis), as an introduced aquaculture species, has successfully broken through the key technologies of large - scale artificial breeding and pest control in China. With the increasing development of the belly - swollen seahorse breeding technology, the exploration of its potential nutritional components is beneficial to the high - value utilization of seahorses. Chinese Patent CN1695656A discloses a traditional Chinese medicine composition with the functions of enhancing sexual function, enhancing physical strength and anti - fatigue, which includes the following components by weight: seahorse 1 - 20, dog kidney 1 - 20, pilose antler 1 - 20, epimedium 2 - 40, sangpiaoxiao 1.5 - 30, semen allii tuberosi 0 - 30, fructus psoraleae 0 - 30, ginseng 2 - 40, cinnamon 0 - 20, angelica sinensis 0 - 40, rehmannia glutinosa 0 - 40. Among them, seahorse is mentioned to have the effects of enhancing estrogen - like and androgen - like effects. However, when seahorse is used as a traditional Chinese medicine, it is usually taken orally in the form of decoction, powder, etc., with complex components, and it is difficult to avoid the metabolic burden and side effects. Currently, there is less research on the specific components that play a role in seahorses, and there is no research and application of belly - swollen seahorse peptide in repairing reproductive system injuries, which is a technical and market gap that needs to be filled. Summary of the Invention

[0004] The technical problem to be solved by the present invention is that currently, there is less research on the specific components that play a role in seahorses, and there is no research and application of belly - swollen seahorse peptide in repairing reproductive system injuries, which is a technical and market gap that needs to be filled.

[0005] To solve the above problems, the present invention provides a belly - swollen seahorse peptide and its application in the preparation of functional products for repairing male reproductive system injuries. By using a green and controllable enzymatic hydrolysis technology, protein polypeptides with nutritional and medicinal values are obtained, and on this basis, 5 active peptide sequences with high biological activities are further studied.

[0006] To achieve the above object, the present invention is realized by the following technical means: An application of a hippocampus abdominalis peptide in the preparation of a functional product for repairing male reproductive system injuries; the hippocampus abdominalis peptide includes polypeptides with amino acid sequences shown in SEQ ID NO.1-5: SEQ ID NO.1: GGPQMPGPM.

[0007] SEQ ID NO.2: NFDTFLPML.

[0008] SEQ ID NO.3: FEGFLPM.

[0009] SEQ ID NO.4: GGGFDFI.

[0010] SEQ ID NO.5: LFGVLF.

[0011] The preparation method of the above-mentioned hippocampus abdominalis peptide includes the following steps: Take fresh hippocampus abdominalis, wash it and then freeze-dry it, and crush it into powder; add distilled water, add 3% compound protease and stir evenly, adjust the pH to 7.5 with HCL solution and NaOH solution respectively, and enzymatically hydrolyze at 50 °C for 5 h; After centrifuging the enzymatic hydrolysate, take the supernatant and filter it through a 0.45 μm microporous filter membrane, and then centrifuge it at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cut-off of 3 kDa. Separate through the ultrafiltration membrane with a molecular weight cut-off of 3 kDa to obtain peptide segments with a molecular weight less than 3 kDa; freeze-dry the peptide solution to obtain hippocampus abdominalis peptide powder.

[0012] Furthermore, add distilled water at a ratio of 1:9 of the solid-liquid ratio.

[0013] Furthermore, the concentrations of the HCL solution and the NaOH solution are both 1 mol / L.

[0014] Furthermore, the enzyme activity of the compound protease is 100 U / mg.

[0015] The above-mentioned hippocampus abdominalis peptide with amino acid sequences shown in SEQ ID NO.1-5.

[0016] Furthermore, the hippocampus abdominalis peptide with amino acid sequences shown in SEQ ID NO.1-5 is artificially synthesized.

[0017] The beneficial effects of the present invention are as follows: (1) By using the green and controllable enzymatic hydrolysis technology, protein polypeptides with nutritional and medicinal values are obtained. The molecular weight of the hippocampus abdominalis peptide is mainly short peptides with < 1000 Da.

[0018] (2) Five active peptide sequences with high biological activities are disclosed. Highly pure peptides are obtained by chemical synthesis, and the biological activities of the peptides are verified using mouse spermatogonia, improving the understanding of the hippocampus abdominalis active peptide as a potential health supplement for improving male reproductive damage.

[0019] (3) Long-term oxidative stress in testicular tissues can promote the apoptosis of testicular Sertoli cells and spermatogenic cells, thereby destroying the testicular structure and spermatogenic function. The hippocampus peptide of the present invention can increase the sperm quality of cyclophosphamide-induced reproductive damage mice, reduce the sperm malformation rate, and maintain the normal morphological structure of the testis and epididymis by reducing the oxidative stress level of testicular tissues and increasing the activity of antioxidant enzyme LDH, thereby protecting the blood-testis barrier of mice from damage and repairing male reproductive system damage. Description of the Drawings

[0020] Figure 1 is the molecular weight distribution of the hippocampus abdominalis peptide in Example 1; where A is the gel exclusion chromatogram; B is the molecular weight distribution range.

[0021] Figure 2 is the amino acid composition of the hippocampus abdominalis peptide in Example 1.

[0022] Figure 3 is the sperm motility and total sperm count of mice; where A is the sperm motility; B is the total sperm count.

[0023] Figure 4 is the sperm morphology diagram of mice.

[0024] Figure 5 is Figure 4 the enlarged view of the corresponding part.

[0025] Figure 6 is the morphology of mouse testis and epididymis tissues.

[0026] Figure 7 is the effect of SHP on the microscopic structure of the blood-testis barrier in mice. Note: The yellow arrow indicates the blood-testis barrier structure, and the red arrow indicates the local incompleteness of the blood-testis barrier.

[0027] Figure 8 is Figure 7 the enlarged view of the corresponding part.

[0028] Figure 9are the sex hormone levels in mouse serum. Note: **Compared with the normal group, p < 0.01; ##Compared with the model group, p < 0.01; where A is the serum testosterone level; B is the serum luteinizing hormone level.

[0029] Figure 10 is the effect of SHP on the antioxidant level in mouse testicular tissue. Note: **Compared with the normal group, p < 0.01; #Compared with the model group, p < 0.05, ##Compared with the model group, p < 0.01; where A is the testicular malondialdehyde level; B is the testicular lactate dehydrogenase level.

[0030] Figure 11 is the effect of peptide segments at different concentrations on the viability of mouse spermatogonial GC-2 cells. Detailed implementation manners

[0031] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0032] In addition, all materials used in the embodiments of the present invention are obtained by purchasing from the market without special instructions.

[0033] Example 1: A pterygopogon kauderni peptide is prepared by the following method: Take 100 g of fresh pterygopogon kauderni, wash it and then freeze-dry it, and crush it into powder. Add distilled water according to the solid-liquid ratio of 1:9 (m / v), add 3% compound protease and stir evenly. Adjust the pH to the optimal range of the protease (pH = 7.5) with HCL solution (1 mol / L) and NaOH solution (1 mol / L) respectively, and carry out enzymatic hydrolysis at 50 °C for 5 h. After the enzymatic hydrolysate is centrifuged (centrifuged at 4 °C and 4000 g / min for 15 min), take the supernatant and filter it through a 0.45 μm microporous membrane, and then centrifuge it at 4000 g / min for 20 min with an ultrafiltration tube with a molecular weight cut-off of 3 kDa, and separate it through an ultrafiltration membrane (molecular weight cut-off 3 kDa) to obtain peptide segments with a molecular weight less than 3 kDa. Freeze-dry the peptide solution to obtain pterygopogon kauderni peptide powder. Among them, the compound protease (1.2 million U / mg, product number: S10155) is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0034] Result verification: Molecular weight determination of Hippocampus abdominalis peptide: The molecular weight distribution of SHP was determined by gel exclusion chromatography. The specific conditions were as follows: Chromatographic column: Superdex peptide PE10 / 300GL; Sample concentration: 2 mg / mL; Injection volume: 100 μL; Mobile phase: 25% acetonitrile (0.1% formic acid); Flow rate: 0.35 mL / min; Column temperature: room temperature; UV detection wavelength: 214 nm.

[0035] Amino acid composition of Hippocampus abdominalis peptide: The total amino acid composition was analyzed using an automatic amino acid analyzer (Biochrom 30 Ltd). 0.1 g of the sample was weighed into an ampoule, 5 mL of 6 mol / mL hydrochloric acid solution was added, and acid hydrolysis was carried out at 110 °C for 24 h. After cooling, it was made up to 25 mL with ultrapure water. Then, 1 mL of the solution was deacidified to dryness and then 1 mL of sample buffer was added. The solution was filtered through a 0.22 μm filter membrane and loaded into a liquid phase vial for on-machine analysis. The amino acid content was expressed as mg / g.

[0036] The molecular weight distribution of the complex protease total hydrolysate of Hippocampus abdominalis peptide was detected by gel chromatography, and the results are as Figure 1 shown. By comparing with the standard molecular weight curve, it was calculated that the peptides with a molecular weight less than 500 Da in the total hydrolysate of Hippocampus abdominalis accounted for 23.96%, and the short peptides with a molecular weight of 500 - 1 kDa had the highest proportion of 48.21%, among which the short peptides with a molecular weight of 1 - 3 kDa accounted for 27.83% ( Figure 1 shown as A - B in it).

[0037] The amino acid composition of Hippocampus abdominalis peptide (SHP, molecular weight < 3 kDa) is as Figure 2 shown. The top 5 amino acids with the highest content in Hippocampus abdominalis peptide were glycine (14.5%), glutamic acid (9.6%), alanine (8.5%), arginine (7.6%), and proline (6.9%) in sequence. Among them, the content of essential amino acids accounted for 35.1%, which was close to the ideal amino acid pattern (EAA / TAA = 40%).

[0038] Animal experiment: 40 healthy male ICR mice (7 weeks old, body weight 30.0 ± 1.0 g) were purchased from Jinan Pengyue Laboratory Animal Technology Co., Ltd. The animal rearing environment was maintained at a temperature of 21°C - 23°C and a humidity of 45% - 55%, with a 12 h light / 12 h dark cycle. During the experiment, the mice had free access to food (AIN-93G type feed) and water. After 7 days of adaptive feeding, 32 mice were randomly selected and intraperitoneally injected with cyclophosphamide (60 mg / kg, dissolved in 0.85% physiological saline) once a day for 7 consecutive days. Another 8 mice were intraperitoneally injected with the same dose of physiological saline as the control group. The experimental groups were as shown in the following table, and a model of reproductive system injury in male mice induced by cyclophosphamide was established. Hippocampal peptide was supplemented by gavage every day for a total of 14 days, and the model group and the control group continued to be gavaged with physiological saline. After the experiment, the mice were fasted for 12 h, anesthetized with isoflurane, and then blood was collected by eye socket puncture, and the mice were sacrificed by cervical dislocation. Bilateral testicular and epididymal tissues of the mice were collected, weighed, and the organ indices of the testis and epididymis were calculated.

[0039] ; Table 1 Animal grouping and dosing: 。

[0040] Sperm index detection: Cut off 2 cm of the caudal epididymis, rinse it 2 - 3 times in physiological saline at 37°C to wash away the blood and fat globules on the tissue surface to avoid affecting sperm observation. Then, cut the epididymis into pieces in an EP tube containing 3.5 ml of M199 culture medium (37°C) to allow sperm to flow out, and then incubate it in an incubator with 5% CO2 for 10 min (37°C), and gently shake it to allow sperm to swim out more fully. Dilute the sperm suspension 20 times, mix well, and then pipette 10 μL of sperm culture medium onto a hemocytometer (the hemocytometer has been preheated to 37°C). Observe the sperm under a microscope at 400×, record the number of motile sperm, and after recording, place it in an oven at 60°C to kill the sperm, and then record the total number of sperm (i.e., the total sperm count). The ratio of the number of motile sperm to the total number of sperm is the sperm motility.

[0041] Pathomorphological analysis of the epididymis and testis: Fix the testicular and epididymal tissues with an animal testicular tissue fixative for 24 h, place them in 75% ethanol, dehydrate them with gradient ethanol, clear them with xylene, and embed them in paraffin. Cut the wax blocks into 4 µm sagittal sections, stain them with HE (hematoxylin - eosin), observe the tissue structure status of the testis in each group under a microscope, and perform image acquisition.

[0042] Ultrastructural observation of the blood-testis barrier: Immediately after killing the mouse, the abdomen was opened and one testicle was pulled out. Two to three small holes were gently punctured at one end of the testicle with a needle. A 1 mL syringe was used to gently pierce the tunica albuginea at the other end. The needle was inserted parallel to the tunica albuginea and the fixative was injected until the fixative overflowed. The testicular tissue was removed and placed in a dish containing electron microscopy fixative. It was quickly cut into 1 cm pieces with a blade. 3 Small tissue blocks of about 100 μg were transferred to cryovials filled with new fixative and stored at 4°C without freezing. Ultrathin sections were prepared and observed under a transmission electron microscope at the Electron Microscopy Experimental Center of Qingdao University Medical College to collect typical images of pathological changes in the blood-testis barrier.

[0043] Determination of serum biochemical indicators: The relative levels of serum reproductive hormones such as testosterone (T), luteinizing hormone (LH), TNF-α, and IL-1β were determined according to the instructions of the Elisa kit.

[0044] Testicular biochemical markers were measured: Testicular tissue (approximately 0.1 g) was removed from a -80°C freezer and placed in a 2 mL enzyme-free EP tube. Pre-chilled saline was added at a 1:9 (m / v) ratio and the testicles were homogenized using a tissue homogenizer to prepare a 10% testicular homogenate. The testicles were then centrifuged at 8000 g for 20 minutes at 4°C, and the supernatant was collected. The lipid peroxidation products malondialdehyde (MDA) and lactate dehydrogenase (LDH) were determined according to the manufacturer's instructions. The testicular protein concentration in the supernatant was calibrated using a BCA assay kit using bovine serum albumin (BSA) as a standard.

[0045] As shown in Table 2, the testicular and epididymal indices of the model group mice were significantly decreased compared with the control group (P < 0.05), indicating successful model establishment. Furthermore, CTX-induced reductions in the relative weights of the testes and epididymis of the mice were observed. Compared with the model group, supplementation with SHP-L increased the testicular and epididymal indices of the mice, but the differences were not statistically significant (P > 0.05). SHP supplementation also inhibited CTX-induced reproductive organ atrophy.

[0046] Table 2 Mouse testis and epididymis index: .

[0047] like Figure 3 As shown in Figures AB, sperm motility in the model group was significantly decreased compared with the control group, while sperm motility in the SHP-L and H groups was significantly increased compared with the model group (P < 0.01). The total sperm count in the model group was significantly lower than that in the control group, while the total sperm count in the SHP-L and H groups showed an increased trend compared with the model group, with a highly significant difference between the SHP-H group and the model group (P < 0.01).

[0048] Sperm morphology is an important apparent indicator for judging the normal reproductive function of mice. As Figure 4 and Figure 5 show, the sperm morphology of the control group mice was complete and the structure was normal; the sperm of the model group mice showed deformities, such as neck bending, coiled tails, and severed heads. Compared with the control group, the quantity and quality of sperm were significantly lower than those of the control group; compared with the model group, SHP treatment could significantly improve sperm quality, increase sperm quantity, and reduce the sperm malformation rate. The effects of different doses of SHP were dose-dependent, and the improvement effect of SHP-H on sperm damage was more significant.

[0049] The testis is composed of seminiferous tubules and interstitial cells. The cross-section of the mouse testis has hundreds of seminiferous tubules. Each tubule is composed of Sertoli cells and germ cells at different developmental stages and is an important site for sperm production. Whether the structure and morphology of the testis are normal determines the quantity and quality of sperm. As Figure 6 shown, the HE staining results of the testis tissues of each group showed that: in the normal group, the seminiferous tubules of the mouse testis were arranged evenly, the shape and structure were clear, the thickness of the tube wall and the peritubular basement membrane was uniform, and the spermatogenic cells and Sertoli cells at all levels were distributed evenly, and there were more sperm in the lumen; compared with the normal group, the seminiferous tubules of the model group mice were distributed scattered, the intertubular space increased, the outline was blurred, the tube wall thickness was uneven, the spermatogenic cells and Sertoli cells in the tube wall decreased to varying degrees, the secondary spermatocytes were severely vacuolated, and the number of sperm in the lumen decreased; compared with the model group, the spermatogenic tablets and SHP treatment could, to a certain extent, restore the morphological abnormalities caused by reproductive damage. After SHP-L treatment, the distribution of seminiferous tubules in the mouse testis tissue tended to be uniform, the structure was clearer, and the formation of vacuoles in spermatocytes decreased; the seminiferous tubules in the SHP-H group were distributed evenly, the structure was clear, and the number of spermatocytes and the number of sperm in the lumen further increased.

[0050] The epididymis has the functions of storing and discharging sperm, promoting sperm maturation, and providing nutrients for sperm. Most mature sperm are stored in the cauda epididymis. Therefore, the structure and morphology of the epididymis can reflect the health of the mouse reproductive system. As Figure 6 shown, in the normal group, the testicular efferent ducts in the head of the epididymis were arranged evenly, the tube wall thickness was uniform, the structure was clear, and the sperm in the lumen were distributed evenly. The cauda epididymis was filled with a large number of sperm; after CTX injury, the structure in the head of the epididymis of the model group mice was severely damaged, the testicular efferent ducts were severely atrophied and unevenly distributed, and there were almost no visible sperm in the lumen. The wall thickness of the cauda epididymis decreased, and the number of internal sperm decreased severely; compared with the model group, the spermatogenic tablets and SHP treatment could, to a certain extent, restore the morphological abnormalities of the mouse epididymis, and the sperm density in the cauda epididymis increased. In the SHP-H group, the tubules in the head of the epididymis were distributed evenly, the structure was clear, and a large number of sperm could be seen in the cavity of the cauda epididymis, approaching the control group level.

[0051] To further observe the ultrastructure of the blood-testis barrier in the mouse testis tissue, detection and observation were carried out by transmission electron microscopy (Figure 7 and Figure 8 ), and it was found that the microstructure of the BTB in the testicular tissues of the control group mice was intact, while in the CTX model group mice, the microstructure of the BTB in the testes showed phenomena such as fractures and vacuoles. Compared with the model group, the microstructure of the BTB in the testes of mice supplemented with SHP was restored to a certain extent, and there was a dose-effect relationship.

[0052] There is a significant correlation between male serum reproductive hormones testosterone (T) and luteinizing hormone (LH) and spermatogenic function of the testis. As Figure 9 shown in A - B below, compared with the control group, the serum T and LH levels in the model group were significantly decreased (P < 0.01), indicating that CTX interfered with the serum hormone levels of mice. Compared with the CTX model group, SHP treatment could restore the serum T and LH levels of mice. It is worth noting that the effect of the SHP - H group was better than that of the positive control (Shengjing Tablets) group, almost reaching a level similar to that of the control group. The above results indicate that SHP can improve the disorder of serum reproductive hormones induced by CTX.

[0053] Oxidative stress is one of the causes of testicular injury. By measuring the oxidative indexes of each group, it was found that compared with the control group, the MDA level in the model group was significantly increased (P < 0.01), and the LDH level was significantly decreased (P < 0.01) ( Figure 10 shown in A - B below), indicating that the antioxidant system of the testis was severely damaged. SHP - H intervention reduced the MDA level, increased the LDH activity, and improved the oxidative stress of the testicular tissues of reproductive injury mice, and the effect was equivalent to that of the positive control (Shengjing Tablets).

[0054] Example 2: Based on the results of Example 1, sequence analysis of the abdominal - swelling seahorse peptide was carried out: the seahorse peptide samples were desalted, and each sample was in triplicate. Using an UltiMate 3000 UHPLC system (Thermo Fisher Scientiiffc), an ACQUITY UPLC Peptide CSH C18 column (2.1×100 mm, 1.7 μm) and a TripleTOF 5600+ system were used to analyze the SHP after dialysis desalting. The parameters were as follows: positive ion mode, spray voltage 3600 eV, capillary temperature 320 °C, signal intensity threshold 1.6e 5 . The peptide sequences obtained by de novo sequencing were searched for seahorse protein peptide sequences in the NCBI database, and the results showed that the peptide sequences were not publicly available. Peptide Ranker was used to predict the possibility that the identified peptide segments had biological activity.

[0055] Table 3 Identification and activity prediction of abdominal - swelling seahorse peptide sequences: ; a From PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0056] The above-mentioned Hippocampus abdominalis bioactive peptides were biosynthesized respectively, and were synthesized by Shanghai Sangon Biotech Co., Ltd. through the Fmoc-solid phase peptide synthesis method. Through HPLC and MS sequence analysis, the purity of the five peptide segments GGPQMPGPM (①), NFDTFLPML (②), FEGFLPM (③), GGGFDFI (④), and LFGVLF (⑤) was greater than 98.89%. Chemically synthesized polypeptides have the characteristics of high purity and good clinical application safety due to the controllability of raw materials and processes. However, during the synthesis process, due to the large number of steps involved and the use of various solvents, it is inevitable to have a certain impact on the biological activity of the polypeptide. Therefore, it is necessary to further verify the biological activity of the synthesized polypeptide.

[0057] Mouse spermatogonial cell line GC-2 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, placed in an incubator at 37 °C and 5% CO2, and passaged when the cells grew to the logarithmic growth phase. The CCK method was used to detect the effect of the above-mentioned synthetic peptide segments on the viability of mouse GC-2 cells: after passage, GC-2 cells (3×10 4 cells / mL) were inoculated into 96-well plates and cultured with the culture solution of the above-mentioned peptide segments at different concentrations (0, 25, 50, 100, 200 μg / mL) for 24 h. 10 μL of CCK-8 reagent was added to each well and cultured for another 4 h. The OD values of each well were measured at 490 nm, and the average OD values of each parallel well were taken. Cell viability % = (OD of sample-added cells - OD of blank) / (OD of control cells - OD of blank) × 100%.

[0058] The results of the CCK-8 colorimetric method are as Figure 11 shown. The hippocampal active peptide sequences at different concentrations significantly promoted the proliferation of mouse GC-2 cells, and the effect was dose-dependent. Among them, the SHP at 50 and 100 μg / mL had the greatest amplitude of enhancing cell viability, and there was no difference between the concentration of 200 μg / mL and 100 μg / mL, indicating that the synthesized pure peptide had a significant promoting effect on cell proliferation.

[0059] Finally, it should be noted that although the above embodiments describe the specific implementation manners of the present invention, they do not limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. And all modifications or equivalent replacements should be included within the protection scope of the present invention.

Claims

1. Application of a ventral-bellied seahorse peptide in preparing a product for repairing male reproductive system damage, characterized in that: The ventral-bellied seahorse peptide comprises polypeptides with amino acid sequences shown in SEQ ID NO.1-5.

2. The preparation method of the abdominal-puffing seahorse peptide according to claim 1, characterized in that It includes the following steps: Take fresh ventral-bellied seahorses, wash them, freeze-dry them, and crush them into powder; add distilled water, add 3% complex protease and stir evenly, adjust the pH to 7.5 with HCL solution and NaOH solution respectively, and enzymatically hydrolyze at 50 °C for 5 h; After the enzymatic hydrolysate is centrifuged, take the supernatant, filter it through a 0.45 μm microporous filter membrane, and then centrifuge it at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cut-off of 3 kDa. Separate through the ultrafiltration membrane with a molecular weight cut-off of 3 kDa to obtain peptide segments with a molecular weight less than 3 kDa; freeze-dry the peptide solution to obtain ventral-bellied seahorse peptide powder.

3. The preparation method according to claim 2, characterized in that: Add distilled water at a material-liquid ratio of 1:

9.

4. The preparation method according to claim 2, wherein: The concentrations of the HCL solution and the NaOH solution are both 1 mol / L.

5. The preparation method according to claim 2, characterized in that: The enzyme activity of the complex protease is 100 U / mg.

6. The ventral-bellied seahorse peptide powder prepared by any method according to claims 2-5.

7. The ventral-belly seahorse peptide according to claim 1, wherein: The amino acid sequences are shown in SEQ ID NO.1-5.

8. The ventral-bulging seahorse peptide according to claim 7, wherein: The ventral-bellied seahorse peptide is artificially synthesized.

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