Sea cucumber ovary peptide for alleviating ovary decline, preparation method and application thereof

Abstract

The application discloses a sea cucumber egg peptide for relieving ovary recession and a preparation method and application thereof, and belongs to the field of active peptides. The sea cucumber egg oligopeptide with improved ovary function is prepared by supercritical extraction combined with complex enzymolysis, has an amino acid sequence as shown in SEQ ID NO. 1, can improve the impaired state of E2, FSH and AMH hormone levels in ovary granulosa cells, and can improve the impairment of granulosa cells through antioxidation, and can be applied in female reproductive health improvement products, and has a wide prospect in the field of biological medicine.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptides, specifically relating to a sea cucumber egg peptide that alleviates ovarian decline and its preparation method. Background Technology

[0002] In recent years, with the continuous improvement of people's living standards, attention to women's reproductive health has also increased. Premature ovarian insufficiency (POI) refers to a condition in women under 40 years of age where ovarian function declines to a certain level. It is mainly characterized by menstrual irregularities such as amenorrhea, oligomenorrhea, or polymenorrhea, elevated gonadotropin levels (FSH>25U / L), and fluctuating estrogen levels. Premature ovarian failure (POF) is the final stage of ovarian function decline, referring to a condition in women under 40 years of age characterized by menstrual changes, infertility, estrogen deficiency, and elevated gonadotropin levels (FSH>40U / L) caused by various factors. Statistics show that the general incidence of POF in different populations worldwide is approximately 1.1%, while the prevalence in Chinese women is approximately 0.5%.

[0003] Clinically, hormone replacement therapy is mainly used to treat this condition and establish a normal menstrual cycle. The effect is immediate, but it cannot improve egg quality, activate the remaining follicle pool, or evaluate its improvement on ovarian function. Long-term use of hormone drugs can also produce certain side effects.

[0004] Sea cucumber eggs are rich in nutrients such as protein, with crude protein accounting for more than 50% of the dry weight. They are also rich in polysaccharides, phospholipids, unsaturated fatty acids and other active substances, making them an ideal raw material for preparing bioactive peptides.

[0005] Several invention patents have been issued regarding the preparation of bioactive peptides from sea cucumber eggs. For example, patent application CN118852346A discloses a bioactive polypeptide from sea cucumber, its preparation method, and its application. This patent prepares anti-inflammatory peptides through enzymatic hydrolysis, purifies them using ultrafiltration fractionation, identifies the peptide sequences by LC-MS / MS, and uses an anti-inflammatory peptide prediction website and molecular docking technology to screen for anti-inflammatory active fragments. Peptides with high scores are synthesized and their anti-inflammatory activity is verified in in vitro cell experiments.

[0006] Patent application CN117534726A discloses an active peptide from sea cucumber viscera, with the amino acid sequence ADDFYYQ. This patent uses sea cucumber flowers as raw material to obtain the amino acid sequence of the active peptide from sea cucumber viscera, and the prepared active peptide from sea cucumber viscera exhibits good ACE inhibitory activity.

[0007] However, there are no reports on developing bioactive peptide products related to female reproductive health using sea cucumber eggs. Studies have shown that water extracts of sea cucumber eggs can increase endometrial thickness in cyclophosphamide-induced mice, while also promoting ovarian development, increasing the number of follicles, and to some extent increasing serum estradiol and progesterone levels, thus playing a preventative role against premature ovarian failure. The water extract contains over 50% sea cucumber egg protein, indicating that sea cucumber egg protein has the potential to improve female ovarian function. Therefore, preparing sea cucumber egg peptides that improve female ovarian function is of great significance for developing novel female reproductive health products. Summary of the Invention

[0008] In order to address the problem of improving ovarian function, in a first aspect, according to some embodiments of this application, there is a sea cucumber egg oligopeptide with an amino acid sequence as shown in SEQ ID NO.1.

[0009] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of this application, including

[0010] S10. Add 0.2% to 0.4% (by volume) of a complex protease to the aqueous solution containing the extract residue of sea cucumber egg protein for enzymatic hydrolysis;

[0011] S20. Inactivate the enzyme in the enzyme hydrolysate, centrifuge to collect the supernatant, and perform membrane separation;

[0012] S30. The membrane solution was dried to obtain peptide powder, and the aqueous solution of the peptide powder was separated and purified using a Sephadex LH-20 column.

[0013] S40. Further purification was performed using a C18 column.

[0014] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of this application, the mass ratio of the complex protease is alkaline protease: trypsin: flavor protease = (3~5):(2~4):1, the enzymatic hydrolysis temperature is 37~45℃, the enzymatic hydrolysis pH is 8.0~9.0, and the enzymatic hydrolysis time is 3~5h.

[0015] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of this application, the molecular weight cutoff for membrane separation is 3 kDa.

[0016] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of this application, the sea cucumber egg oligopeptides were separated and purified using a Sephadex LH-20 column with a mobile phase of 25%~35% methanol and a flow rate of 0.5~1.0 mL / min. The absorbance of the eluent was measured at 280 nm, and the chromatographic peaks with a retention time of 15~16 min were collected.

[0017] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of this application, the mobile phase A of the C18 column is 0.07% trifluoroacetic acid water (v / v), the mobile phase B is acetonitrile, and the gradient elution conditions are: 0-10 min, 10% B; 10-15 min, 10%-15% B; 15-25 min, 15% B-30% B; 25-35 min, 30% B-35% B; the flow rate is 0.8-1.0 mL / min, the detection wavelength is 280 nm, and the chromatographic peak with a retention time of 13-14 min is collected to obtain sea cucumber egg oligopeptides.

[0018] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of this application, the Sephadex LH-20 column is a 3.0cm×100cm Sephadex LH-20 column; the C18 column is a 4.6mm×250mm, 5μm C18 column.

[0019] According to the method for preparing sea cucumber egg oligopeptides in some embodiments of this application, the aqueous solution containing the extract residue of sea cucumber egg protein in step S10 is a solution prepared by mixing the extract residue with deionized water in a ratio of 1:10.

[0020] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of this application, the enzyme inactivation temperature in step S20 is 90°C and the time is 15 min.

[0021] According to the method for preparing sea cucumber egg oligopeptides in some embodiments of this application, the centrifugation speed in step S20 is 8000 r / min.

[0022] According to the method for preparing sea cucumber egg oligopeptides in some embodiments of this application, the drying in step S30 is spray drying.

[0023] According to the method for preparing sea cucumber egg oligopeptides in some embodiments of this application, the concentration of peptide powder in the aqueous solution of peptide powder in step S30 is 100 mg / ml.

[0024] According to the method for preparing sea cucumber egg oligopeptides in some embodiments of this application, the preparation of the extract residue containing sea cucumber egg protein is achieved by the following method: crushing sea cucumber eggs, degreasing and deodorizing them, and retaining the extract residue containing sea cucumber egg protein.

[0025] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of this application, the sea cucumber eggs are fresh sea cucumber eggs, which are washed, freeze-dried, pulverized and passed through an 80-mesh sieve, and degreased and deodorized by CO2 supercritical extraction.

[0026] Sea cucumber egg oligopeptides prepared according to any of the preparation methods described in some embodiments of this application.

[0027] The application of sea cucumber egg oligopeptides in the preparation of food or functional food according to some embodiments of this application.

[0028] The use of sea cucumber oocyte oligopeptides according to some embodiments of this application in the preparation of medicaments for the prevention or treatment of premature ovarian failure or ovarian degeneration, or for increasing ovarian FSH (follicle-stimulating hormone) levels, or increasing ovarian E2 (estradiol) levels, or increasing ovarian AMH (anti-Müllerian hormone) levels, or increasing ovarian ROS (reactive oxygen species) levels.

[0029] Beneficial Effects: This invention uses sea cucumber eggs as raw material to obtain sea cucumber egg bioactive peptides that improve ovarian function. The amino acid sequence is Gly-Ser-Lys-Val-Thr-Thr-Gly, and a search of online bioactive peptide databases such as BIOPEP and EROP-Moscow confirms this sequence as a novel small-molecule bioactive peptide. This bioactive peptide improves the damaged levels of E2, FSH, and AMH hormones in ovarian granulosa cells and improves granulosa cell damage through antioxidant effects. It has broad prospects in the biomedical field and can be applied to products improving female reproductive health. Furthermore, this invention first uses supercritical fluid extraction to remove lipid components, and then hydrolyzes the obtained sea cucumber egg protein, which helps improve enzymatic hydrolysis efficiency and increase the yield of the bioactive peptides. Attached Figure Description

[0030] Figure 1 This is a comparison chart of E2 levels.

[0031] Figure 2 This is a comparison chart of FSH levels.

[0032] Figure 3 This is a comparison chart of AMH levels.

[0033] Figure 4 This is a comparison chart of ROS content. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments do not constitute a limitation on the scope of protection of the present invention.

[0035] The sea cucumber egg peptide of this invention, which alleviates ovarian decline, has the amino acid sequence Gly-Ser-Lys-Val-Thr-Thr-Gly. This invention utilizes sea cucumber eggs as raw material, and obtains sea cucumber egg oligopeptides that improve ovarian function through complex enzymatic hydrolysis and multi-stage purification. The method for preparing the above-mentioned sea cucumber egg oligopeptides includes:

[0036] S1. Fresh sea cucumber eggs are washed, freeze-dried, pulverized, and passed through a 70-90 mesh sieve, preferably an 80 mesh sieve. They are then degreased and deodorized using supercritical CO2 extraction, retaining the extract residue containing sea cucumber egg protein. Further, the sea cucumber is either a sea cucumber (spiny sea cucumber) or a spiny sea cucumber (spiny sea cucumber).

[0037] S2. Prepare an aqueous solution by mixing the extract residue from step S1 with deionized water at a ratio of 1:10. Add 0.2-0.4% of the solution volume of a complex protease. The mass ratio of the complex protease is alkaline protease: trypsin: flavor protease = 3-5:2-4:1. Enzymatically hydrolyze for 3-5 hours at a temperature of 37-45℃ and a pH of 8.0-9.0. Inactivate the enzyme at 90℃ for 15 minutes. Centrifuge at 8000 r / min to remove the residue and obtain the supernatant. Pass the supernatant through a 3 kDa ultrafiltration membrane, collect the permeate, and spray dry to obtain sea cucumber egg peptide powder.

[0038] S3. Dissolve sea cucumber ovopeptide powder in water to prepare a concentration of 100 mg / ml. Separate and purify the powder using Sephadex LH-20 column chromatography (3.0 × 100 cm). The mobile phase is 25-35% methanol, the flow rate is 0.5-1.0 mL / min, and the absorbance of the eluent is measured at 280 nm. Collect the desired peaks based on the absorbance values, and collect the chromatographic peaks with a retention time of 15-16 min.

[0039] S4. Further purification was performed using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Elite C18 column (4.6 mm × 250 mm, 5 μm); mobile phase A was 0.07% trifluoroacetic acid in water (v / v); mobile phase B was acetonitrile; gradient elution conditions were: 0–10 min, 10% B; 10–15 min, 10%–15% B; 15–25 min, 15% B–30% B; 25–35 min, 30% B–35% B; flow rate was 0.8–1.0 mL / min; detection wavelength was 280 nm; chromatographic peaks with retention times of 13–14 min were collected, concentrated, and freeze-dried to obtain sea cucumber egg active peptides.

[0040] The 3.0×100cm Sephadex LH-20 column indicates that the column has an inner diameter of 3.0cm, a length of 100cm, and uses Sephadex LH-20 packing material. The 4.6mm×250mm, 5μm C18 column indicates that the column has an inner diameter of 4.6mm, a length of 250mm, a particle size of 5μm, and uses C18 packing material.

[0041] Example 1. Preparation of sea cucumber ovary oligopeptide (SOO), the preparation method includes the following steps:

[0042] S1. Fresh sea cucumber eggs were washed, freeze-dried and pulverized, passed through an 80-mesh sieve, and degreased and deodorized using supercritical CO2 extraction (extraction vessel temperature 35℃, extraction pressure 25MPa, CO2 flow rate 20L / h, extraction time 3h), while retaining the extract residue containing sea cucumber egg protein.

[0043] S2. Take 100g of the extract residue from S1 and 1000mL of deionized water to prepare an aqueous solution. Add 2g of a complex protease (0.2% of the solution volume), which includes alkaline protease, trypsin, and flavor protease in a ratio of 3:2:1. Enzymatically hydrolyze the solution at 45℃ and pH 8.0 for 4 hours, inactivate the enzyme at 90℃ for 15 minutes, centrifuge at 8000r / min to remove the residue and obtain the supernatant. Pass the supernatant through a 3 kDa ultrafiltration membrane, collect the permeate, and spray dry to obtain sea cucumber egg peptide powder.

[0044] S3. Dissolve sea cucumber ovopeptide powder in water to prepare a concentration of 100 mg / ml. Separate and purify the powder using Sephadex LH-20 column chromatography (3.0 × 100 cm). The mobile phase is 30% methanol, the flow rate is 1.0 mL / min, and the absorbance of the eluent is measured at 280 nm. Collect the chromatographic peaks at 18-19 minutes.

[0045] S4. Further purification was performed using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Elite C18 column (4.6 mm × 250 mm, 5 μm); mobile phase A was 0.07% trifluoroacetic acid in water (v / v); mobile phase B was acetonitrile; gradient elution conditions were: 0–10 min, 10% B; 10–15 min, 10%–15% B; 15–25 min, 15% B–30% B; 25–35 min, 30% B–35% B; flow rate was 0.8–1.0 mL / min; detection wavelength was 280 nm; chromatographic peaks with retention times of 13–14 min were collected, concentrated, and freeze-dried to obtain sea cucumber egg active peptides.

[0046] The amino acid sequence of the active peptide obtained by high performance liquid chromatography-mass spectrometry was determined to be SEQ ID NO.1, with the amino acid sequence being Gly-Ser-Lys-Val-Thr-Thr-Gly and a molecular weight of 648.

[0047] Example 2. Preparation of Sea Cucumber Ovary Aqueous Extract (SOAE). The preparation method includes the following steps: Weigh a certain amount of sea cucumber eggs, homogenize them using a tissue homogenizer, add distilled water at a mass ratio of 1:3, extract using ultrasound-assisted extraction at 30℃ for 25 min, extract by stirring in a 30℃ water bath for 2 h, and centrifuge at 4000 r / min for 5 min, and collect the supernatant. Add distilled water to the precipitate at a mass ratio of 1:2, repeat the above operation, combine the supernatants, filter, concentrate the filtrate to 1 / 3 by rotary evaporation, and freeze-dry to obtain the sea cucumber egg aqueous extract.

[0048] Experimental description:

[0049] Experimental Example (1) Cell Experiment: An ovarian granulosa cell injury model was constructed by selecting human ovarian granulosa cells and dividing them into 4 groups, including a blank control group, a cisplatin model group, a sea cucumber egg active peptide group, and a sea cucumber egg water extract group. Human ovarian granulosa cells were completely cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum) and passaged at 37°C under 5% CO2 conditions. Logarithmic growth phase cells were taken and adjusted to a density of 2×10⁻⁶ cells. 5 100 μL / well was seeded into each well of a 96-well cell culture plate and cultured for 3–4 h. The cisplatin model group was treated with 20 μM cisplatin solution for 24 h. The sea cucumber egg active peptide group and the sea cucumber egg water extract group were treated with cisplatin solution followed by the addition of sea cucumber egg active peptide / sea cucumber egg water extract to a final concentration of 200 μg / mL, and cultured for another 24 h in a 5% CO2, 37℃ cell culture incubator. The supernatants from each group were collected, and the levels of FSH (follicle-stimulating hormone), E2 (estradiol), and AMH (anti-Müllerian hormone) in the supernatants were detected using an ELISA kit. The total intracellular ROS content was detected using a probe method, and the average optical density was calculated using ImageJ to analyze the ROS (reactive oxygen species) content.

[0050] Experimental Example (2) Animal Experiment: Rats were randomly divided into a control group (n=8) and a premature ovarian failure (POF) model group (n=24). The POF model group received an intraperitoneal injection of 120 mg / kg cyclophosphamide on day 1 and continued to be fed for 15 days. Blood was collected from the tail vein to detect sex hormone levels. Successful modeling was indicated by decreased AMH and E2 and increased FSH. The model rats were divided into a model group, a sea cucumber egg oligopeptide group, and a sea cucumber egg water extract group, with 8 rats in each group. The sea cucumber egg oligopeptide group and the sea cucumber egg water extract group were administered 20 g / kg by gavage once a day for 4 consecutive weeks. On the second day after the last administration, abdominal aortic blood was collected from each group of rats, and the levels of ovarian hormones FSH, E2, and AMH were detected by ELISA. Six rats from each group were sacrificed by cervical dislocation, and ovarian tissue was collected, fixed with paraformaldehyde, embedded, sectioned, and the number of atretic follicles was observed and recorded.

[0051] Experimental Example (1) Cell Experiment Results are as follows Figures 1-4 As shown, Figure 1 This is a comparison chart of the E2 level. Figure 2 This is a comparison chart of FSH levels. Figure 3 This is a comparison chart of AMH levels. Figure 4 The graph shows a comparison of ROS levels. Compared to the control group, the model group exhibited a significant increase in FSH levels and a decrease in E2 and AMH levels, indicating that cisplatin, a platinum-based chemotherapy drug, affected the secretory function of human ovarian granulosa cells, consistent with the hormonal levels observed in premature ovarian failure. After treatment with sea cucumber egg bioactive peptides and sea cucumber egg water extract, both E2 and AMH levels improved, while FSH levels decreased. Furthermore, the effect of sea cucumber egg bioactive peptides was superior to that of sea cucumber egg water extract, suggesting that sea cucumber egg bioactive peptides have an ameliorative effect on the recovery of hormone levels after ovarian function impairment. Simultaneously, the ROS level in the model group was significantly higher than that in the control group, indicating oxidative damage to cells. The ROS level in the cell group treated with sea cucumber egg bioactive peptides significantly decreased, indicating that the sea cucumber egg bioactive peptides cleared the excessive production of ROS.

[0052] The results of the animal experiment in Experiment Example (2) are shown in Table 1. Table 1 records the levels of FSH (follicle-stimulating hormone), E2 (estradiol), AMH (anti-Müllerian hormone) and the number of atretic follicles.

[0053] Table 1

[0054]

[0055] Compared with the control group, a P<0.05; compared with the model group, b P<0.05; compared with the SOO group, c P<0.05; compared with the SOAE group, d P<0.05

[0056] As shown in Table 1, similar to the results of the human ovarian cell experiment, the sea cucumber egg oligopeptide group exhibited a good effect in improving the levels of hormones related to ovarian function. Furthermore, the number of atretic follicles, an important indicator of ovarian function, was significantly increased in the model group rats compared to the control group, a condition also improved in the sea cucumber egg oligopeptide group. Among all indicators, the sea cucumber egg oligopeptide group showed better results than the sea cucumber egg water extract group.

[0057] Based on the above embodiments and experimental results, it can be seen that the sea cucumber egg oligopeptide of the present invention exhibits a good effect on improving ovarian function in both cell and animal experiments, and has good application prospects in the fields of biomedicine. Finally, it should be noted that the above examples are only a few specific embodiments of the present invention. All derivatives that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A low molecular weight oligopeptide from sea cucumber eggs, characterized in that, The amino acid sequence is shown as SEQ ID NO.

1.

2. The method for preparing the low molecular weight oligopeptide of sea cucumber egg as claimed in claim 1, characterized in that, Comprise S10. Adding complex protease solution of 0.2%-0.4% volume to the water solution of the extract residual phase containing sea cucumber egg protein for enzymolysis; S20. Inactivating the enzyme of the enzymolysis solution, centrifuging to obtain supernatant, and performing membrane separation; S30. Drying the membrane liquid to obtain peptide powder, and separating and purifying the water solution of the peptide powder by using Sephadex LH-20 chromatographic column; S40. Further purifying by using C18 chromatographic column; The mass ratio of the complex protease is alkaline protease: trypsin: flavor protease=(3-5):(2-4):1, the enzymolysis temperature is 37-45℃, the enzymolysis pH is 8.0-9.0, and the enzymolysis time is 3-5h; The molecular weight cut-off of the membrane separation is 3 kDa; The Sephadex LH-20 chromatographic column is used for separation and purification, the mobile phase is 25%-35% methanol, the flow rate is 0.5-1.0 mL / min, the absorbance of the eluent is measured at 280 nm, and the chromatographic peak with a retention time of 15-16 min is collected; The mobile phase A of the C18 chromatographic column is 0.07% trifluoroacetic acid water (v / v), the mobile phase B is acetonitrile, the gradient elution condition is: 0-10 min, 10% B; 10-15 min, 10%-15% B; 15-25 min, 15% B-30% B; 25-35 min, 30% B-35% B; the flow rate is 0.8-1.0 mL / min, the detection wavelength is 280 nm, the chromatographic peak with a retention time of 13-14 min is collected, and sea cucumber egg oligopeptide is obtained; The Sephadex LH-20 chromatographic column is a Sephadex LH-20 chromatographic column with a size of 3.0 cm*100 cm; and the C18 chromatographic column is a C18 chromatographic column with a size of 4.6 mm*250 mm and a particle size of 5 μm.

3. The preparation method according to claim 2, characterized in that, The water solution of the extract residual phase containing sea cucumber egg protein in step S10 is a solution prepared by the extract residual phase and deionized water in a ratio of 1:

10.

4. The preparation method according to claim 2, characterized in that, The inactivation temperature of the enzyme in step S20 is 90℃, and the time is 15 min.

5. The preparation method according to claim 2, characterized in that, The centrifugation speed in step S20 is 8000 r / min.

6. The preparation method according to claim 2, characterized in that, The drying in step S30 is spray drying.

7. The preparation method according to claim 2, characterized in that, The concentration of the peptide powder in the water solution of the peptide powder in step S30 is 100 mg / ml.

8. The preparation method according to claim 2, characterized in that, The preparation of the extract residual phase containing sea cucumber egg protein is realized by the following manner: crushing sea cucumber eggs, degreasing and deodorizing treatment, and leaving the extract residual phase containing sea cucumber egg protein.

9. The production method according to claim 8, characterized by, The sea cucumber eggs are fresh sea cucumber eggs, which are crushed and freeze-dried after washing, and are treated by CO2 supercritical extraction method for degreasing and deodorizing.

10. A sea cucumber egg oligopeptide prepared by the preparation method of any one of claims 2-9.

11. The use of the sea cucumber egg oligopeptide of claim 1 or 10 in the preparation of a drug for preventing or treating premature ovarian failure.

Images