A bulbus cavernosus hippocampus peptide and application thereof in preparation of a product for repairing damaged functions of male reproductive system

Abstract

The present application relates to the preparation and application field of biological peptide, and particularly relates to a plectinum peptide and application of the plectinum peptide in preparation of a product for repairing male reproductive system damage function. The present application utilizes green controllable enzymolysis technology to obtain protein polypeptides with nutritional health care and medicinal values, and further researches on the basis to obtain an active peptide sequence with high biological activity, obtains the active peptide with high purity through a chemical synthesis mode, and verifies the biological activity of the peptide by using mouse spermatogonia.

Description

Technical Field

[0001] This invention relates to the field of preparation and application of bioactive peptides, and particularly to a bloated hippocampal peptide and its application in the preparation of functional products for repairing damage to the male reproductive system. Background Technology

[0002] Male reproductive injury is a type of reproductive system disorder induced by multiple factors, mainly manifested as structural damage or functional impairment of the reproductive system. Its core harm lies in its potential negative impact on spermatogenesis, normal sexual function, and fertility, seriously threatening male reproductive health. Currently, clinical treatment for male reproductive injury primarily relies on antioxidant intervention and assisted reproductive technology, playing a fundamental role in improving patients' reproductive function. In recent years, significant progress has been made in nutrition-related research. Studies have confirmed that scientific nutritional support strategies and the application of specific functional foods can effectively enhance the body's antioxidant capacity and increase the number of spermatogenic cells. Further animal experiments (using mice as a model) have shown that this intervention has a clear positive effect on improving the state of reproductive system damage and restoring sexual function, providing new research directions and practical evidence for the adjuvant treatment of male reproductive injury.

[0003] Hippocampus abdominalis is a new species introduced to my country in recent years. With the continuous development of the marine aquaculture industry, the improvement of living standards, and the popularization and promotion of the concept of "health and wellness," people's demand for health preservation is increasing. Exploring its potential nutritional components is beneficial for the high-value utilization of seahorses. Currently, research on the specific active components in Hippocampus abdominalis only includes the compound product and a few peptides obtained by the inventors in their previous research, namely, a Hippocampus peptide disclosed in Chinese Patent CN120392955A and its application in the preparation of functional products for repairing damage to the male reproductive system. However, current research on Hippocampus peptides has not yet reached a sufficient level of exploration, and the discovery of existing functional peptides remains relatively limited. Given the increasing demand for novel functional peptides due to technological development, further research efforts are needed to discover more Hippocampus peptide resources that meet these needs. Summary of the Invention

[0004] The technical problem this invention aims to solve is that current research on bloating hippocampal peptides has not yet reached a sufficient level of exploration, and the discovery of existing functional peptides remains relatively limited. Given the increasing demand for novel functional peptides driven by technological advancements, further research efforts are needed to uncover more bloating hippocampal peptide resources that meet these needs.

[0005] To address the aforementioned issues, this invention provides 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. Furthermore, a highly bioactive peptide sequence was obtained through further research.

[0006] To achieve the above objectives, the present invention utilizes the following technical means: a bloating hippocampal peptide, wherein the amino acid sequence of the bloating hippocampal peptide is the polypeptide shown in SEQ ID NO.1:

[0007] SEQ ID NO.1:

[0008] SGPINF.

[0009] Furthermore, the bloated hippocampal peptide with the amino acid sequence shown in SEQ ID NO.1 is artificially synthesized.

[0010] The above-mentioned expanding hippocampal peptide is used in the preparation of products for repairing damage to the male reproductive system. If testicular tissue is under prolonged oxidative stress, it accelerates the apoptosis process of Sertoli cells and spermatogenic cells. This cellular damage further disrupts testicular tissue structure and impairs spermatogenesis. The hippocampal peptide proposed in this invention exerts its efficacy through a dual mechanism: on the one hand, it reduces the degree of oxidative stress in testicular tissue; on the other hand, it increases the activity of the antioxidant enzyme lactate dehydrogenase (LDH). Based on these two effects, the hippocampal peptide can effectively improve sperm quality in mice with reproductive damage induced by cyclophosphamide, ultimately achieving a repair effect on damage to the male reproductive system.

[0011] Furthermore, the concentration of bloated hippocampal peptide in products for repairing damage to the male reproductive system is 50~100 μg / mL.

[0012] The method for preparing the powder containing the above-mentioned swelling hippocampal peptide includes the following steps:

[0013] Fresh bloated seahorses were taken, washed, freeze-dried, and pulverized into powder. Distilled water was added, and 3% compound protease was added and stirred evenly. The pH was adjusted to 7.5 with HCl solution and NaOH solution, respectively, and enzymatic hydrolysis was carried out at 50°C for 5 h.

[0014] After centrifugation, the supernatant of the enzymatic hydrolysate was filtered through a 0.45 μm microporous membrane and then centrifuged at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The peptides with a molecular weight cutoff of 3 kDa were obtained by separation through the ultrafiltration membrane. The peptide solution was freeze-dried to obtain expanded hippocampal peptide powder.

[0015] Furthermore, distilled water is added at a material-to-liquid ratio of 1:9.

[0016] Furthermore, the concentrations of both the HCl solution and the NaOH solution are 1 mol / L.

[0017] Furthermore, the enzyme activity of the complex protease is 1.2 million U / mg.

[0018] The beneficial effects of this invention are as follows:

[0019] A highly bioactive peptide sequence was disclosed, and a high-purity peptide fragment was obtained through chemical synthesis. The bioactivity of the peptide fragment was verified using mouse spermatogonia. This improved our understanding of the active peptide from the bloated hippocampus as a potential health supplement to improve male reproductive damage. Attached Figure Description

[0020] Figure 1 This is the amino acid composition of the expanded hippocampal peptide from Example 1.

[0021] Figure 2 The values ​​represent mouse sperm motility and total sperm count; Note: **Compared with the normal group, p < 0.01; ##Compared with the model group, p < 0.01; where A represents sperm motility; B represents total sperm count.

[0022] Figure 3 The values ​​represent the serum sex hormone levels in mice. Note: **Compared with the normal group, p < 0.01; ##Compared with the model group, p < 0.01; where A represents serum testosterone levels; B represents serum luteinizing hormone levels.

[0023] Figure 4 The values ​​represent the antioxidant levels 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 represents the malondialdehyde level in the testes; B represents the lactate dehydrogenase level in the testes.

[0024] Figure 5 The effect of different concentrations of peptides on the viability of mouse spermatogonia GC-2 cells; Note: * p < 0.05 compared with the control group; ** p < 0.01 compared with the control group. Detailed Implementation

[0025] 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.

[0026] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0027] Example 1:

[0028] A type of bloating hippocampal peptide powder is prepared by the following method:

[0029] 100 g of fresh swollen seahorse was washed, freeze-dried, and pulverized into powder. Distilled water was added at a material-to-liquid ratio of 1:9 (m / v), and 3% complex protease was added and stirred until homogeneous. The pH was adjusted to the optimal range for the protease (pH=7.5) using HCl solution (1 mol / L) and NaOH solution (1 mol / L), respectively. Enzymatic hydrolysis was carried out at 50°C for 5 h. After centrifugation (4°C, 4000 g / min for 15 min), the supernatant was filtered through a 0.45 μm microporous membrane and then centrifuged at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. Peptides with a molecular weight cutoff of less than 3 kDa were obtained by ultrafiltration. The peptide solution was freeze-dried to obtain swollen seahorse peptide powder. The complex protease (1.2 million U / mg, product code: S10155) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0030] Result verification:

[0031] Amino acid composition of expanded hippocampal peptides: Total amino acid composition was analyzed using a Biochrom 30 Ltd. automated amino acid analyzer. 0.1 g of sample was weighed into an ampoule, and 5 mL of 6 mol / mL hydrochloric acid solution was added. The mixture was acid-hydrolyzed at 110 °C for 24 h. After cooling, the volume was adjusted to 25 mL with ultrapure water. One mL of this solution was then deacidified and dried before adding 1 mL of sample buffer. The solution was filtered through a 0.22 μm filter and then transferred to a liquid chromatography vial for analysis. Amino acid content was expressed in mg / g.

[0032] The amino acid composition of the expanded hippocampal peptide (molecular weight < 3 kDa) is as follows: Figure 1 As shown, the five most abundant amino acids in the expanded hippocampal peptide are, in descending order: glycine (14.5%), glutamic acid (9.6%), alanine (8.5%), arginine (7.6%), and proline (6.9%). The content of essential amino acids is 35.1%, close to the ideal amino acid pattern (EAA / TAA = 40%).

[0033] Animal Experiments: Healthy male ICR mice (7 weeks old, weighing 30.0±1.0 g) were purchased from Jinan Pengyue Experimental Animal Technology Co., Ltd. The animal housing environment was maintained at 21°C~23°C and 45%~55% humidity, with a 12-hour light / 12-hour dark cycle. Mice had free access to food (AIN-93G type feed) and water during the experiment. After 7 days of acclimatization, the mice were divided into 5 groups: normal group, model group, spermatogenic tablet group, low-dose hippocampal peptide group, and high-dose hippocampal peptide group. Except for the normal group, all other groups of mice were intraperitoneally injected with cyclophosphamide (60 mg / kg, dissolved in 0.85% saline) once daily for 7 consecutive days. The normal group served as a control, receiving the same dose of saline intraperitoneally. The experimental groups are shown in the table below. A cyclophosphamide-induced male mouse reproductive system damage model was established through intraperitoneal injection. Hippocampal peptide was supplemented daily by gavage for 14 days. The model group and control group continued to be administered saline by gavage. After the experiment, the mice were fasted for 12 hours, anesthetized with isoflurane, enucleated to collect blood, and euthanized by cervical dislocation. Bilateral testicular and epididymal tissues were collected, weighed, and their organ indices were calculated.

[0034] .

[0035] Table 1. Animal grouping and drug dosage:

[0036] .

[0037] Sperm index detection: A 2 cm section of the epididymis tail was removed and rinsed 2-3 times in 37°C physiological saline to remove blood and fat globules from the tissue surface to avoid affecting sperm observation. The epididymis was then cut into pieces in an EP tube containing 3.5 mL of M199 culture medium (37°C) to allow sperm to flow out. The tube was then incubated in a 5% CO2 incubator for 10 min (37°C), with gentle agitation to encourage further sperm motility. The sperm suspension was diluted 20-fold and thoroughly mixed. 10 μL of the sperm culture medium was then dropped into a hemocytometer (preheated to 37°C). Sperm were observed under a 400× microscope, and the number of motile sperm was recorded. After recording, the tube was placed in a 60°C oven to kill the sperm, and the total number of sperm was recorded again. The ratio of motile sperm to the total number of sperm is the sperm motility.

[0038] Serum biochemical indicators were measured: serum reproductive hormones testosterone (T) and luteinizing hormone (LH) were measured according to the ELISA kit instructions.

[0039] Testicular biochemical assays: Testicular tissue (approximately 0.1 g) was removed from a -80°C freezer and placed in a 2 mL enzyme-free EP tube. Pre-cooled physiological saline was added at a ratio of 1:9 (m / v), and the tissue was homogenized at low temperature using a tissue homogenizer to prepare a 10% testicular homogenate. The homogenate was then centrifuged at 8000 g for 20 min at 4°C, and the supernatant was collected. Following the instructions of a standard kit, the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH), lipid peroxidation products, were determined. The testicular protein concentration of the supernatant from the testicular homogenate was determined using a BCA kit with bovine serum albumin (BSA) as a standard.

[0040] As shown in Table 2, compared with the normal group, the testicular and epididymal indices of mice in the model group decreased by 26% and 44%, respectively (P < 0.05), indicating successful model establishment. Compared with the model group, the testicular and epididymal indices of mice in the low-dose hippocampal peptide group showed an increasing trend, but the difference was not statistically significant (P > 0.05). In contrast, the testicular and epididymal indices of mice in the high-dose hippocampal peptide group increased by 32% and 39%, respectively (P < 0.05), indicating that high-dose hippocampal peptide supplementation inhibited CTX-induced reproductive organ atrophy.

[0041] Table 2. Mouse testicular and epididymal indices:

[0042] .

[0043] like Figure 2 As shown in Figures A and B, compared with the normal group, the sperm motility of mice in the model group decreased by 56% (P < 0.01); compared with the model group, the sperm motility of mice in the hippocampal peptide group increased by 1.5 and 1.9 times, respectively (P < 0.01). Compared with the normal group, the total sperm count of mice in the model group decreased by 67% (P < 0.01); while compared with the model group, the total sperm count of mice in each hippocampal peptide group showed an increasing trend, and there was a highly significant difference between the high-dose hippocampal peptide group and the model group (P < 0.01).

[0044] Serum reproductive hormones testosterone (T) and luteinizing hormone (LH) are significantly correlated with testicular spermatogenesis, such as... Figure 3 As shown in Figures A and B, compared with the normal group, the serum T and LH levels in the model group mice were significantly reduced (P < 0.01), decreasing by 40.6% and 39.1%, respectively. Compared with the model group, there were no significant changes in serum hormone levels in the low-dose hippocampal peptide group, but the serum T and LH levels in the high-dose hippocampal peptide group were increased by 1.5 times, respectively (P < 0.01). These results indicate that hippocampal peptide can improve CTX-induced serum reproductive hormone disorders in mice.

[0045] Oxidative stress is one of the causes of testicular injury. Oxidative indicators were measured in each group. The results showed that, compared with the normal group, the model group had a 139.7% increase in testicular malondialdehyde (MDA) levels (P < 0.01) and a 50.2% decrease in testicular lactate dehydrogenase (LDH) levels (P < 0.01). Figure 4 As shown in Figure AB, the antioxidant system of the testes was severely damaged. Compared with the model group, the MDA levels in the testes of mice in the low-dose and high-dose hippocampal peptide groups decreased by 39% and 51%, respectively (P < 0.05), while the LDH levels increased by 1.2 and 1.8 times, respectively (P < 0.05). These results indicate that hippocampal peptide improved the level of CTX-induced oxidative stress in mouse testicular tissue, thereby alleviating reproductive damage in mice.

[0046] Example 2:

[0047] Sequence analysis of the expanded hippocampal peptides was performed based on the results of Example 1: The hippocampal peptide samples were desalted, with three copies of each sample. The SHP of the dialysis-desalted samples was analyzed using an UltiMate 3000 UHPLC system (Thermo Fisher Scientific) with an ACQUITY UPLC peptide CSH C18 column (2.1 × 100 mm, 1.7 μm) and a TripleTOF 5600+ system. The parameters were as follows: positive ion mode, spray voltage 3600 eV, capillary temperature 320 °C, and signal intensity threshold 1.6 eV. 5 The peptide sequences obtained from de novo sequencing were searched for hippocampal protein peptide sequences in the NCBI database. PeptideRanker was used to predict the bioactivity of the identified peptide sequences. Further screening identified one peptide, SGPINF, with a residual local confidence score greater than 90% and a PeptideRanker score greater than 0.85. Comparison with previously identified bioactive peptides in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / peptide_data.php) revealed that this single peptide sequence had not been previously reported and was therefore considered a novel bioactive peptide sequence. The results are shown in Table 3.

[0048] Table 3. Identification and activity prediction of peptide sequences from the distended hippocampus:

[0049] ;

[0050] a Source: PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0051] The aforementioned bioactive peptide from the distended hippocampus was biosynthesized by Shanghai Sangon Biotech Co., Ltd. using the Fmoc-peptide solid-phase synthesis method. HPLC and MS sequence analysis showed that the purity of the SGPINF peptide was greater than 98.89%. Chemically synthesized peptides, due to the controllability of raw materials and processes, exhibit high purity and good safety for clinical applications. However, the synthesis process involves numerous steps and uses a variety of solvents, which inevitably affects the bioactivity of the peptides. Therefore, further verification of the bioactivity of the synthesized peptides is necessary.

[0052] Mouse spermatogonial cell line GC-2 was cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged after reaching the logarithmic growth phase. The effect of the synthesized peptides on mouse GC-2 cell viability was detected using the CCK assay: After passage, GC-2 cells (3 × 10⁶ cells / year) were... 4 Cells were seeded at 100 μg / mL in 96-well plates and cultured for 24 h with different concentrations of SGPINF peptide (0, 25, 50, 100 μg / mL). 10 μL of CCK-8 reagent was added to each well, and the plates were cultured for another 4 h. The OD value of each well was measured at 490 nm, and the average OD value of all parallel wells was taken. Cell viability % = (OD of sampled cells - OD of blank cells) / (OD of control cells - OD of blank cells) × 100% (OD value of control wells at 0 μg / mL).

[0053] The results of the CCK-8 colorimetric method are as follows: Figure 5 As shown, different concentrations of the hippocampal active peptide sequence SGPINF significantly promoted the proliferation of mouse GC-2 cells (P < 0.05), with a dose-dependent effect. Compared with the control group, the 25 μg / mL peptide significantly increased cell viability by 10.9% (P < 0.05); while 50 and 100 μg / mL SGPINF showed the greatest increase in cell viability, increasing it by 33% and 28.9% respectively compared with the control group (P < 0.01), but there was no significant difference between the two concentrations, indicating that the synthesized pure peptide has a significant promoting effect on cell proliferation.

[0054] 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 bloating hippocampal peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the expanded hippocampal peptide according to claim 1 in the preparation of products that promote spermatogonial cell proliferation.

3. The application as described in claim 2, characterized in that: The concentration of the expanded hippocampal peptide in the product promoting spermatogonial cell proliferation is 50~100μg / mL.

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