Sea cucumber ovum oligopeptide composition for increasing female AMH value and improving ovarian function and application of sea cucumber ovum oligopeptide composition

Abstract

The invention relates to a sea cucumber ovum oligopeptide composition for increasing female AMH value and improving ovarian function and application of the sea cucumber ovum oligopeptide composition, and belongs to the field of medicines, and the sea cucumber ovum oligopeptide composition comprises, by weight, 15-25 parts of sea cucumber ovum oligopeptide; 10 to 15 parts of mussel oligopeptide; and 10-20 parts of a polyphenol extract with homology of medicine and food. The sea cucumber ovum oligopeptide has the effects of improving the levels of hormones such as AMH, improving the energy metabolism and oxidative stress levels of oocytes and ovarian granular cells and promoting the proliferation of ovarian stem cells. The medicinal and edible polyphenol extract has the effects of reducing chronic ovarian cell inflammation, improving ovarian tissue blood supply, increasing uterus visceral organ coefficient and relieving ovarian immune injury, and compared with direct intake of an existing traditional Chinese medicine formula, the medicinal and edible polyphenol extract has the advantage that the absorption rate is remarkably improved. All the components improve the ovarian function from different aspects, the ovarian reserve improving effect better than that of single or few components is achieved under the synergistic effect of all the components, and the composition is safe and free of toxic and side effects.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a sea cucumber egg oligopeptide composition and its application for increasing female AMH values ​​and improving ovarian function. Background Art

[0002] Ovarian function gradually declines with age, especially after age 35, when the decline accelerates. This is manifested by decreased anti-Müllerian hormone (AMH) levels, diminished ovarian reserve, and imbalances in estrogen and progesterone secretions, leading to a range of symptoms such as menstrual irregularities, decreased fertility, hot flashes, night sweats, and loose skin. AMH, secreted by the granulosa cells of the preantral and small antral follicles in the ovaries, is a key indicator of ovarian reserve. A decrease in AMH levels is closely associated with a decrease in the number and quality of ovarian follicles. Ovarian decline not only affects women's reproductive health but also has a significant impact on their physical and mental health and quality of life.

[0003] Currently, research on female ovarian failure is increasing, but most studies focus on only a few indicators, including the six sex hormones, follicle count, uterine organ index, and ovarian blood flow. No products are currently available that comprehensively improve these indicators. Clinical treatments for female ovarian failure are limited, primarily relying on hormone replacement therapy, which carries certain risks. For example, hormone replacement therapy can increase the risk of breast cancer, endometrial cancer, and other diseases, and long-term use can cause side effects. While there are a number of products that use traditional Chinese medicine formulas to nourish yin and tonify the kidneys to improve ovarian decline, the specific active ingredients are still unclear and their absorption rates are poor. For example, Chinese patent publication number CN101919963A discloses a traditional Chinese medicine composition, its preparation method, and its use. This composition includes the following ingredients: Ligustrum lucidum fruit, Morus alba fruit, Cistanche deserticola, Salvia miltiorrhiza, Cyathula officinalis, roasted Cyperus rotundus, White Peony Root, Anemarrhena asphodeloides, and Lilium bulbs. For example, Chinese patent publication number CN102274468A discloses a traditional Chinese medicine composition for regulating female endocrine function and its preparation method. This composition is made from Astragalus membranaceus, Angelica sinensis, Codonopsis pilosula, Ligusticum chuanxiong, Epimedium, Patrinia patriniae, Licorice root, Curcuma aromatica, Polygala tenuifolia, and Evodia rutaecarpa. Therefore, developing safe and effective products to improve female ovarian function is of great practical significance.

[0004] Sea cucumber eggs are rich in various nutrients, including protein, polysaccharides, and fatty acids. Studies have found that enzymatic hydrolysis of sea cucumber eggs into oligopeptides reveals excellent biological activity and absorbability. Furthermore, some plant extracts and functional nutrients have demonstrated benefits in endocrine regulation, antioxidant activity, and cell metabolism. Summary of the Invention

[0005] In order to solve the problems related to female ovarian failure, the present invention provides a sea cucumber egg oligopeptide composition and its application for increasing female AMH value and improving ovarian function.

[0006] The present invention provides a sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function, which has a good effect on improving hormone endocrine levels (especially AMH), follicle number, uterine organ index, ovarian blood flow, etc., and there is no recurrence in the short term after discontinuation of use.

[0007] The technical solutions adopted by the present invention to solve the technical problems are as follows: The present invention provides a sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function, comprising the following components in parts by weight: 15-25 parts of sea cucumber egg oligopeptide; 10-15 parts of mussel oligopeptides; 10-20 parts of edible and medicinal polyphenol extracts.

[0008] As a preferred embodiment, the preparation method of the sea cucumber egg oligopeptide is: Step S1: fresh sea cucumber eggs are washed, freeze-dried, and crushed, sieved, and then subjected to a CO2 supercritical extraction method for defatting and removing fishy smell, retaining the residual phase containing sea cucumber egg protein; Step S2: the residual phase of the extraction and deionized water are mixed uniformly at a volume ratio of 1:10-20 to prepare an aqueous solution, 0.2%-0.4% of the solution volume of a composite protease is added, and enzymatic hydrolysis is carried out at 37°C-45°C and pH 8.0-9.0 for 3h-5h, the enzyme is inactivated at 80°C-90°C for 10-15 minutes, and the residue is removed by centrifugation at 6000 r / min-10000 r / min to obtain a supernatant, which is filtered through an ultrafiltration membrane and the permeate is collected, and the sea cucumber egg peptide powder is obtained after spray drying; Step S3: Dissolve the sea cucumber egg peptide powder in water to prepare a solution with a concentration of 10 mg / ml-100 mg / ml, separate and purify it by chromatography, measure the absorbance of the eluate at 280 nm, collect the chromatographic peak at 16 min-17 min, concentrate and freeze-dry to obtain the sea cucumber egg oligopeptide.

[0009] As a preferred embodiment, the sea cucumber is Apostichopus japonicus or Apostichopus japonicus.

[0010] As a preferred embodiment, the composite protease consists of alkaline protease, trypsin, and flavor protease, and the mass ratio of the alkaline protease, trypsin, and flavor protease is (3-5): (2-4):1.

[0011] As a preferred embodiment, in the chromatography method, a Sephadex LH-20 column is used, the mobile phase is 25%-35% methanol, and the flow rate is 0.5 mL / min-1.0 mL / min.

[0012] As a preferred embodiment, the edible and medicinal polyphenol extract comprises the following components in parts by weight: 6-12 parts of angelica; 5-10 parts of Pueraria root; 5-10 parts dandelion; 5-10 parts of Eucommia ulmoides leaves; 5-10 parts of dried orange peel.

[0013] As a preferred embodiment, the preparation method of the edible and medicinal polyphenol extract is as follows: Step S1: raw material pretreatment; Select angelica, kudzu root, dandelion, eucommia leaf and red orange peel according to weight, wash and dry them, and then grind them to obtain raw material powder with a particle size of 20-40 mesh; Step S2: membrane-assisted enzymatic extraction; The raw material powder is mixed with citric acid-sodium citrate buffer solution at a material-liquid ratio of (1g:10 mL) to (1g:20 mL), and a composite enzyme is added at 0.5%-1.5% of the weight of the raw material powder. Enzymatic hydrolysis is carried out at 40°C-50°C for 1-2 hours. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is filtered through an ultrafiltration membrane with a molecular weight cutoff of 5000Da-10000Da to remove macromolecular impurities to obtain a preliminary extract. Step S3: aqueous two-phase extraction and enrichment; ammonium sulfate and polyethylene glycol are added to the primary extract to achieve a mass concentration of ammonium sulfate of 15% to 25% and a mass concentration of polyethylene glycol of 10% to 20%, and the mixture is thoroughly stirred to form a two-phase aqueous system; the two-phase aqueous system is allowed to stand at 20°C to 30°C for 30 to 60 minutes to allow polyphenols to accumulate in the upper phase; and the upper phase is separated to obtain a polyphenol-enriched solution; Step S4: freeze drying; The concentrated polyphenol enriched liquid is freeze-dried to obtain dry powder of the medicinal and edible polyphenol extract.

[0014] As a preferred embodiment, the complex enzyme consists of cellulase and pectinase, and the mass ratio of the cellulase to the pectinase is 1:1-3.

[0015] The present invention provides a use of a sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function in the preparation of a drug for improving female ovarian function.

[0016] As a preferred embodiment, the functions of the sea cucumber egg oligopeptide composition include: (1) improving the hormone secretion level of ovarian granulosa cells; (2) improving the energy metabolism and oxidative stress level of oocytes and ovarian granulosa cells; (3) promoting the proliferation of ovarian stem cells; (4) reducing chronic inflammation of ovarian cells; (5) improving blood supply to ovarian tissue; (6) increasing the organ coefficient of the uterus; (7) relieving immune damage to the ovary.

[0017] The beneficial effects of the present application are: 1) In the sea cucumber egg oligopeptide composition for improving female AMH values and ovarian function provided by the present application, the sea cucumber egg oligopeptide has the effects of improving hormone levels such as AMH, improving the energy metabolism and oxidative stress level of oocytes and ovarian granulosa cells, and promoting the proliferation of ovarian stem cells (OSCs). From the three dimensions of cell energy supply, structure protection, and reserve regeneration, the function of follicle generation-development-ovulation-hormone secretion of the ovary is improved, not only solving the symptoms of ovarian function decline (hormone disorder, symptoms discomfort), but also improving the root cause (insufficient follicle reserve, cell function damage), which is suitable for people with premature ovarian failure and decreased ovarian reserve function.

[0018] 2) In the sea cucumber egg oligopeptide composition for improving female AMH values and ovarian function provided by the present application, the pharmacognosy polyphenol extract has the effects of reducing chronic inflammation of ovarian cells, improving blood supply to ovarian tissue, increasing the organ coefficient of the uterus, relieving immune damage to the ovary, and reducing oxidative damage, and the absorption rate of the pharmacognosy polyphenol extract is significantly improved compared with directly taking existing traditional Chinese medicine prescriptions.

[0019] 3) In the sea cucumber egg oligopeptide composition for improving female AMH values and ovarian function provided by the present application, each component improves ovarian function from different aspects. After the synergy of angelica polyphenols and mussel oligopeptides, the blood supply to the ovary is improved, the synergy of eucommia leaf polyphenols and sea cucumber egg oligopeptides promotes the proliferation of OSCs, the synergy of wampee polyphenols and sea cucumber egg oligopeptides promotes AMH secretion, etc. The synergy of each component produces better ovarian reserve improvement effect than taking single or fewer components, and has no toxic side effects. DETAILED DESCRIPTION

[0020] The sea cucumber egg oligopeptide composition for improving female AMH values and ovarian function provided by the present application includes the following components in weight parts: Sea cucumber egg oligopeptide 15-25 parts; 10-15 parts of mussel oligopeptides; 10-20 parts of edible and medicinal polyphenol extracts.

[0021] Preferably, the sea cucumber egg oligopeptide composition comprises the following components in parts by weight: 25 parts of sea cucumber egg oligopeptide; 15 parts of mussel oligopeptides; 20 portions of polyphenol extracts of medicinal and edible origin.

[0022] Preferably, the edible and medicinal polyphenol extract comprises the following components in parts by weight: 6-12 parts of angelica; 5-10 parts of Pueraria root; 5-10 parts dandelion; 5-10 parts of Eucommia ulmoides leaves; 5-10 parts of dried orange peel.

[0023] More preferably, the edible and medicinal polyphenol extract comprises the following components in parts by weight: 12 parts of angelica; 10 parts of Pueraria root; 10 parts dandelion; 10 parts of Eucommia ulmoides leaves; 10 parts of dried orange peel.

[0024] Among them, the effects of each component of the polyphenol extracts of medicinal and edible origin are as follows: Angelica sinensis has the effect of promoting blood circulation and unblocking meridians. Its polyphenolic components, such as ferulic acid and ligustilide, can inhibit the NF-κB inflammatory pathway in ovarian tissue, reduce TNF-α and IL-6 levels, improve local blood perfusion, and provide sufficient nutrition for follicular development. Furthermore, the present invention found that when combined with mussel oligopeptides, this effect of improving ovarian blood supply is further enhanced.

[0025] Pueraria root has the effect of endocrine regulation: it contains isoflavones such as puerarin, which can simulate the activity of weak estrogen, directly act on ovarian granulosa cells, promote E2 secretion, inhibit FSH receptor desensitization, and enhance the stimulating effect of gonadotropin on follicles.

[0026] Dandelion has anti-inflammatory and immunomodulatory effects: it contains polyphenols such as inulin and chlorogenic acid, which can inhibit the overactivation of ovarian macrophages, reduce the release of IL-1β, and alleviate autoimmune ovarian damage.

[0027] Eucommia leaf: The present invention found that the synergistic effect of Eucommia leaf polyphenols and sea cucumber egg oligopeptides can enhance the effect of promoting OSCs proliferation and increase the follicle reserve.

[0028] Exocarpium Citri Grandis: The present application finds that the polyphenols in Exocarpium Citri Grandis have the effect of increasing uterine organ index, and can improve the effect of promoting AMH secretion in cooperation with sea cucumber egg oligopeptide, and more effectively improve the hormone level.

[0029] Preferably, the preparation method of the sea cucumber egg oligopeptide is as follows: Step S1: The fresh sea cucumber eggs are cleaned, freeze-dried, crushed, and passed through an 80-mesh sieve, and then subjected to defatting and deodorizing treatment by CO2 supercritical extraction, and the residual phase containing sea cucumber egg protein is retained.

[0030] Preferably, the sea cucumber is Apostichopus japonicus or Stichopus japonicus.

[0031] Step S2: The residual phase obtained above and deionized water are mixed uniformly at a volume ratio of 1:10-20 to prepare an aqueous solution, and 0.2%-0.4% of a complex protease (prepared by mixing alkaline protease, trypsin, and flavor protease at a mass ratio of (3-5):(2-4):1) is added to the solution, and then subjected to enzymolysis at 37°C-45°C and pH 8.0-9.0 for 3h-5h, and then subjected to enzyme inactivation at 80°C-90°C for 10 minutes-15 minutes, and then subjected to centrifugation at 6000 r / min-10000 r / min to remove the residue and obtain the supernatant, and then subjected to filtration through a 3 kDa ultrafiltration membrane and collection of the permeate, and then subjected to spray drying to obtain sea cucumber egg peptide powder.

[0032] Step S3: The sea cucumber egg peptide powder is dissolved in water to prepare a solution with a concentration of 10mg / ml-100mg / ml, and then subjected to separation and purification by Sephadex LH-20 column chromatography (3.0x100cm) with a mobile phase of 25%-35% methanol at a flow rate of 0.5mL / min-1.0mL / min, and then subjected to determination of absorbance of the eluate at 280nm, and then subjected to collection of the chromatographic peak at 16min-17min, and then subjected to freeze-drying after concentration to obtain sea cucumber egg oligopeptide.

[0033] Preferably, the preparation method of the polyphenol extract of food and medicine is as follows: Step S1: Pretreatment of raw materials; The raw materials (Angelica sinensis, Pueraria lobata, Taraxacum mongolicum, Eucommia ulmoides leaf, and Exocarpium Citri Grandis) are selected according to weight fraction, washed, dried, and then subjected to crushing treatment to obtain raw material powder with a particle size of 20-40 mesh; Step S2: Membrane-assisted enzymatic extraction; The raw material powder is mixed with a citric acid-sodium citrate buffer solution (pH 4.5-5.5) at a material-liquid ratio of (1 g:10 mL) to (1 g:20 mL), and a composite enzyme (composed of cellulase and pectinase in a mass ratio of 1:1-3) is added at a concentration of 0.5%-1.5% by mass of the raw material powder. The mixture is enzymatically hydrolyzed at 40°C-50°C for 1-2 hours. After the enzymatic hydrolysis is completed, the hydrolyzate is filtered through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da-10000 Da to remove macromolecular impurities, thereby obtaining a preliminary extract. Step S3: aqueous two-phase extraction and enrichment; ammonium sulfate and polyethylene glycol (PEG-4000) are added to the primary extract to a concentration of 15% to 25% ammonium sulfate and 10% to 20% polyethylene glycol, and the mixture is thoroughly stirred to form a two-phase aqueous system. The two-phase aqueous system is allowed to stand at 20°C to 30°C for 30 to 60 minutes to allow polyphenols to accumulate in the upper phase (PEG phase). The upper phase is separated to obtain a polyphenol-enriched solution. Step S4: freeze drying; The concentrated polyphenol enriched liquid is placed in a freeze dryer for freeze drying to obtain dry powder of the medicinal and edible polyphenol extract.

[0034] The present invention also provides a use of a sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function in the preparation of a drug for improving female ovarian function.

[0035] Preferably, the functions of the sea cucumber egg oligopeptide composition mainly include: (1) Improve the hormone secretion level of ovarian granulosa cells; (2) Improve the energy metabolism and oxidative stress levels of oocytes and ovarian granulosa cells; (3) Promote the proliferation of ovarian stem cells; (4) Reduce chronic inflammation of ovarian cells; (5) Improve blood supply to ovarian tissue; (6) Increase the uterine organ coefficient; (7) Alleviate ovarian immune damage.

[0036] The present invention finds that sea cucumber egg oligopeptides and edible and medicinal polyphenol extracts can improve ovarian function from different aspects, and the components work together to produce an effect that is better than directly taking a traditional Chinese medicine prescription.

[0037] Preferably, the preparation method of the drug for improving female ovarian function is as follows: The above ingredients are weighed according to the ratio and mixed to ensure that they are fully and evenly dispersed. The preparation is further formed according to the different dosage form requirements (such as tablets, capsules, granules, etc.).

[0038] Specifically, when preparing tablets, appropriate amounts of binders (such as hydroxypropyl methylcellulose), lubricants (such as magnesium stearate) and other auxiliary materials can be added to the mixed material, and the mixture can be pressed into tablets after granulation and drying.

[0039] Specifically, when preparing capsules, the uniformly mixed materials can be directly filled into the capsule shell.

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] Example 1 Preparation of Sea Cucumber Egg Oligopeptide

[0042] (1) Fresh sea cucumber eggs were washed, freeze-dried, and crushed, passed through an 80-mesh sieve, and subjected to a CO2 supercritical extraction process for degreasing and deodorization, retaining the residual phase containing sea cucumber egg protein; (2) The above-obtained extraction residual phase was mixed with deionized water in a volume ratio of 1:10 to prepare an aqueous solution, and a composite protease (the mass ratio of alkaline protease, trypsin, and flavor protease was 3:2:1) of 0.3% by volume of the solution was added. The mixture was enzymatically hydrolyzed at 40°C and pH 8.0 for 4 h, and the enzyme was inactivated at 90°C for 15 min. The residue was removed by centrifugation at 8000 r / min to obtain a supernatant, which was filtered through a 3 kDa ultrafiltration membrane and the permeate was collected. The sea cucumber egg peptide powder was obtained after spray drying. (3) The sea cucumber egg peptide powder was dissolved in water and prepared into a solution with a concentration of 100 mg / ml. The solution was separated and purified by Sephadex LH-20 column chromatography (3.0×100 cm). The mobile phase was 35% methanol, the flow rate was 1.0 mL / min, and the absorbance of the eluent was measured at 280 nm. The chromatographic peak at 16-17 min was collected according to the absorbance value. After concentration, the solution was freeze-dried to obtain sea cucumber egg oligopeptide (SCOP).

[0043] Example 2 Preparation of edible and medicinal polyphenol extracts (aqueous two-phase extraction method)

[0044] Step S1: raw material pretreatment; Select the medicinal and edible raw materials (12 parts of angelica, 10 parts of kudzu root, 10 parts of dandelion, 10 parts of eucommia leaf, 10 parts of tangerine peel) according to weight, wash and dry them, and then grind them to obtain a raw material powder with a particle size of 20 mesh; Step S2: membrane-assisted enzymatic extraction; The raw material powder was mixed with a citric acid-sodium citrate buffer solution with a pH value of 4.5 at a solid-liquid ratio of 1:10 (g / mL), and a composite enzyme (composed of cellulase and pectinase at a mass ratio of 1:1) was added at 1.5% by mass of the raw material powder. The mixture was enzymatically hydrolyzed at 50°C for 2 hours. After the enzymatic hydrolysis was completed, the hydrolyzate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da to remove macromolecular impurities, thereby obtaining a preliminary extract. Step S3: aqueous two-phase extraction and enrichment; Ammonium sulfate and polyethylene glycol (PEG-4000) were added to the primary extract to a concentration of 25% ammonium sulfate and 20% polyethylene glycol, respectively. The mixture was thoroughly stirred to form a two-phase aqueous system. The two-phase aqueous system was allowed to stand at 30°C for 60 minutes to allow polyphenols to accumulate in the upper phase (PEG phase). The upper phase was separated to obtain a polyphenol-enriched solution. Step S4: freeze drying; The concentrated polyphenol enriched liquid is placed in a freeze dryer for freeze drying to obtain a medicine-food polyphenol extract dry powder (PE).

[0045] Example 3 Preparation of edible and medicinal water extract (conventional water extraction method)

[0046] The edible and medicinal ingredients (12 parts Angelica sinensis, 10 parts Pueraria root, 10 parts Taraxacum mongolicum, 10 parts Eucommia ulmoides leaves, and 10 parts Citrus aurantium) were weighed according to their weight, washed, air-dried, and then pulverized to obtain a 20-mesh powder. 100 g of the powder was added to 1500 mL of purified water and heated at 90°C for 1.5 hours, followed by filtration. The residue was extracted once with 1000 mL of purified water, and the two filtrates were combined. The filtrate was centrifuged (4000 rpm for 10 minutes), and the supernatant was freeze-dried to obtain the edible and medicinal water extract (WE) powder.

[0047] Example 4 Preparation of Angelica Sinensis Polyphenol Extract (ASPE), Eucommia ulmoides Leaf Polyphenol Extract (EULPE), and Citrus aurantium Polyphenol Extract (ECGPE) (Aqueous Two-Phase Extraction Method)

[0048] According to the preparation method of Example 2, Angelica sinensis polyphenol extract (ASPE), Eucommia ulmoides leaf polyphenol extract (EULPE) and Citrus aurantium polyphenol extract (ECGPE) were prepared respectively.

[0049] Example 5 Preparation of Sea Cucumber Egg Oligopeptide Composition for Increasing Female AMH Values ​​and Improving Ovarian Function

[0050] Sea cucumber egg oligopeptide (Example 1), mussel oligopeptide, edible and medicinal polyphenol extract (Example 2), and edible and medicinal water extract (Example 3) were mixed according to the ratio in Table 1 to ensure that the components were fully and evenly dispersed to obtain composite powders SCOPMIX1-SCOPMIX5.

[0051] Table 1

[0052] Example 6 Mouse ovarian stem cell (OSCs) proliferation experiment Mouse ovarian stem cells (OSCs) were cultured at a rate of 1 × 10 4 Cells were seeded into 96-well plates at a density of cells / well and cultured for 24 hours until adherence.

[0053] The experimental groups were divided into a control group (DMEM / F12 basal medium alone), a SCOP group (SCOP was added to DMEM / F12 basal medium to a SCOP concentration of 20 μg / mL), an EULPE group (EULPE was added to DMEM / F12 basal medium to a SCOP concentration of 20 μg / mL), a SCOP+EULPE1 group (SCOP final concentration of 20 μg / mL, EULPE final concentration of 5 μg / mL), a SCOP+EULPE2 group (SCOP final concentration of 20 μg / mL, EULPE final concentration of 10 μg / mL), and a SCOP+EULPE3 group (SCOP final concentration of 20 μg / mL, EULPE final concentration of 20 μg / mL). After adding samples to each group, cells were incubated at 37°C for 24 hours. OD values ​​at 450 nm were measured using a microplate reader using the CCK-8 assay. The cell proliferation rate of each group was calculated using the following formula:

[0054] The results are shown in Table 2. Compared with the control group, the number of mouse OSCs in the SCOP group, the SCOP+EULPE1 group, the SCOP+EULPE2 group, and the SCOP+EULPE3 group all increased significantly, while the EULPE group showed no significant change. Furthermore, the proliferation rates of the SCOP+EULPE2 and SCOP+EULPE3 groups were significantly higher than those of the SCOP group. This indicates that the sea cucumber egg oligopeptide composition prepared by the present invention has the effect of promoting the proliferation of mouse ovarian stem cells (OSCs), and this effect is further enhanced in the presence of a certain concentration of Eucommia leaf polyphenol extract (EULPE).

[0055] Table 2

[0056] Example 7 Human ovarian granulosa cell injury intervention experiment Human ovarian granulosa cells were selected and cultured (the cells were seeded in T25 culture flasks with DMEM / F12 culture medium and cultured in a 37°C, 5% CO2, saturated humidity incubator). The cultured cells were divided into a blank control group (only DMEM / F12 basal culture medium (without any damaging agent or intervention drug) was added and cultured until the end of the experiment), a cisplatin model group (cisplatin was diluted with DMEM / F12 basal culture medium to a final concentration of 5-20 μM cisplatin working solution, and cultured in an incubator for 24 hours), a SCOPMIX1 group (composition powder SCOPMIX1 was added to DMEM / F12 basal culture medium to make the concentration of composition powder SCOPMIX1 in the culture medium to be 20 μg / mL), a SCOPMIX2 group (composition powder SCOPMIX2 was added to DMEM / F12 basal culture medium to make the concentration of composition powder SCOPMIX2 in the culture medium to be 20 μg / mL), and a SCOPMIX3 group (DMEM / F12 The following groups were used: SCOPMIX3 group (composition powder SCOPMIX3 was added to the basal culture medium so that the concentration of composition powder SCOPMIX3 in the culture medium was 20 μg / mL), SCOPMIX4 group (composition powder SCOPMIX4 was added to the DMEM / F12 basal culture medium so that the concentration of composition powder SCOPMIX4 in the culture medium was 20 μg / mL), SCOPMIX5 group (composition powder SCOPMIX5 was added to the DMEM / F12 basal culture medium so that the concentration of composition powder SCOPMIX5 in the culture medium was 20 μg / mL), SCOP group (SCOP was added to the DMEM / F12 basal culture medium so that the concentration of SCOP in the culture medium was 20 μg / mL), ECGPE group (ECGPE was added to the DMEM / F12 basal culture medium so that the concentration of ECGPE in the culture medium was 20 μg / mL), and SCOP+ECGPE group (the final concentration of SCOP was 20 μg / mL and the final concentration of ECGPE was 10 μg / mL). The expression levels of AMH, FSH, and E2 in the culture supernatant of each group were detected by ELISA. The total intracellular ROS content was detected by probe method, and the average optical density was calculated using Image J to analyze the ROS content. The expression level of the cellular inflammatory factor TNF-α was detected by immunoblotting. The mitochondrial membrane potential was detected by fluorescence microplate reader.

[0057] The results are shown in Table 2. In terms of hormone endocrine regulation, compared with the blank control group, the AMH and E2 levels in the cisplatin model group were significantly decreased (P < 0.05), and the FSH level was significantly increased (P < 0.05). The AMH level measurements in the SCOP, ECGPE, and SCOP + ECGPE groups indicate that the tangerine peel polyphenol extract (ECGPE) can further enhance the AMH secretion-promoting effect of the sea cucumber egg oligopeptide composition. In the SCOPMIX1 group, all three hormone levels were significantly improved compared to the cisplatin model group (P < 0.05). Results from the SCOPMIX2, SCOPMIX3, SCOPMIX4, and SCOPMIX5 groups showed that the mussel oligopeptides in the combination significantly improved FSH levels. In the SCOPMIX2 group, which lacked the sea cucumber egg oligopeptides, AMH and E2 levels were significantly lower than in the SCOPMIX1 group, indicating that the sea cucumber egg oligopeptides effectively improved these two hormone levels. Furthermore, when the sea cucumber egg oligopeptides were combined with the edible-medicinal polyphenol extract, their regulatory effects on endocrine hormone secretion were further enhanced. Furthermore, in the SCOPMIX5 group, where PE was replaced with WE, levels were comparable to those in the SCOPMIX4 group, indicating that the edible-medicinal water extract had no significant effect on the hormonal regulation of the sea cucumber egg oligopeptides. Regarding ROS levels, both the edible-medicinal polyphenol extract and the sea cucumber egg oligopeptides showed significant improvements, with the edible-medicinal polyphenol extract demonstrating a superior effect than the sea cucumber egg oligopeptides. In terms of anti-inflammatory effects and mitochondrial damage repair, the cisplatin model group showed a significant increase in relative TNF-α levels compared to the blank control group, while the relative membrane potential level significantly decreased, indicating increased inflammation and mitochondrial damage. Among the intervention groups, the relative TNF-α levels of the SCOPMIX1, SCOPMIX2, and SCOPMIX3 groups, which contained polyphenol extracts derived from both medicinal and edible sources, were significantly decreased compared to the cisplatin model group. However, the mitochondrial membrane potential levels of the SCOPMIX1, SCOPMIX3, SCOPMIX4, and SCOPMIX5 groups, which contained SCOP, were significantly restored. Furthermore, these experimental results demonstrate that the sea cucumber egg oligopeptide composition, through the synergistic action of its components with different functional targets, has a beneficial effect on the repair of hormone secretion, oxidative stress, inflammatory factor (TNF-α) release, and mitochondrial damage in ovarian granulosa cells.

[0058] Table 3

[0059] Example 8 Intervention Experiment on Premature Ovarian Failure Rats

[0060] Rats were randomly divided into a control group (no cyclophosphamide injection, conventional feeding), a model group (one-time intraperitoneal injection of cyclophosphamide, cyclophosphamide dose of 120 mg / kg body weight, dissolved in physiological saline at a concentration of 20 mg / mL, and conventional feeding), SCOPMIX1 group (same as the model group after modeling, fed with composition powder SCOPMIX1 in addition to conventional feeding, the feeding amount is 20 mg / kg•d), SCOPMIX2 group (model group after modeling, fed with composition powder SCOPMIX2 in addition to conventional feeding, the feeding amount is 20 mg / kg•d), SCOPMIX3 group (model group after modeling, fed with composition powder SCOPMIX3 in addition to conventional feeding, the feeding amount is 20 mg / kg•d), SCOPMIX4 group (model group after modeling, fed with composition powder SCOPMIX4 in addition to conventional feeding, the feeding amount is 20 mg / kg•d), SCOPMIX5 group (model group after modeling, fed with composition powder SCOPMIX5 in addition to conventional feeding, the feeding amount is 20 mg / kg•d), 5 rats in each group, and the experimental groups were given medication continuously for 4 weeks. Rats were intraperitoneally injected with 120 mg / kg cyclophosphamide and maintained for 15 days. Blood was collected from the tail vein for the assessment of sex hormone levels. Successful modeling was indicated by decreased AMH and E2 and increased FSH. Two days after the final dose, abdominal aortic blood was collected from each group, and levels of the ovarian hormones FSH, E2, and AMH were measured using ELISA. Doppler ultrasound was used to measure the RI value of the ovarian vessels to observe blood supply. Ovarian tissue was collected, and the ovarian organ index was recorded, along with the number of follicles at different stages.

[0061] The results showed that the SCOPMIX1 group had the best effect in alleviating ovarian decline. Compared with the model group, the SCOPMIX1 group showed increased AMH and E2 levels, decreased FSH levels, increased ovarian organ index, decreased ovarian vascular RI values, decreased number of atretic follicles, and increased number of antral follicles. The SCOPMIX4 group, which removed PE, and the SCOPMIX5 group, which replaced PE with WE, both showed less improvement than the SCOPMIX1 group, and the ovarian vascular RI values ​​did not change significantly compared with the model group.

[0062] The present invention provides a sea cucumber egg oligopeptide composition for increasing AMH levels and improving ovarian function in women. While improving AMH and other hormone levels, the sea cucumber egg oligopeptide also improves energy metabolism and oxidative stress levels in oocytes and ovarian granulosa cells, promoting the proliferation of ovarian stem cells (OSCs). This improves oocyte and granulosa cell quality, including mitochondrial function, inflammatory response, and oxidative stress levels, a feature not found in existing products for treating female ovarian failure. Furthermore, the present invention further extracts and prepares the effective ingredients for improving ovarian failure from a traditional Chinese medicine formula, thereby improving the absorption and utilization rate of the formula.

[0063] The present invention discloses a sea cucumber egg oligopeptide composition and its application for improving female AMH values ​​and ovarian function. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. A sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function, characterized in that: The following components are included in parts by weight: 15-25 parts of sea cucumber egg oligopeptide; 10-15 parts of mussel oligopeptides; 10-20 parts of edible and medicinal polyphenol extracts.

2. A sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function according to claim 1, characterized in that: The preparation method of the sea cucumber egg oligopeptide is: Step S1: fresh sea cucumber eggs are washed, freeze-dried, and crushed, sieved, and then subjected to a CO2 supercritical extraction method for defatting and removing fishy smell, retaining the residual phase containing sea cucumber egg protein; Step S2: the residual phase of the extraction and deionized water are mixed uniformly at a volume ratio of 1:10-20 to prepare an aqueous solution, 0.2%-0.4% of the solution volume of a composite protease is added, and enzymatic hydrolysis is carried out at 37°C-45°C and pH 8.0-9.0 for 3h-5h, the enzyme is inactivated at 80°C-90°C for 10-15 minutes, and the residue is removed by centrifugation at 6000 r / min-10000 r / min to obtain a supernatant, which is filtered through an ultrafiltration membrane and the permeate is collected, and the sea cucumber egg peptide powder is obtained after spray drying; Step S3: Dissolve the sea cucumber egg peptide powder in water to prepare a solution with a concentration of 10 mg / ml-100 mg / ml, separate and purify it by chromatography, measure the absorbance of the eluate at 280 nm, collect the chromatographic peak at 16 min-17 min, concentrate and freeze-dry to obtain the sea cucumber egg oligopeptide.

3. A sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function according to claim 2, characterized in that: The sea cucumber is an imitation sea cucumber or a rice sea cucumber.

4. A sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function according to claim 2, characterized in that: The composite protease consists of alkaline protease, trypsin and flavor protease, and the mass ratio of the alkaline protease, trypsin and flavor protease is (3-5): (2-4):

1.

5. A sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function according to claim 2, characterized in that: In the chromatography method, a Sephadex LH-20 column is used, the mobile phase is 25%-35% methanol, and the flow rate is 0.5 mL / min-1.0 mL / min.

6. A sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function according to claim 1, characterized in that: The medicinal and edible polyphenol extract comprises the following components in parts by weight: 6-12 parts of angelica; 5-10 parts of Pueraria root; 5-10 parts dandelion; 5-10 parts of Eucommia ulmoides leaves; 5-10 parts of dried orange peel.

7. A sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function according to claim 1 or 6, characterized in that: The preparation method of the edible and medicinal polyphenol extract is as follows: Step S1: raw material pretreatment; Select angelica, kudzu root, dandelion, eucommia leaf and red orange peel according to weight, wash and dry them, and then grind them to obtain raw material powder with a particle size of 20-40 mesh; Step S2: membrane-assisted enzymatic extraction; The raw material powder is mixed with citric acid-sodium citrate buffer solution at a material-liquid ratio of (1g:10 mL) to (1g:20 mL), and a composite enzyme is added at 0.5%-1.5% of the weight of the raw material powder. Enzymatic hydrolysis is carried out at 40°C-50°C for 1-2 hours. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is filtered through an ultrafiltration membrane with a molecular weight cutoff of 5000Da-10000Da to remove macromolecular impurities to obtain a preliminary extract. Step S3: aqueous two-phase extraction and enrichment; ammonium sulfate and polyethylene glycol are added to the primary extract to a concentration of 15% to 25% by mass of ammonium sulfate and 10% to 20% by mass of polyethylene glycol, and the mixture is thoroughly stirred to form a two-phase aqueous system; the two-phase aqueous system is allowed to stand at 20° C. to 30° C. for 30 to 60 minutes to allow polyphenols to accumulate in the upper phase; and the upper phase is separated to obtain a polyphenol-enriched solution. Step S4: freeze drying; The concentrated polyphenol enriched liquid is freeze-dried to obtain dry powder of the medicinal and edible polyphenol extract.

8. A sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function according to claim 7, characterized in that: The complex enzyme consists of cellulase and pectinase, and the mass ratio of the cellulase to the pectinase is 1:1-3.

9. Use of a sea cucumber egg oligopeptide composition for increasing female AMH values ​​and improving ovarian function as claimed in any one of claims 1 to 8 in the preparation of a drug for improving female ovarian function.

10. The use according to claim 9, characterized in that The functions of the sea cucumber egg oligopeptide composition include: (1) Improve the hormone secretion level of ovarian granulosa cells; (2) Improve the energy metabolism and oxidative stress levels of oocytes and ovarian granulosa cells; (3) Promote the proliferation of ovarian stem cells; (4) Reduce chronic inflammation of ovarian cells; (5) Improve blood supply to ovarian tissue; (6) Increase the uterine organ coefficient; (7) Alleviate ovarian immune damage.

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