A sea cucumber ovary oligopeptide composition for improving female amh values and ovarian function and application thereof

Abstract

The application relates to a sea cucumber egg oligopeptide composition for improving female AMH values and ovarian function and an application thereof, and belongs to the medical field. The sea cucumber egg oligopeptide composition comprises, in terms of weight parts, 15-25 parts of sea cucumber egg oligopeptide, 10-15 parts of mussel oligopeptide and 10-20 parts of a medicinal and edible polyphenol extract. The sea cucumber egg oligopeptide has the effects of improving hormone levels such as AMH, improving energy metabolism and oxidative stress levels of oocytes and ovarian granulosa cells and promoting ovarian stem cell proliferation. The medicinal and edible polyphenol extract has the effects of reducing chronic inflammation of ovarian cells, improving blood supply of ovarian tissues, increasing the organ coefficient of the uterus, and relieving immune damage of the ovary, and the absorption rate of the medicinal and edible polyphenol extract is significantly improved compared with direct intake of an existing traditional Chinese medicine formula. The components improve ovarian function from different aspects, and the components synergistically produce an improvement effect on ovarian reserve which is superior to that of intake of single or few components, and the components are safe and have no toxic side effects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a sea cucumber egg oligopeptide composition for improving female AMH values and ovarian function and application thereof. BACKGROUND

[0002] With age, female ovarian function gradually declines, especially after the age of 35, the decline in ovarian function accelerates, which is manifested as a decrease in anti-Mullerian hormone (AMH) levels, a decrease in ovarian reserve function, an imbalance in the secretion of estrogen, progesterone and other hormones, and a series of symptoms such as menstrual disorders, decreased fertility, hot flashes, night sweats, and skin relaxation. Anti-Mullerian hormone (AMH) is secreted by granulosa cells of preantral follicles and small antral follicles in the ovary, and is an important indicator for evaluating ovarian reserve function. The decrease in AMH values is closely related to the decrease in the number and quality of ovarian follicles. Ovarian function decline not only affects women's reproductive health, but also has a serious impact on their physical and mental health and quality of life.

[0003] At present, research on female ovarian decline is gradually increasing, but most of them only focus on a few of the six sex hormones, follicle number, uterine organ index, and ovarian blood flow, and there is no product with comprehensive improvement effect on these indicators. The treatment methods for female ovarian decline in clinical practice are limited, mainly using hormone replacement therapy, which has certain risks. For example, hormone replacement therapy increases the risk of breast cancer, endometrial cancer and other diseases, and long-term use may have side effects. There are not a few products that use traditional Chinese medicine prescriptions to nourish yin and tonify kidney to improve ovarian decline, but the specific functional components are not clear, and the absorption rate is poor. For example, Chinese Patent No. CN101919963A discloses a traditional Chinese medicine composition and its preparation method and application, which comprises the following raw materials: female privet fruit, mulberry fruit, cistanche, salvia miltiorrhiza, radix cyathulae, fried cyperus rotundus, radix paeoniae alba, anemarrhena asphodeloides and lily; Chinese Patent No. CN102274468A discloses a traditional Chinese medicine composition for regulating female endocrine and its preparation method, which is prepared from radix angelicae sinensis, radix angelicae sinensis, radix angelicae sinensis, radix angelicae sinensis, radix angelicae sinensis, radix angelicae sinensis, radix angelicae sinensis, radix angelicae sinensis, radix angelicae sinensis and radix angelicae sinensis. Therefore, it is of great practical significance to develop safe and effective products for improving female ovarian function.

[0004] Sea cucumber eggs are rich in protein, polysaccharides, fatty acids and other nutrients. Studies have found that sea cucumber eggs have good biological activity and absorption after being prepared into oligopeptides by enzymatic hydrolysis. At the same time, some plant extracts and functional nutrients have certain effects on regulating endocrine, antioxidant, improving cell metabolism, etc. SUMMARY

[0005] In order to solve the problem related to female ovarian decline, the application provides a sea cucumber egg oligopeptide composition for improving female AMH value and improving ovarian function and application thereof.

[0006] The sea cucumber egg oligopeptide composition for improving female AMH value and improving ovarian function provided by the application has good improvement effect on hormone endocrine level (especially AMH), follicle number, uterine organ index, ovarian blood flow, etc., and has no recurrence in a short period after stopping use.

[0007] The technical solution adopted by the application to solve the technical problem is as follows:

[0008] The sea cucumber egg oligopeptide composition for improving female AMH value and improving ovarian function provided by the application comprises the following components in weight parts:

[0009] Sea cucumber egg oligopeptide 15-25 parts;

[0010] Mussel oligopeptide 10-15 parts;

[0011] Medicinal and edible polyphenol extract 10-20 parts.

[0012] As a preferred embodiment, the preparation method of the sea cucumber egg oligopeptide is as follows:

[0013] Step S1: Fresh sea cucumber eggs are washed, freeze-dried and crushed, screened, and then subjected to degreasing and deodorizing treatment by CO2 supercritical extraction, and the extraction residual phase containing sea cucumber egg protein is reserved;

[0014] Step S2: The extraction residual phase and deionized water are mixed uniformly at a volume ratio of 1:10-20 to prepare an aqueous solution, 0.2%-0.4% of a complex protease solution is added, and the solution is subjected to enzymolysis at 37℃-45℃ and pH 8.0-9.0 for 3h-5h, and then the enzyme is inactivated at 80℃-90℃ for 10 minutes-15 minutes, and the supernatant is obtained by centrifugation at 6000 r / min-10000 r / min, and the filtrate is collected by filtering through an ultrafiltration membrane, and sea cucumber egg peptide powder is obtained by spray drying;

[0015] Step S3: The sea cucumber egg peptide powder is dissolved in water to prepare a solution with a concentration of 10 mg / ml-100 mg / ml, and then subjected to separation and purification by chromatography, and the absorbance of the eluent is measured at 280 nm, and the chromatographic peak at 16 min-17 min is collected, and then the sea cucumber egg oligopeptide is obtained by freeze-drying after concentration.

[0016] As a preferred embodiment, the sea cucumber is Apostichopus japonicus or Stichopus japonicus.

[0017] As a preferred embodiment, the complex protease is composed 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.

[0018] As a preferred embodiment, in the chromatography, a Sephadex LH-20 column is used, and the mobile phase is 25%-35% methanol, and the flow rate is 0.5 mL / min-1.0 mL / min.

[0019] As a preferred embodiment, the medicinal and edible polyphenol extract includes the following components in terms of weight fraction:

[0020] Angelica 6-12 parts;

[0021] Radix Paeoniae 5-10 parts;

[0022] Dandelion 5-10 parts;

[0023] Eucommia ulmoides leaf 5-10 parts;

[0024] Citrus grandis (L.) Osbeck 5-10 parts.

[0025] As a preferred embodiment, the preparation method of the medicinal and edible polyphenol extract is:

[0026] Step S1: raw material pretreatment;

[0027] Angelica, Radix Paeoniae, Dandelion, Eucommia ulmoides leaf and Citrus grandis (L.) Osbeck are selected according to weight fraction, washed, dried and then crushed to obtain raw material powder with a particle size of 20-40 mesh;

[0028] Step S2: membrane-assisted enzymatic extraction;

[0029] The raw material powder is mixed with a citric acid-sodium citrate buffer solution at a solid-liquid ratio of (1g:10 mL)-(1g:20 mL), and 0.5%-1.5% of the complex enzyme is added, and the enzyme is hydrolyzed at 40-50°C for 1-2 hours. After the enzyme hydrolysis is completed, the enzyme solution is filtered through an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Da to remove macromolecular impurities and obtain a preliminary extract;

[0030] Step S3: two-phase aqueous extraction and enrichment;

[0031] Ammonium sulfate and polyethylene glycol are added to the preliminary extract, so that the mass concentration of ammonium sulfate reaches 15%-25% and the mass concentration of polyethylene glycol reaches 10%-20%, after uniform stirring, a two-water-phase system is formed; the two-water-phase system is placed at 20-30 DEG C for 30-60 minutes, so that polyphenols are enriched in the upper phase; the upper phase is separated, and a polyphenol enrichment solution is obtained;

[0032] Step S4: freeze-drying;

[0033] The concentrated polyphenol enrichment solution is freeze-dried to obtain a dry powder of the homology-of-medicine-and-food polyphenol extract.

[0034] As a preferred embodiment, the complex enzyme is composed of cellulase and pectinase, and the mass ratio of the cellulase and the pectinase is 1:1-3.

[0035] The application provides an application of a sea cucumber ovary oligopeptide composition for improving female AMH values and improving ovary functions in preparation of a medicine for improving female ovary functions.

[0036] As a preferred embodiment, the functions of the sea cucumber ovary oligopeptide composition include:

[0037] (1) improving ovary granulosa cell hormone secretion levels;

[0038] (2) improving energy metabolism and oxidative stress levels of oocytes and ovary granulosa cells;

[0039] (3) promoting ovary stem cell proliferation;

[0040] (4) reducing chronic inflammation of ovary cells;

[0041] (5) improving blood supply of ovary tissues;

[0042] (6) increasing uterine organ coefficients;

[0043] (7) relieving ovary immune damage.

[0044] The application has the following beneficial effects:

[0045] 1) The sea cucumber egg oligopeptide composition for improving female AMH value and improving ovarian function provided by the present application, the sea cucumber egg oligopeptide has the effects of improving AMH and other hormone levels, 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 ovarian follicle generation-development-ovulation-hormone secretion function is improved, not only solving the symptoms of ovarian function decline (hormone disorder, symptoms discomfort), but also improving the root cause (insufficient ovarian follicle reserve, cell function damage), and is suitable for people with premature ovarian failure, ovarian reserve function decline and the like.

[0046] 2) The sea cucumber egg oligopeptide composition for improving female AMH value and improving ovarian function provided by the present application, the pharmacognosy polyphenol extract has the effects of reducing chronic inflammation of ovarian cells, improving blood supply of ovarian tissue, increasing uterine organ coefficient, relieving immune damage of 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.

[0047] 3) The sea cucumber egg oligopeptide composition for improving female AMH value and improving ovarian function provided by the present application, each component improves ovarian function from different aspects, the blood supply of ovary is improved after the synergy of angelica polyphenols and mussel oligopeptide, the proliferation of OSCs is promoted after the synergy of eucommia leaf polyphenols and sea cucumber egg oligopeptide, AMH secretion is promoted after the synergy of wampee polyphenols and sea cucumber egg oligopeptide, and the like, the improvement of ovarian reserve effect is better than taking single or few components under the synergy of each component, and the safety has no toxic side effects. DETAILED DESCRIPTION

[0048] The sea cucumber egg oligopeptide composition for improving female AMH value and improving ovarian function provided by the present application includes the following components in weight parts:

[0049] Sea cucumber egg oligopeptide 15-25 parts;

[0050] Mussel oligopeptide 10-15 parts;

[0051] Pharmacognosy polyphenol extract 10-20 parts.

[0052] Preferably, the sea cucumber egg oligopeptide composition includes the following components in weight parts:

[0053] Sea cucumber egg oligopeptide 25 parts;

[0054] Mussel oligopeptide 15 parts;

[0055] Pharmacognosy polyphenol extract 20 parts.

[0056] Preferably, the medicinal and edible polyphenol extract comprises the following components by weight:

[0057] Angelica sinensis 6-12 parts;

[0058] 5-10 portions of kudzu root;

[0059] 5-10 portions of dandelion;

[0060] 5-10 parts of Eucommia ulmoides leaves;

[0061] 5-10 parts of dried tangerine peel.

[0062] More preferably, the medicinal and edible polyphenol extract comprises the following components by weight:

[0063] 12 parts of Angelica sinensis;

[0064] 10 portions of kudzu root;

[0065] 10 portions of dandelion;

[0066] 10 portions of Eucommia ulmoides leaves;

[0067] 10 parts of dried tangerine peel.

[0068] The efficacy of each component in the polyphenol extract, which is both medicinal and edible, is as follows:

[0069] Angelica sinensis has the effect of promoting blood circulation and clearing the meridians. It contains polyphenolic components such as ferulic acid and ligustilide, which 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 follicle development. Furthermore, this invention found that its effect on improving ovarian blood supply is further enhanced when synergistically combined with mussel oligopeptides.

[0070] Kudzu root has endocrine-regulating effects: it contains isoflavones such as puerarin, which can mimic the activity of weak estrogen and act directly on ovarian granulosa cells to promote E2 secretion, while inhibiting FSH receptor desensitization and enhancing the stimulatory effect of gonadotropins on follicles.

[0071] Dandelion has anti-inflammatory and immunomodulatory effects: it contains polyphenols such as inulin and chlorogenic acid, which can inhibit excessive activation of ovarian macrophages, reduce IL-1β release, and alleviate autoimmune ovarian damage.

[0072] Eucommia ulmoides leaves: This invention has found that the polyphenols in Eucommia ulmoides leaves, in synergy with sea cucumber egg oligopeptides, can enhance their effect on promoting OSC proliferation and increase the follicle reserve.

[0073] Huajuhong: This invention has discovered that the polyphenols in Huajuhong can increase the uterine organ index, and when combined with sea cucumber egg oligopeptides, they can enhance the AMH secretion-promoting effect and more effectively improve hormone levels.

[0074] Preferably, the preparation method of the sea cucumber egg oligopeptide is as follows:

[0075] Step S1: fresh sea cucumber eggs are washed, freeze-dried, crushed, and passed through an 80-mesh sieve, and a CO2 supercritical extraction method is used for degreasing and deodorizing treatment, and the extraction residual phase containing sea cucumber egg protein is retained.

[0076] Preferably, the sea cucumber is Apostichopus japonicus or Stichopus japonicus.

[0077] Step S2: the above-mentioned extraction residual phase and deionized water are mixed uniformly at a volume ratio of 1:10-20 to prepare an aqueous solution, 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 the solution is subjected to enzymolysis 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 minutes-15 minutes, and the supernatant is obtained by centrifugation at 6000 r / min-10000 r / min, the supernatant is filtered through a 3 kDa ultrafiltration membrane, and the permeate is collected, and sea cucumber egg peptide powder is obtained by spray drying.

[0078] 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 the solution is separated and purified by a dextran gel Sephadex LH-20 column chromatography (3.0x100cm), the mobile phase is 25%-35% methanol, the flow rate is 0.5mL / min-1.0mL / min, the absorbance of the eluate is measured at 280nm, the chromatographic peak collected at 16min-17min is concentrated and freeze-dried to obtain sea cucumber egg oligopeptide.

[0079] Preferably, the preparation method of the polyphenol extract of medicinal and edible materials is as follows:

[0080] Step S1: pretreatment of raw materials;

[0081] According to the weight fraction, medicinal and edible raw materials (Angelica sinensis, Pueraria lobata, Taraxacum mongolicum, Eucommia ulmoides leaf, and Citrus grandis) are selected, washed, dried, and crushed to obtain raw material powder with a particle size of 20-40 mesh;

[0082] Step S2: membrane-assisted enzymatic extraction;

[0083] The raw material powder is mixed with a citric acid-sodium citrate buffer solution (pH 4.5-5.5) according to a solid-liquid ratio of (1g:10 mL)-(1g:20 mL), 0.5%-1.5% of a composite enzyme (prepared by mixing cellulase and pectinase according to a mass ratio of 1:1-3) is added to the raw material powder, and enzymolysis is carried out at 40-50 DEG C for 1-2 hours; after the enzymolysis is completed, the enzymolysis solution is filtered through an ultrafiltration membrane with a molecular weight cut-off of 5000-10000 Da to remove macromolecular impurities, and a preliminary extraction solution is obtained;

[0084] Step S3: aqueous two-phase extraction and enrichment;

[0085] Ammonium sulfate and polyethylene glycol (PEG-4000) are added to the preliminary extraction solution, so that the mass concentration of ammonium sulfate reaches 15%-25% and the mass concentration of polyethylene glycol reaches 10%-20%, and after uniform stirring, an aqueous two-phase system is formed; the aqueous two-phase system is allowed to stand at 20-30 DEG C for 30-60 minutes, so that the polyphenols are enriched in the upper phase (PEG phase); the upper phase is separated to obtain a polyphenol enrichment solution;

[0086] Step S4: freeze-drying;

[0087] The concentrated polyphenol enrichment solution is placed in a freeze dryer for freeze-drying to obtain a dry powder of the homology-of-medicine-and-food polyphenol extract.

[0088] The application also provides a use of the sea cucumber egg oligopeptide composition for improving female AMH values and improving ovarian function in the preparation of a medicine for improving female ovarian function.

[0089] Preferably, the functions of the sea cucumber egg oligopeptide composition mainly include:

[0090] (1) improving the hormone secretion level of ovarian granulosa cells;

[0091] (2) improving the energy metabolism and oxidative stress level of oocytes and ovarian granulosa cells;

[0092] (3) promoting the proliferation of ovarian stem cells;

[0093] (4) reducing chronic inflammation of ovarian cells;

[0094] (5) improving blood supply to ovarian tissue;

[0095] (6) increasing the uterine organ coefficient;

[0096] (7) relieving immune damage to the ovary.

[0097] The present application finds that sea cucumber egg oligopeptide and polyphenol extract with medicinal and edible properties can improve ovarian function from different aspects, and the components synergistically produce better effects than directly taking traditional Chinese medicine formula.

[0098] Preferably, the preparation method of the female ovarian function improving medicine is as follows:

[0099] The above ingredients are weighed and mixed according to the ratio, so that the ingredients are evenly dispersed. According to the requirements of different dosage forms (such as tablets, capsules, granules, etc.), further preparation forming operation is carried out.

[0100] Specifically, when preparing tablets, a suitable amount of adhesive (such as hydroxypropyl methyl cellulose), lubricant (such as magnesium stearate) and other auxiliary materials can be added to the mixed material, and then the material is granulated, dried and compressed into tablets.

[0101] Specifically, when preparing capsules, the uniformly mixed material can be directly filled into the capsule shell.

[0102] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0103] Example 1: Preparation of sea cucumber egg oligopeptide

[0104] (1) Fresh Apostichopus japonicus egg was 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 was retained;

[0105] (2) The residual phase obtained above was prepared into an aqueous solution at a volume ratio of 1:10 with deionized water, and 0.3% of a compound protease (alkaline protease, trypsin and flavor protease at a mass ratio of 3:2:1) was added to the solution. Enzymolysis was carried out at 40°C and pH 8.0 for 4 hours, and then the enzyme was inactivated at 90°C for 15 minutes. The supernatant was obtained by centrifugation at 8000 r / min, and the residue was removed. The supernatant was filtered through a 3 kDa ultrafiltration membrane, and the permeate was collected. After spray drying, sea cucumber egg peptide powder was obtained;

[0106] (3) The sea cucumber egg peptide powder was dissolved in water to prepare a solution with a concentration of 100 mg / ml. Sephadex LH-20 column chromatography (3.0x100 cm) was used for separation and purification, with 35% methanol as the mobile phase, a flow rate of 1.0 mL / min, and the absorbance of the eluent at 280 nm. The 16-17 min chromatographic peak was collected according to the absorbance value, concentrated and freeze-dried to obtain sea cucumber egg oligopeptide (SCOP).

[0107] Preparation of a medicinal and edible polyphenol extract (aqueous two-phase extraction method)

[0108] Step S1: raw material pretreatment;

[0109] According to the weight fraction, medicinal and edible raw materials (Angelica 12 parts, Pueraria 10 parts, Dandelion 10 parts, Eucommia leaf 10 parts, and Citron 10 parts) were selected, washed, dried, and then crushed to obtain raw material powder with a particle size of 20 mesh;

[0110] Step S2: membrane-assisted enzymatic extraction;

[0111] 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 1.5% of the raw material powder was added to the complex enzyme (prepared by mixing cellulase and pectinase at a mass ratio of 1:1). The enzyme was incubated at 50°C for 2 hours. After the enzyme reaction was completed, the enzyme solution was filtered through an ultrafiltration membrane with a molecular weight cutoff of 10000 Da to remove macromolecular impurities, and a preliminary extract was obtained.

[0112] Step S3: aqueous two-phase extraction and enrichment;

[0113] Ammonium sulfate and polyethylene glycol (PEG-4000) were added to the preliminary extract to achieve a mass concentration of 25% for ammonium sulfate and 20% for polyethylene glycol. After thorough stirring, an aqueous two-phase system was formed. The aqueous two-phase system was allowed to stand at 30°C for 60 minutes, and the polyphenols were enriched in the upper phase (PEG phase). The upper phase was separated to obtain a polyphenol enrichment solution.

[0114] Step S4: freeze-drying;

[0115] The concentrated polyphenol enrichment solution was placed in a freeze-drier to obtain a medicinal and edible polyphenol extract dry powder (PE).

[0116] Preparation of a medicinal and edible water extract (ordinary water extraction method)

[0117] According to the weight fraction, medicinal and edible raw materials (Angelica 12 parts, Pueraria 10 parts, Dandelion 10 parts, Eucommia leaf 10 parts, and Citron 10 parts) were selected, washed, dried, and then crushed to obtain raw material powder with a particle size of 20 mesh; 100g of raw material powder was added to 1500mL of purified water, heated at 90°C for 1.5 hours, and filtered. The residue was extracted with 1000mL of purified water for 1 hour, and the two filtrates were combined. The filtrate was centrifuged at 4000r / min for 10 minutes, and the supernatant was freeze-dried to obtain a medicinal and edible water extract dry powder (WE).

[0118] Preparation of Angelica sinensis polyphenol extract (ASPE), Eucommia ulmoides leaf polyphenol extract (EULPE) and Citrus grandis (L.) Osbeck polyphenol extract (ECGPE) (aqueous two-phase extraction method)

[0119] Angelica sinensis polyphenol extract (ASPE), Eucommia ulmoides leaf polyphenol extract (EULPE) and Citrus grandis (L.) Osbeck polyphenol extract (ECGPE) were prepared according to the preparation method of Example 2.

[0120] Example 5 Preparation of sea cucumber egg oligopeptide composition for improving female AMH value and improving ovarian function

[0121] The sea cucumber egg oligopeptide (Example 1), mussel oligopeptide, polyphenol extract of medicinal and edible homologous substances (Example 2), and medicinal and edible homologous water extract (Example 3) were mixed according to the ratio in Table 1 to make the composition powder SCOPMIX1-SCOPMIX5.

[0122] Table 1

[0123]

[0124] Example 6 Mouse ovarian stem cell (OSC) proliferation experiment

[0125] Mouse ovarian stem cells (OSCs) were inoculated in a 96-well plate at a density of 1 x 10 4 cells / well and cultured for 24 hours to adhere.

[0126] The experimental groups were divided into a control group (only DMEM / F12 basic medium was added), a SCOP group (SCOP was added to the DMEM / F12 basic medium to make the SCOP concentration in the medium 20 μg / mL), an EULPE group (EULPE was added to the DMEM / F12 basic medium to make the EULPE concentration in the medium 20 μg / mL), a SCOP+EULPE1 group (the final concentration of SCOP was 20 μg / mL and the final concentration of EULPE was 5 μg / mL), a SCOP+EULPE2 group (the final concentration of SCOP was 20 μg / mL and the final concentration of EULPE was 10 μg / mL), and a SCOP+EULPE3 group (the final concentration of SCOP was 20 μg / mL and the final concentration of EULPE was 20 μg / mL). After adding the samples, each group was cultured in a 37°C incubator for 24 hours. The OD value at 450 nm was detected by a microplate reader by CCK-8 method. The cell proliferation rate of each group was calculated according to the following formula:

[0127]

[0128] 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 was significantly increased, while the EULPE group had no obvious change, and the increase rate of the SCOP+EULPE2 group and the SCOP+EULPE3 group was significantly higher than that of the SCOP group. It is indicated that the sea cucumber egg oligopeptide composition prepared in the application has the effect of promoting the proliferation of mouse ovarian stem cells (OSCs), and in the presence of a certain concentration of eucommia leaf polyphenol extract (EULPE), the effect is further enhanced.

[0129] Table 2

[0130]

[0131] Example 7 Intervention experiment of human ovarian granulosa cell damage

[0132] Human ovarian granulosa cells were selected, cultured (cells were inoculated in T25 culture bottles, DMEM / F12 medium was used as culture medium, and the culture was placed in a 37℃, 5% CO2, saturated humidity incubator for culture), and the cultured cells were divided into a blank control group (only DMEM / F12 basic medium (without any damage agent or intervention drug) was added, and the culture was continued to the end of the experiment), a cisplatin model group (cisplatin was diluted with DMEM / F12 basic medium to prepare a cisplatin working solution with a final concentration of 5-20 μM, and the culture was continued in the incubator for 24 hours), an SCOPMIX1 group (composition powder SCOPMIX1 was added to DMEM / F12 basic medium to make the concentration of composition powder SCOPMIX1 in the medium 20 μg / mL), an SCOPMIX2 group (composition powder SCOPMIX2 was added to DMEM / F12 basic medium to make the concentration of composition powder SCOPMIX2 in the medium 20 μg / mL), an SCOPMIX3 group (composition powder SCOPMIX3 was added to DMEM / F12 basic medium to make the concentration of composition powder SCOPMIX3 in the medium 20 μg / mL), an SCOPMIX4 group (composition powder SCOPMIX4 was added to DMEM / F12 basic medium to make the concentration of composition powder SCOPMIX4 in the medium 20 μg / mL), an SCOPMIX5 group (composition powder SCOPMIX5 was added to DMEM / F12 basic medium to make the concentration of composition powder SCOPMIX5 in the medium 20 μg / mL), an SCOP group (SCOP was added to DMEM / F12 basic medium to make the concentration of SCOP in the medium 20 μg / mL), an ECGPE group (ECGPE was added to DMEM / F12 basic medium to make the concentration of ECGPE in the medium 20 μg / mL), and an 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 ROS content in the cells was detected by a probe method, the average optical density was calculated using Image J, and the ROS content was analyzed; the expression level of inflammatory factor TNF-α in the cells was detected by immunoblotting; and the mitochondrial membrane potential was detected by a fluorescence microplate reader.

[0133] Results are shown in Table 2. In terms of hormone endocrine regulation, compared with the blank control group, the AMH level and E2 level of the cisplatin model group significantly decreased (P<0.05), and the FSH level significantly increased (P<0.05). The AMH level determination results of the SCOP group, the ECGPE group and the SCOP+ECGPE group showed that the citrus grandis (L.) Osbeck polyphenol extract (ECGPE) could further improve the AMH secretion effect of the sea cucumber egg oligopeptide composition. The three hormone levels in the SCOPMIX1 group were significantly improved compared with the cisplatin model group (P<0.05). From the results of the SCOPMIX2, SCOPMIX3, SCOPMIX4 and SCOPMIX5 groups, it can be seen that the mussel oligopeptide in the composition has a significant effect on improving the FSH level. The AMH level and E2 level in the SCOPMIX2 group lacking sea cucumber egg oligopeptide were significantly lower than those in the SCOPMIX1 group, indicating that the sea cucumber egg oligopeptide has an effective effect on improving the levels of the two hormones, and the sea cucumber egg oligopeptide and the edible and medicinal polyphenol extract have a further improved effect on the regulation of hormone endocrine. At the same time, the SCOPMIX5 group with PE replaced by WE and the SCOPMIX4 group were at the same level, indicating that the edible and medicinal water extract does not have a significant promoting effect on the hormone regulation ability of the sea cucumber egg oligopeptide. In terms of ROS level, the edible and medicinal polyphenol extract and the sea cucumber egg oligopeptide both showed a significant improvement effect, and the effect of the edible and medicinal polyphenol extract was better than that of the sea cucumber egg oligopeptide. In terms of anti-inflammatory effect and mitochondrial damage repair, compared with the blank control group, the relative level of TNF-α in the cisplatin model group significantly increased, and the relative level of membrane potential significantly decreased, indicating an increase in inflammation level and mitochondrial damage. In the intervention groups, the relative level of TNF-α in the SCOPMIX1 group, the SCOPMIX2 group and the SCOPMIX3 group containing the edible and medicinal polyphenol extract was significantly lower than that in the cisplatin model group, while the mitochondrial membrane potential level of the SCOPMIX1 group, the SCOPMIX3 group, the SCOPMIX4 group and the SCOPMIX5 group containing SCOP significantly increased. In addition, the above test results showed that the sea cucumber egg oligopeptide composition had a good repair effect on the hormone secretion level, oxidative stress level, inflammatory factor (TNF-α) release level and mitochondrial damage of the ovarian granulosa cells through the synergistic effect of the components with different functional targets.

[0134] Table 3

[0135]

[0136] Example 8 Intervention experiment of premature ovarian failure rats

[0137] The rats are randomly divided into a control group (no cyclophosphamide injection, normal feeding), a model group (a one-time intraperitoneal injection of cyclophosphamide, the dose of cyclophosphamide is 120 mg / kg of body weight, dissolved in normal saline, the concentration is 20 mg / mL, normal feeding), a SCOPMIX1 group (after modeling, in addition to normal feeding, the composition powder SCOPMIX1 is fed, the feeding amount is 20 mg / kg·d), a SCOPMIX2 group (after modeling, in addition to normal feeding, the composition powder SCOPMIX2 is fed, the feeding amount is 20 mg / kg·d), a SCOPMIX3 group (after modeling, in addition to normal feeding, the composition powder SCOPMIX3 is fed, the feeding amount is 20 mg / kg·d), a SCOPMIX4 group (after modeling, in addition to normal feeding, the composition powder SCOPMIX4 is fed, the feeding amount is 20 mg / kg·d), and a SCOPMIX5 group (after modeling, in addition to normal feeding, the composition powder SCOPMIX5 is fed, the feeding amount is 20 mg / kg·d), 5 rats in each group, and the experimental groups are continuously administered for 4 weeks. The rats are intraperitoneally injected with 120 mg / kg of cyclophosphamide, and continue to be fed for 15 days, tail vein blood is collected, sex hormone levels are detected, and the successful modeling is marked by the decrease of AMH and E2 and the increase of FSH. On the second day after the last administration, abdominal aortic blood of rats in each group is collected, and the levels of ovarian hormones FSH, E2 and AMH are detected by ELISA; the RI value of ovarian blood vessels is detected by Doppler ultrasound to observe the blood supply; ovarian tissues are collected, the ovarian organ index is recorded, and the number of follicles at different stages is observed.

[0138] The results show that the ovarian regression alleviation effect of the SCOPMIX1 group is the best, compared with the model group, the AMH and E2 levels in the SCOPMIX1 group increase, the FSH level decreases, the ovarian organ index increases, the ovarian vascular RI value decreases, the number of atretic follicles decreases, and the number of antral follicles increases. The improvement effect of the SCOPMIX4 group without PE and the SCOPMIX5 group with WE instead of PE is inferior to that of the SCOPMIX1 group, and the ovarian vascular RI value does not change significantly compared with the model group.

[0139] The sea cucumber ovary oligopeptide composition for improving the AMH value of women and improving ovarian function provided by the application has the functions of improving the energy metabolism and oxidative stress level of oocytes and ovarian granulosa cells, promoting the proliferation of ovarian stem cells (OSCs), and solving the problems that the existing products for improving the ovarian regression of women do not have the functions of improving the mitochondrial function, inflammatory response, oxidative stress level and the quality of oocytes and granulosa cells. Meanwhile, the application further extracts and prepares the functional components for improving ovarian regression from the traditional Chinese medicine formula, and improves the absorption rate and utilization rate of the traditional Chinese medicine formula.

[0140] The application discloses a sea cucumber ovulatory oligopeptide composition for improving female AMH values and improving ovarian function and application thereof, and those skilled in the art can refer to the content of the present application and appropriately improve process parameters. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as being included in the present application. The product of the present application has been described through a preferred embodiment, and relevant personnel can obviously change or appropriately change and combine the product described in the present application without departing from the content, spirit and scope of the present application, so as to realize and apply the present application.

Claims

1. A sea cucumber egg oligopeptide composition, characterized in that, Composed of the following components in parts by weight: Sea cucumber egg oligopeptide 15-25 parts; Mussel oligopeptide 10-15 parts; and Medicinal and edible polyphenol extract dry powder 10-20 parts; The preparation method of the sea cucumber egg oligopeptide is: Step S1: Fresh sea cucumber eggs are washed, freeze-dried, crushed, and sieved, and then subjected to defatting and deodorizing treatment by CO2 supercritical extraction, and the residual phase containing sea cucumber egg protein is retained; Step S2: The residual phase and deionized water are mixed uniformly at a volume ratio of 1:10-20 to prepare an aqueous solution, 0.2%-0.4% of a complex protease is added to the solution, and the solution is subjected to enzymolysis at 37℃-45℃ and pH 8.0-9.0 for 3-5 hours, and then the enzyme is inactivated at 80℃-90℃ for 10-15 minutes, and the supernatant is obtained by centrifugation at 6000 r / min-10000 r / min, and the filtrate is collected by ultrafiltration membrane filtration, and sea cucumber egg peptide powder is obtained by spray drying; The complex protease is composed 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, Step S3: The sea cucumber egg peptide powder is dissolved in water to prepare a solution with a concentration of 10 mg / ml-100 mg / ml, and then subjected to separation and purification by chromatography, and then subjected to freeze-drying after concentration to obtain sea cucumber egg oligopeptide; The preparation method of the medicinal and edible polyphenol extract dry powder is: Step S1: Raw material pretreatment: According to parts by weight, angelica, radix puerariae, dandelion, eucommia leaf, and citrus grandis (L.) Osbeck are selected, washed, dried, and crushed to obtain raw material powder with a particle size of 20-40 mesh; The weight parts of the raw material are as follows: Angelica 6-12 parts; Radix puerariae 5-10 parts; Dandelion 5-10 parts; Eucommia leaf 5-10 parts; Citrus grandis (L.) Osbeck 5-10 parts; Step S2: Membrane-assisted enzymatic extraction: The raw material powder is mixed with a citric acid-sodium citrate buffer solution at a solid-liquid ratio of 1g:10mL-1g:20mL, and 0.5%-1.5% of a complex enzyme is added, and the mixture is subjected to enzymolysis at 40℃-50℃ for 1-2 hours; after the enzymolysis is completed, the enzymolysis solution is filtered through an ultrafiltration membrane with a molecular weight cut-off of 5000Da-10000Da to remove macromolecular impurities and obtain a preliminary extract; Step S3: Two-phase aqueous extraction and enrichment; Ammonium sulfate and polyethylene glycol are added to the preliminary extract to achieve a mass concentration of 15%-25% for ammonium sulfate and 10%-20% for polyethylene glycol, and the mixture is stirred uniformly to form a two-phase aqueous system; the two-phase aqueous system is allowed to stand at 20℃-30℃ for 30 minutes-60 minutes to enrich polyphenols in the upper phase; the upper phase is separated to obtain a polyphenol-rich liquid; Step S4: Freeze-drying; The concentrated polyphenol-rich liquid is subjected to freeze-drying to obtain medicinal and edible polyphenol extract dry powder, The complex enzyme is composed of cellulase and pectinase, and the mass ratio of the cellulase and pectinase is 1:1-3.

2. The sea cucumber egg oligopeptide composition according to claim 1, characterized in that, The sea cucumber is Acaudina molpadioides or Stichopus japonicus.

3. The sea cucumber egg oligopeptide composition according to claim 1 or 2, characterized in that, The chromatography is carried out by using a Sephadex LH-20 column, a 25%-35% methanol as a mobile phase, a flow rate of 0.5 mL / min-1.0 mL / min, and a 280 nm wavelength for measuring the absorbance of the eluent, and the sea cucumber egg oligopeptide is obtained by collecting a chromatographic peak at 16 min-17 min, concentrating, and then freeze-drying.

4. Use of the sea cucumber egg oligopeptide composition according to any one of claims 1-3 in the preparation of a medicine for improving female ovaries.

5. Use according to claim 4, characterized in that, The functions of the sea cucumber egg oligopeptide composition are selected from any one or more of the following combinations: (1) increasing the secretion level of anti-Mullerian hormone AMH; (2) promoting the proliferation of ovarian stem cells; (3) reducing the release level of ovarian granulosa cell inflammatory factor TNF-α; (4) repairing the mitochondrial damage of granulosa cells.

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