Sea cucumber ovum peptide for relieving ovarian recession as well as preparation method and application thereof

Abstract

The invention relates to a sea cucumber ovum peptide for relieving ovarian recession and a preparation method and application thereof, and belongs to the field of active peptides, the sea cucumber ovum oligopeptide with the ovarian improving function is prepared through combination of supercritical extraction and composite enzymolysis, the amino acid sequence of the sea cucumber ovum oligopeptide is shown as SEQIDNO.1, and the sea cucumber ovum oligopeptide has the effects of improving the damaged state of E2, FSH and AMH hormone levels in ovarian granular cells and improving the ovarian recession. The damage of granular cells is improved through the anti-oxidation effect, and the compound can be applied to female reproductive health improvement products and has wide prospects in the fields of biological medicines and health foods.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of active peptides, and particularly relates to a sea cucumber egg peptide for relieving ovary recession and a preparation method thereof. BACKGROUND

[0002] In recent years, with the continuous improvement of people's living standards, the concern for women's reproductive health has also increased. Premature ovarian decline (POI) is a disease state when the ovary of a woman under the age of 40 declines to a certain extent, mainly manifested as menstrual disorders such as amenorrhea, oligomenorrhea or frequent menstruation, elevated gonadotropin levels (FSH > 25 U / L), and fluctuating estrogen levels. Premature ovarian failure (POF) is the end stage of ovarian function decline, which refers to a disease characterized by menstrual changes, infertility, estrogen deficiency, and elevated gonadotropin levels (FSH > 40 U / L) in women under the age of 40 due to various causes. According to statistics, the general incidence of this disease in different populations around the world is about 1.1% of women worldwide, and the prevalence of POF in Chinese women is about 0.5%. Clinically, hormone replacement therapy is mainly used for treatment to establish a normal menstrual cycle, with immediate effect, but it cannot improve egg quality, activate the remaining follicle bank, or evaluate the improvement of ovarian function. Long-term use of hormone drugs can also produce certain side effects. Sea cucumber eggs are rich in nutrients such as protein, with crude protein accounting for more than 50% of dry weight, and are also rich in polysaccharides, phospholipids, unsaturated fatty acids, and other active substances, making them an ideal raw material for preparing bioactive peptides. Sea cucumber eggs are usually processed into functional foods in the form of sea cucumber egg peptide powder, which exhibits excellent calcium and iron binding capacity and absorption promoting efficacy, as well as physiological activities such as antioxidant, antibacterial, antitumor, and immune enhancement.

[0003] Currently, there are some invention patents for preparing active peptides from sea cucumber eggs. For example, patent application CN118852346A discloses a sea cucumber active polypeptide and its preparation method and application. The patent prepared an anti-inflammatory polypeptide through enzymatic hydrolysis, purified by ultrafiltration fractionation, identified the polypeptide sequence by LC-MS / MS, and screened anti-inflammatory active fragments using an anti-inflammatory peptide prediction website and molecular docking technology. In vitro cell experiments verified the anti-inflammatory activity of the polypeptide with higher scores.

[0004] Patent application CN117534726A discloses a sea cucumber internal organ active peptide with the amino acid sequence ADDFYYQ. The patent uses sea cucumber flowers as raw materials to obtain the amino acid sequence of the sea cucumber internal organ active peptide, and the prepared sea cucumber internal organ active peptide has good ACE inhibitory activity.

[0005] However, there have been no reports on the development of active peptide products related to female reproductive health using sea cucumber eggs. Studies have shown that sea cucumber egg aqueous extracts can increase endometrial thickness in cyclophosphamide-induced mice, promote ovarian development, increase the number of follicles, and, to a certain extent, elevate serum estradiol and progesterone levels, potentially preventing and treating premature ovarian failure. The content of sea cucumber egg protein in the aqueous extract exceeds 50%, indicating that sea cucumber egg protein has the potential to improve female ovarian function. The preparation of sea cucumber egg peptides with the potential to improve female ovarian function is of great significance for the development of new female reproductive health products. Summary of the Invention

[0006] In order to solve the problem of improving ovarian function, in a first aspect, according to the sea cucumber egg oligopeptide in some embodiments of the present application, its amino acid sequence is shown as SEQ ID NO.1.

[0007] According to the preparation method of sea cucumber egg oligopeptide in some embodiments of the present application, the method comprises: S10. To the aqueous solution containing the remaining phase of the sea cucumber egg protein extract, 0.2% to 0.4% of the solution volume of the composite protease was added for enzymatic hydrolysis; S20. Inactivate the enzyme in the hydrolyzate, centrifuge, and collect the supernatant for membrane separation; S30 membrane liquid dried to obtain peptide powder, the peptide powder aqueous solution was separated and purified using SephadexLH-20 column; S40 was further purified using a C18 column.

[0008] According to the preparation method of sea cucumber egg oligopeptide in some embodiments of the present application, the mass ratio of the composite protease is alkaline protease: trypsin: flavor protease = (3~5): (2~4): 1, the enzymatic hydrolysis temperature is 37~45°C, the enzymatic hydrolysis pH is 8.0~9.0, and the enzymatic hydrolysis time is 3~5h.

[0009] According to the preparation method of sea cucumber egg oligopeptide in some embodiments of the present application, the molecular weight cut-off for membrane separation is 3 kDa.

[0010] According to the preparation method of sea cucumber egg oligopeptides in some embodiments of the present application, 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.

[0011] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, 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 the sea cucumber egg oligopeptide is obtained.

[0012] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, 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.

[0013] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, the aqueous solution of the extraction residual phase containing sea cucumber egg protein in step S10 is a solution prepared by mixing the extraction residual phase and deionized water at a ratio of 1:10.

[0014] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, the temperature for enzyme inactivation in step S20 is 90°C, and the time is 15 min.

[0015] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, the centrifugal speed in step S20 is 8000 r / min.

[0016] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, the drying in step S30 is spray drying.

[0017] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, the concentration of the peptide powder in the aqueous solution of the peptide powder in step S30 is 100 mg / ml.

[0018] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, the preparation of the extraction residual phase containing sea cucumber egg protein is realized by the following manner: the sea cucumber eggs are crushed, degreased and deodorized, and the extraction residual phase containing sea cucumber egg protein is obtained.

[0019] According to the preparation method of the sea cucumber egg oligopeptide in some embodiments of the present application, the sea cucumber eggs are fresh sea cucumber eggs, which are washed, freeze-dried, crushed and passed through an 80-mesh sieve, and then are degreased and deodorized by using a CO2 supercritical extraction method.

[0020] The sea cucumber egg oligopeptide prepared by any one of the preparation methods in some embodiments of the present application.

[0021] The application of the sea cucumber egg oligopeptide according to some embodiments of the application in the preparation of food or functional food.

[0022] The application of the sea cucumber egg oligopeptide according to some embodiments of the application in the preparation of a medicine for preventing or treating premature ovarian failure disease or ovarian decline disease or increasing the FSH (follicle stimulating hormone) content of the ovary or increasing the E2 (estradiol) content of the ovary or increasing the AMH (anti-Mullerian hormone) content of the ovary or increasing the ROS (reactive oxygen species) content of the ovary.

[0023] Beneficial effects: The sea cucumber egg active peptide with improved ovarian function is obtained by taking sea cucumber eggs as raw materials, and the amino acid sequence of the sea cucumber egg active peptide is Gly-Ser-Lys-Val-Thr-Thr-Gly. The sequence is a new small molecule active peptide after searching the online database BIOPEP and the active peptide database EROPE-Moscow. The active peptide has the effects of improving the impaired state of E2, FSH and AMH hormone levels in ovarian granulosa cells, and improving the damage of granulosa cells through antioxidant effect, and can be applied in female reproductive health improvement products, and has broad prospects in the fields of biological medicine and health food. In addition, the removal of lipid components by supercritical extraction and the hydrolysis of the obtained sea cucumber egg protein can help to improve the enzymatic efficiency and increase the yield of the active peptide. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is an E2 level comparison chart.

[0025] Figure 2 It is an FSH level comparison chart.

[0026] Figure 3 It is an AMH level comparison chart.

[0027] Figure 4 It is a ROS content comparison chart. DETAILED DESCRIPTION

[0028] The application will be further described below in combination with the drawings and examples, and it should be noted that the examples do not constitute a limitation on the scope of protection required by the application.

[0029] The sea cucumber egg peptide for relieving ovarian decline of the application has the amino acid sequence Gly-Ser-Lys-Val-Thr-Thr-Gly. The sea cucumber egg is used as raw material in the application, and a sea cucumber egg oligopeptide with improved ovarian function is obtained through complex enzymolysis and multi-stage purification. The method for preparing the sea cucumber egg oligopeptide according to the application comprises: S1. The fresh sea cucumber egg is washed, freeze-dried, crushed, and passed through a 70-90 mesh, preferably 80 mesh sieve. The sea cucumber egg is subjected to a defatting and deodorizing treatment using a CO2 supercritical extraction method, and the residual phase containing sea cucumber egg protein is retained. Further, the sea cucumber is one of Apostichopus japonicus or Stichopus japonicus.

[0030] S2. The residual phase in step S1 is mixed with deionized water to prepare an aqueous solution at a ratio of 1:10. A complex protease is added at a volume of 0.2-0.4% of the solution, and the mass ratio of the complex protease is alkaline protease: trypsin: flavor protease = 3-5: 2-4: 1. The enzyme is hydrolyzed at a temperature of 37-45℃ and a pH of 8.0-9.0 for 3-5 hours. The enzyme is inactivated at 90℃ for 15 minutes, and the supernatant is obtained by centrifugation at 8000 r / min to remove the residue. The permeate is collected by passing through a 3 kDa ultrafiltration membrane and is spray-dried to obtain sea cucumber egg peptide powder. S3. The sea cucumber egg peptide powder is dissolved in water to prepare a solution at a concentration of 100 mg / ml. The solution is separated and purified by a dextran gel Sephadex LH-20 column chromatography (3.0 x 100 cm) method. 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. The required peak is collected according to the absorbance value, and the chromatographic peak with a retention time of 15-16 min is collected.

[0031] S4. Further purification is performed by high performance liquid chromatography. The chromatographic conditions are as follows: an Yilite C18 chromatographic column (4.6 mm x 250 mm, 5 μm), a mobile phase A of 0.07% trifluoroacetic acid water (v / v), a mobile phase B of acetonitrile, a gradient elution condition of 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; a flow rate of 0.8-1.0 mL / min, a detection wavelength of 280 nm, and collection of the chromatographic peak with a retention time of 13-14 min. After concentration, the sea cucumber egg active peptide is obtained by freeze-drying.

[0032] Wherein, the Sephadex LH-20 chromatographic column of 3.0 x 100 cm indicates that the inner diameter of the chromatographic column is 3.0 cm, the length is 100 cm, and the Sephadex LH-20 filler. The C18 chromatographic column of 4.6 mm x 250 mm, 5 μm indicates that the inner diameter of the chromatographic column is 4.6 mm, the length is 250 mm, the particle size is 5 μm, and the C18 filler.

[0033] Example 1. Sea cucumber egg oligopeptide (Seacucumber Ovary Oligopeptide, SOO) is prepared by the following steps: S1. Fresh sea cucumber egg was washed, freeze-dried, crushed, and passed through an 80-mesh sieve. The sea cucumber egg was subjected to defatting and deodorizing treatment by CO2 supercritical extraction (extraction kettle temperature 35℃, extraction pressure 25 MPa, CO2 flow rate 20 L / h, extraction time 3 h), and the residual phase containing sea cucumber egg protein was reserved; S2. 100 g of the residual phase in S1 was mixed with 1000 mL of deionized water to prepare an aqueous solution, and 2 g of complex protease was added to the solution at a volume fraction of 0.2%. The complex protease included alkaline protease, trypsin, and flavor protease at a ratio of 3:2:1. Enzymolysis was performed at 45℃ and pH 8.0 for 4 h, and the enzyme was inactivated at 90℃ for 15 min. The supernatant was obtained by centrifugation at 8000 r / min, and the residue was removed. The supernatant was passed through a 3 kDa ultrafiltration membrane, and the permeate was collected and spray-dried to obtain sea cucumber egg peptide powder. S3. The sea cucumber egg peptide powder was dissolved in water to prepare a solution with a concentration of 100 mg / ml. The solution was separated and purified by dextran gel Sephadex LH-20 column chromatography (3.0×100 cm), and the mobile phase was 30% methanol at a flow rate of 1.0 mL / min. The absorbance of the eluate was measured at 280 nm, and the chromatographic peak collected at 18-19 min.

[0034] S4. Further purification was performed by high performance liquid chromatography (HPLC) under the following conditions: an Elite C18 column (4.6 mm×250 mm, 5 μm), mobile phase A: 0.07% trifluoroacetic acid water (v / v), mobile phase B: acetonitrile, gradient elution conditions: 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: 0.8-1.0 mL / min; detection wavelength: 280 nm. The chromatographic peak with a retention time of 13-14 min was collected, concentrated, and freeze-dried to obtain the sea cucumber egg active peptide.

[0035] The amino acid sequence of the sea cucumber egg active peptide obtained by high performance liquid chromatography-mass spectrometry (HPLC-MS) was determined to be SEQ ID NO. 1, the amino acid sequence was Gly-Ser-Lys-Val-Thr-Thr-Gly, and the molecular weight was 648.

[0036] Example 2. Sea cucumber egg aqueous extract (SOAE) was prepared by the following method: a certain amount of sea cucumber egg was weighed, crushed by a tissue homogenizer, and distilled water was added at a mass ratio of 1:3. Ultrasonic-assisted extraction was performed at 30℃ for 25 min, and extraction was performed at 30℃ for 2 h with stirring. The supernatant was obtained by centrifugation at 4000 r / min for 5 min. The precipitate was added with distilled water at a mass ratio of 1:2, and the above operation was repeated. The supernatants were combined, filtered, and concentrated by rotary evaporation to 1 / 3. The sea cucumber egg aqueous extract was obtained by freeze-drying.

[0037] Experimental illustration: Experimental example (1) cell experiment: the ovarian granulosa cell damage model is constructed, and the construction method is that human ovarian granulosa cells are selected and divided into four 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. The human ovarian granulosa cells are subcultured in a complete DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS (Fetal Bovine Serum) under the condition of 5% CO2 and 37°C. The logarithmic growth phase cells are adjusted to a density of 2×10 5 / ml, 100 μL per well is inoculated into a 96-well cell culture plate for adherent culture for 3-4 h. The cisplatin model group is treated with a 20 μM cisplatin solution for 24 h, and the sea cucumber egg active peptide group and the sea cucumber egg water extract group are treated with the sea cucumber egg active peptide / sea cucumber egg water extract after the cisplatin solution treatment, so that the final concentration is 200 μg / mL, and then the cells are continuously cultured in a 5% CO2 and 37°C cell incubator for 24 h. The supernatants of each group are collected, the content levels of FSH (follicle-stimulating hormone), E2 (estradiol) and AMH (anti-Mullerian hormone) in the supernatants are detected by an ELISA kit, the total ROS content in the cells is detected by a probe method, the average optical density is calculated by using Image J, and the ROS (reactive oxygen species) content is analyzed.

[0038] Experimental example (2) animal experiment: the rats are randomly divided into a control group of 8 and an ovarian premature aging rat model group of 24, wherein the ovarian premature aging rat model group is injected with 120 mg / kg cyclophosphamide in the abdominal cavity on the first day, and is continuously bred for 15 d, tail vein blood is collected, the sex hormone level is detected, and the successful modeling is marked by the decrease of AMH and E2 and the increase of FSH. The model rats are divided into a model group, a sea cucumber egg oligopeptide group and a sea cucumber egg water extract group, 8 in each group. The sea cucumber egg oligopeptide group and the sea cucumber egg water extract group are respectively administered by gavage at 20 g / kg once a day for 4 weeks. On the second day after the last administration, the abdominal aortic blood of the rats in each group is collected, and the ovarian hormone FSH, E2 and AMH levels are detected by an ELISA method; 6 rats in each group are executed by cervical dislocation, the ovarian tissues are collected, fixed with paraformaldehyde, embedded, sliced, and the number of atretic follicles is observed and recorded.

[0039] The cell experiment results of experimental example (1) are shown in Figures 1 to 4 , Figure 1 is a comparison chart of E2 levels, Figure 2 is a comparison chart of FSH levels, Figure 3 is a comparison chart of AMH levels, Figure 4For the ROS content comparison chart, compared with the control group, the FSH level of the model group increased significantly, and the E2 level and AMH level decreased, indicating that the platinum-based chemotherapy drug cisplatin affected the secretion function of human ovarian granulosa cells, which was consistent with the hormone level performance of premature ovarian failure. After the treatment of sea cucumber egg active peptide and sea cucumber egg water extract, the E2 and AMH levels were improved, and the FSH level decreased, and the effect of sea cucumber egg active peptide was better than that of sea cucumber egg water extract, indicating that sea cucumber egg active peptide had an improving effect on the recovery of hormone levels after ovarian function damage. At the same time, the ROS level in the model group was significantly higher than that in the control group, indicating that the cells were damaged by oxidation, and the ROS level in the cell group treated with sea cucumber egg active peptide decreased significantly, indicating that sea cucumber egg active peptide cleared the excessive production of ROS.

[0040] The animal experiment results of the experimental example (2) are shown in Table 1, and Table 1 records the content levels of FSH (follicle-stimulating hormone), E2 (estradiol), AMH (anti-Mullerian hormone) and the number of atretic follicles.

[0041] Table 1 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 As can be seen from Table 1, similar to the results of the human ovarian cell experiment, the sea cucumber egg oligopeptide group showed a good effect of improving the hormone levels related to ovarian function. In addition, as one of the important manifestations of ovarian function, the number of atretic follicles in the model group of rats increased significantly compared with the control group, and this situation was also improved in the sea cucumber egg oligopeptide group. Among the various indicators, the effect of the sea cucumber egg oligopeptide group was better than that of the sea cucumber egg water extract group.

[0042] From the above examples and experimental examples, it can be seen that the sea cucumber egg oligopeptide of the present application shows a good effect of improving ovarian function in cell experiments and animal experiments, and has a good application prospect in the fields of functional food, biological medicine, etc. Finally, it should be noted that the above enumeration is only a few specific embodiments of the present application. All derivatives that can be directly derived or inferred from the content disclosed by those skilled in the art should be considered as the protection scope of the present application.

Claims

1. A sea cucumber egg oligopeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. The method for preparing the sea cucumber egg oligopeptide according to claim 1, wherein include S10. To the aqueous solution containing the remaining phase of the sea cucumber egg protein extract, 0.2% to 0.4% of the solution volume of the composite protease was added for enzymatic hydrolysis; S20. Inactivate the enzyme in the hydrolyzate, centrifuge, and collect the supernatant for membrane separation; S30 membrane liquid dried to obtain peptide powder, the peptide powder aqueous solution was separated and purified using SephadexLH-20 column; S40 was further purified using a C18 column.

3. The preparation method according to claim 2, characterized in that The mass ratio of the composite protease is alkaline protease: trypsin: flavor protease = (3~5): (2~4): 1, the enzymatic hydrolysis temperature is 37~45°C, the enzymatic hydrolysis pH is 8.0~9.0, and the enzymatic hydrolysis time is 3~5h.

4. The preparation method according to claim 2, characterized in that The molecular weight cut-off for membrane separation was 3 kDa.

5. The preparation method according to claim 2, characterized in that Sephadex LH-20 column was used for separation and purification. The mobile phase was 25%~35% methanol at a flow rate of 0.5~1.0 mL / min. The absorbance of the eluent was measured at 280 nm, and the chromatographic peak with a retention time of 15~16 min was collected.

6. The preparation method according to claim 2, characterized in that The mobile phase A of the C18 chromatographic 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 the sea cucumber egg oligopeptide.

7. The preparation method according to claim 2, characterized in that The Sephadex LH-20 chromatographic column is a 3.0 cm × 100 cm Sephadex LH-20 chromatographic column; the C18 chromatographic column is a 4.6 mm × 250 mm, 5 μm C18 chromatographic column.

8. The preparation method according to claim 2, characterized in that Preferably, the aqueous solution containing the residual phase of the extraction of sea cucumber egg protein in step S10 is a solution prepared by mixing the residual phase of the extraction with deionized water in a ratio of 1:10; Preferably, the temperature for inactivating the enzyme in step S20 is 90° C. and the time is 15 min; Preferably, the centrifugal speed in step S20 is 8000 r / min; Preferably, the drying in step S30 is spray drying; Preferably, the concentration of the peptide powder in the aqueous solution of the peptide powder in step S30 is 100 mg / ml; Preferably, the preparation of the residual phase containing sea cucumber egg protein is achieved by: crushing the sea cucumber eggs, defatting and removing the fishy smell, and retaining the residual phase containing sea cucumber egg protein; More preferably, the sea cucumber eggs are fresh sea cucumber eggs, which are freeze-dried, crushed and passed through an 80-mesh sieve after cleaning, and are defatted and deodorized using a CO2 supercritical extraction method.

9. A sea cucumber egg oligopeptide prepared by the preparation method according to any one of claims 2 to 8.

10. Use of the sea cucumber egg oligopeptide according to claim 1 or 9 in the preparation of food or functional food; or use of the sea cucumber egg oligopeptide according to claim 1 or 9 in the preparation of a drug for preventing or treating premature ovarian failure or ovarian decline, or for increasing the ovarian FSH (follicle-stimulating hormone) content, or increasing the ovarian E2 (estradiol) content, or increasing the ovarian AMH (anti-Mullerian hormone) content, or increasing the ovarian ROS (reactive oxygen species) content.

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