Composition for relieving ovarian function decline based on cell autophagy and DNA methylation level

Abstract

The invention relates to the technical field of medicinal and edible compositions, in particular to a composition for relieving ovarian function decline based on cell autophagy and DNA methylation level. The composition for relieving ovarian function decline comprises 10-50 parts of collagen tripeptide, 1-5 parts of gamma-aminobutyric acid, 1-6 parts of a red grape yeast compound, 0.5-2 parts of a yeast extract, 1-6 parts of a sophora flower extract, 0.5-2 parts of golden camellia, 0.5-6 parts of a turmeric extract, 0.1-2.7 parts of blueberry anthocyanin, 1-8 parts of ginseng, 1-6 parts of an angelica sinensis extract, 1-6 parts of a poria cocos extract and 0.1-0.3 part of sodium hyaluronate. According to the composition disclosed by the invention, medicinal and edible substances are taken as main raw materials, and the raw material components are regulated and controlled, so that the composition can improve ovarian granular cell excessive autophagy, lower the methylation level and reduce ovarian granular cell damage on the basis of activating a PI3K / Akt / mTOR pathway, thereby relieving ovarian function decline.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine-food homologous compositions, and in particular to a composition for alleviating ovarian dysfunction based on cell autophagy and DNA methylation levels. Background Art

[0002] Ovarian failure is a common, progressive endocrine disorder of the female reproductive system. Clinically, it manifests as irregular menstrual cycles, amenorrhea, decreased fertility, multiple miscarriages, and infertility. Ovarian function declines not only with aging but is also associated with genetics, surgery, trauma, infection, environmental factors, and stress. Its pathogenesis is primarily linked to endocrine disruption, inflammatory damage, oxidative damage, mitochondrial dysfunction, telomerase abnormalities, apoptosis, and autophagy caused by an imbalance in the hypothalamic-pituitary-ovarian axis (HPOA).

[0003] Abnormal apoptosis of ovarian cells is associated with follicular atresia, which may be the primary mechanism of ovarian dysfunction. Granulosa cells, a key component of the follicular microenvironment, normally maintain ovarian function and regulate follicular development through autophagy homeostasis. However, excessive autophagy can induce follicular atresia in granulosa cells and interfere with ovarian function. Therefore, abnormal activation of autophagy in granulosa cells may contribute to decreased ovarian function.

[0004] Traditional Chinese medicine (TCM) treatment has shown some effectiveness in regulating autophagy in ovarian granulosa cells. Current research focuses on autophagy-related regulatory genes such as ATG6, ATG8, and ATG5, as well as B-cell lymphoma-2 (Bcl-2) and forkhead box protein O (FoxO). Signaling pathways include the p53 / AMPK / mTOR pathway and the MAPK pathway. Research also focuses on mitochondrial autophagy and endoplasmic reticulum autophagy.

[0005] However, the above studies mainly focused on Siwu Decoction, plant extracts such as leonurine, and Western medicines such as metformin. There is no research on the role of preparations with medicinal and edible materials as the main raw materials in regulating ovarian granulosa cell autophagy.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] One purpose of the present invention is to provide a composition for alleviating ovarian dysfunction based on cell autophagy and DNA methylation levels. The composition uses polypeptides and plant extracts as main ingredients, and can improve excessive autophagy of ovarian granulosa cells, downregulate methylation levels, and reduce ovarian granulosa cell damage based on activating the PI3K / Akt / mTOR pathway, thereby alleviating ovarian dysfunction.

[0008] Another object of the present invention is to provide a composition or agent for alleviating ovarian dysfunction for use in preparing a preparation for preventing and / or alleviating ovarian dysfunction or ovarian damage.

[0009] In order to achieve the above-mentioned objectives of the present invention, the first aspect of the present invention provides a composition for alleviating ovarian dysfunction based on cell autophagy and DNA methylation levels, comprising the following components by weight: 10-50 parts of collagen tripeptide, 1-5 parts of γ-aminobutyric acid, 1-6 parts of red grape yeast complex, 0.5-2 parts of yeast extract, 1-6 parts of Sophora japonica extract, 0.5-2 parts of Camellia chrysantha, 0.5-6 parts of Turmeric extract, 0.1-2.7 parts of blueberry anthocyanins, 1-8 parts of ginseng, 1-6 parts of Angelica sinensis extract, 1-6 parts of Poria cocos extract and 0.1-0.3 parts of sodium hyaluronate.

[0010] In a specific embodiment of the present invention, in the composition, the protein content is ≥33wt%, the peptide content is ≥30wt%, the crude polysaccharide content is ≥8wt%, and the γ-aminobutyric acid content is ≥0.8wt%.

[0011] The second aspect of the present invention provides a reagent comprising the composition for alleviating ovarian dysfunction provided by the first aspect of the present invention.

[0012] In a specific embodiment of the present invention, the reagent further comprises an auxiliary material acceptable to functional foods or health foods.

[0013] In a specific embodiment of the present invention, the agent includes at least one of granules, tablets, pills, syrups, capsules and oral liquids.

[0014] The third aspect of the present invention provides use of the composition of the first aspect or the reagent of the second aspect in the preparation of a preparation for preventing and / or alleviating ovarian dysfunction or ovarian damage.

[0015] In a specific embodiment of the present invention, the preparation is used to enhance the activity of ovarian granulosa cells induced by triptolide.

[0016] In a specific embodiment of the present invention, the preparation is used to upregulate the hormone secretion level of ovarian granulosa cells induced by triptolide. Furthermore, the hormones include E2 (estradiol) and P (progesterone).

[0017] In a specific embodiment of the present invention, the preparation inhibits the excessive autophagy of ovarian granulosa cells induced by triptolide by mediating the PI3K / Akt / mTOR pathway.

[0018] In a specific embodiment of the present invention, the preparation is used to upregulate the relative expression levels of p-PI3K, p-Akt and p-mTOR proteins in ovarian granulosa cells induced by triptolide.

[0019] In a specific embodiment of the present invention, the preparation is used to reduce the methylation level of ovarian granulosa cells induced by triptolide.

[0020] In a specific embodiment of the present invention, the preparation is used to down-regulate the mRNA expression of DNMT3a and DNMT3b in ovarian granulosa cells induced by triptolide, and to up-regulate the mRNA expression of TET1 and TET2 in ovarian granulosa cells induced by triptolide.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The composition of the present invention uses medicinal and edible substances as the main raw materials. By regulating the raw material components, the composition can improve excessive autophagy of ovarian granulosa cells, downregulate methylation levels, and reduce ovarian granulosa cell damage based on activating the PI3K / Akt / mTOR pathway, thereby preventing and / or alleviating ovarian dysfunction or ovarian damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a comparative diagram of the alleviating effects of the compositions provided by different embodiments of the present invention on the inhibition of TP-induced human ovarian granulosa cell (KGN) activity; Figure 2 This is the effect of the composition provided in Example 1 of the present invention on the hormone secretion level of KGN cells treated with TP; wherein (A) is the ELISA method for detecting E2 levels, and (B) is the ELISA method for detecting P levels; Figure 3 Effects of the composition provided in Example 1 of the present invention on the autophagy level and expression of autophagy-related proteins in KGN cells treated with TP; wherein, (A) is a comparison of MDC levels in each group, (B) is a comparison of the expression of autophagy-related proteins in KGN cells in each group, and (C) is a Western blot assay for detecting autophagy-related proteins. Figure 4Effects of the composition provided in Example 1 of the present invention on the expression of PI3K / Akt / mTOR pathway-related proteins in KGN cells treated with TP; wherein, (A) is a comparison of the expression of PI3K / Akt / mTOR pathway-related proteins in KGN cells of each group; (B) is a Western blot assay for the detection of PI3K / Akt / mTOR pathway-related proteins in cells; Figure 5 LY294002 was used to verify the effect of the composition provided in Example 1 of the present invention on the PI3K / Akt / mTOR signaling pathway in KGN cells. (A) is a comparison of the expression of PI3K / Akt / mTOR pathway-related proteins in KGN cells of each group; (B) is a Western blot assay for the expression of PI3K / Akt / mTOR pathway-related proteins in cells. Figure 6 This is the effect of the composition provided in Example 1 of the present invention on the methylation level of KGN cells treated with TP. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Those who do not specify specific conditions in the embodiments are carried out according to normal conditions or the conditions recommended by the manufacturer. Those whose reagents or instruments are not specified by the manufacturer are conventional products that can be purchased commercially.

[0025] Triptolide (TP) is an epoxyditerpenoid first isolated from Tripterygium wilfordii extract and is one of the main active ingredients in Tripterygium wilfordii. Clinical data indicate that Tripterygium wilfordii is toxic to the female reproductive system, manifesting as premature ovarian failure, decreased menstruation, and amenorrhea. Studies have shown that exposure to TP in female test subjects prolongs the estrous cycle, reduces ovarian and uterine weights, and is accompanied by decreased serum estradiol and progesterone levels. Furthermore, uterine and ovarian structural damage is associated with blurred uterine glandular, myometrial, and peripapillary structures, decreased columnar cell height and luminal diameter, disorganized uterine epithelial cells, and decreased uterine gland number. Cytological studies have shown that TP significantly promotes apoptosis and oxidative stress in ovarian granulosa cells in vitro. Increased ovarian oxidative stress can induce granulosa cell apoptosis and subsequent follicular atresia.

[0026] The present invention provides a composition with medicinal and edible substances as main raw materials, the main targets of which are ovarian granulosa cell autophagy and DNA methylation, and can alleviate ovarian damage induced by TP.

[0027] The first aspect of the present invention provides a composition for alleviating ovarian dysfunction based on cell autophagy and DNA methylation levels, comprising the following components by weight: 10 to 50 parts of collagen tripeptide, 1 to 5 parts of γ-aminobutyric acid, 1 to 6 parts of red grape yeast complex, 0.5 to 2 parts of yeast extract, 1 to 6 parts of Sophora japonica extract, 0.5 to 2 parts of Camellia chrysantha, 0.5 to 6 parts of Turmeric extract, 0.1 to 2.7 parts of blueberry anthocyanins, 1 to 8 parts of ginseng, 1 to 6 parts of Angelica sinensis extract, 1 to 6 parts of Poria cocos extract and 0.1 to 0.3 parts of sodium hyaluronate.

[0028] The composition of the present invention uses medicinal and edible substances as the main raw materials. By regulating the raw material components, the composition can improve excessive autophagy of ovarian granulosa cells, downregulate methylation levels, and reduce ovarian granulosa cell damage based on activating the PI3K / Akt / mTOR pathway, thereby preventing and / or alleviating ovarian dysfunction or ovarian damage.

[0029] For example, in different embodiments, the amounts of each component may be as follows, calculated by weight: The amount of collagen tripeptide can be 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts or a range consisting of any two thereof; The amount of γ-aminobutyric acid can be 1 part, 2 parts, 3 parts, 4 parts, 5 parts or a range consisting of any two parts thereof; The amount of the red grape yeast complex can be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts or a range consisting of any two parts thereof; The amount of yeast extract can be 0.5 parts, 0.8 parts, 1 part, 1.5 parts, 2 parts or a range consisting of any two thereof; The amount of Sophora japonica flower extract can be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts or a range consisting of any two parts thereof; The dosage of Camellia chrysantha can be 0.5 parts, 0.8 parts, 1 part, 1.5 parts, 2 parts or any two thereof; The amount of turmeric extract can be 0.5 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts or a range consisting of any two parts thereof; The amount of blueberry anthocyanin can be 0.1 parts, 0.5 parts, 1 parts, 1.5 parts, 2 parts, 2.5 parts, 2.7 parts or a range consisting of any two thereof; The dosage of ginseng can be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts or a range consisting of any two of them; The amount of the angelica extract can be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts or a range consisting of any two parts thereof; The amount of Poria cocos extract can be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts or a range consisting of any two parts thereof; The amount of sodium hyaluronate used can be 0.1 part, 0.15 part, 0.2 part, 0.25 part, 0.3 part or a range consisting of any two parts thereof.

[0030] In a specific embodiment of the present invention, the composition includes the following components in parts by weight: 10-50 parts of collagen tripeptide, 1-4 parts of γ-aminobutyric acid, 1-6 parts of red grape yeast complex, 0.5-2 parts of yeast extract, 1-6 parts of Sophora japonica flower extract, 0.5-2 parts of Camellia chrysantha, 0.5-6 parts of Turmeric extract, 0.1-2.7 parts of blueberry anthocyanins, 1-8 parts of ginseng, 1-4 parts of Angelica sinensis extract, 1-6 parts of Poria cocos extract and 0.1-0.3 parts of sodium hyaluronate.

[0031] In a specific embodiment of the present invention, the composition includes, by mass percentage, 50% to 57% of collagen tripeptide, 4% to 5.8% of gamma-aminobutyric acid, 4% to 6% of red grape yeast complex, 2% to 3% of yeast extract, 5% to 9% of Sophora japonica flower extract, 2% to 3% of Camellia chrysantha, 2% to 6% of Turmeric extract, 0.5% to 3% of blueberry anthocyanins, 5% to 9% of ginseng, 4% to 6% of Angelica sinensis extract, 4% to 6% of Poria cocos extract, and 0.3% to 0.6% of sodium hyaluronate.

[0032] For example, in different embodiments, the amounts of the components in the composition can be as follows, calculated by mass percentage: The dosage of collagen tripeptide can be 50%, 52%, 54%, 55%, 57% or a range consisting of any two thereof; The amount of γ-aminobutyric acid can be 4%, 4.2%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, 5.8% or a range consisting of any two thereof; The amount of the red grape yeast complex can be 4%, 4.5%, 5%, 5.5%, 6% or a range consisting of any two thereof; The amount of yeast extract can be 2%, 2.2%, 2.5%, 2.8%, 3% or a range consisting of any two thereof; The amount of Sophora japonica flower extract can be 5%, 6%, 7%, 8%, 9% or a range consisting of any two thereof; The dosage of Camellia chrysantha can be 2%, 2.2%, 2.5%, 2.8%, 3% or any two thereof; The amount of turmeric extract can be 2%, 3%, 4%, 5%, 6% or a range consisting of any two thereof; The amount of blueberry anthocyanin can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or a range consisting of any two thereof; The dosage of ginseng can be 5%, 6%, 7%, 8%, 9% or a range consisting of any two thereof; The amount of the angelica extract can be 4%, 4.5%, 5%, 5.5%, 6% or a range consisting of any two thereof; The amount of Poria cocos extract can be 4%, 4.5%, 5%, 5.5%, 6% or a range consisting of any two thereof; The amount of sodium hyaluronate may be 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6% or a range consisting of any two thereof.

[0033] In a specific embodiment of the present invention, in the composition, the protein content is ≥33wt%, the peptide content is ≥30wt%, the crude polysaccharide content is ≥8wt%, and the γ-aminobutyric acid is ≥0.8wt%.

[0034] In a specific embodiment of the present invention, the purity of the γ-aminobutyric acid used in the composition is ≥20 wt%.

[0035] In a specific embodiment of the present invention, the content of resveratrol in the red grape yeast complex is ≥50 wt %.

[0036] In a specific embodiment of the present invention, the content of curcumin in the turmeric extract is ≥3 wt%.

[0037] In a specific embodiment of the present invention, the content of total anthocyanins in blueberry anthocyanins is ≥40 wt%.

[0038] In a specific embodiment of the present invention, the saponin content in ginseng is ≥5wt%.

[0039] In a specific embodiment of the present invention, the angelica extract is a water extract of angelica.

[0040] In a specific embodiment of the present invention, the content of polysaccharides in the Poria cocos extract is ≥10 wt %.

[0041] In a specific embodiment of the present invention, the molecular weight of sodium hyaluronate is 8.02×10 4 ~4.01×10 6 Da, such as 80200 Da, 90000 Da, 200000 Da, 2000000 Da, 4010000 Da or a range consisting of any two thereof.

[0042] The second aspect of the present invention provides a reagent comprising the composition for alleviating ovarian dysfunction provided by the first aspect of the present invention.

[0043] In a specific embodiment of the present invention, the reagent further comprises an auxiliary material acceptable to functional foods or health foods.

[0044] In a specific embodiment of the present invention, the agent comprises at least one of granules, tablets, pills, syrups, capsules and oral liquids.

[0045] The reagents of the present invention may include but are not limited to health foods, functional foods, etc. When preparing the corresponding reagents, corresponding excipients can be added according to actual needs, and the composition and the excipients can be mixed to prepare the reagents in the corresponding dosage form.

[0046] The third aspect of the present invention provides use of the composition of the first aspect or the practice of the second aspect in the preparation of a preparation for preventing and / or alleviating ovarian dysfunction or ovarian damage.

[0047] In a specific embodiment of the present invention, the preparation is used to enhance the activity of ovarian granulosa cells induced by TP.

[0048] In a specific embodiment of the present invention, the preparation is used to upregulate the hormone secretion level of ovarian granulosa cells induced by TP. Further, the hormones include E2 and P.

[0049] In a specific embodiment of the present invention, the preparation inhibits excessive autophagy of ovarian granulosa cells induced by TP by mediating the PI3K / Akt / mTOR pathway.

[0050] In a specific embodiment of the present invention, the preparation is used to upregulate the relative expression levels of p-PI3K, p-Akt and p-mTOR proteins in ovarian granulosa cells induced by TP.

[0051] In a specific embodiment of the present invention, the formulation is used to reduce the methylation level of ovarian granulosa cells induced by TP.

[0052] In a specific embodiment of the present invention, the preparation is used to down-regulate the mRNA expression of DNMT3a and DNMT3b in ovarian granulosa cells induced by TP, and to up-regulate the mRNA expression of TET1 and TET2 in ovarian granulosa cells induced by TP.

[0053] The composition of the present invention can act in the following ways to reduce ovarian granulosa cell damage, thereby achieving the effect of preventing and / or alleviating ovarian dysfunction or ovarian damage: 1. The composition of the present invention can alleviate TP-induced inhibition of ovarian granulosa cell proliferation and enhance the proliferation activity of ovarian granulosa cells; 2. The composition of the present invention can upregulate the secretion levels of E2 and P in ovarian granulosa cells induced by TP and maintain the function of ovarian granulosa cells; 3. The composition of the present invention can activate the PI3K / Akt / mTOR signaling pathway, reduce the level of TP-induced autophagy in ovarian granulosa cells, and maintain cell homeostasis; 4. The composition of the present invention can downregulate the mRNA expression of DNMT3a and DNMT3b in ovarian granulosa cells induced by TP, and upregulate the mRNA expression of TET1 and TET2 in ovarian granulosa cells induced by TP, thereby reducing the methylation level of ovarian granulosa cells, reducing the 5mC level in cells, and increasing the 5hmC level, thereby alleviating cell damage and maintaining cell homeostasis.

[0054] The various raw materials used in the specific embodiments of the present invention can be corresponding commercially available products. The specific information of some raw materials is as follows, but not limited thereto: Collagen tripeptide, manufactured by Wuhan Tiantianhao Biological Products Co., Ltd.; GABA, 20% GABA; Red grape yeast complex, red grape yeast complex powder with a resveratrol content of 50wt%; Camellia chrysantha, commonly available in the market; Turmeric extract, a turmeric extract having a curcumin content of 5 wt%; Blueberry anthocyanins, containing 40 wt% of total anthocyanins; Ginseng, ginseng powder having a saponin content of 6 wt%; Poria cocos extract, a Poria cocos extract having a polysaccharide content of 15 wt%; Sodium hyaluronate, average molecular weight is 200,000 Da.

[0055] Example 1 This embodiment provides a composition for alleviating ovarian dysfunction, the active ingredients of which include the following components in parts by weight: 40 parts of collagen tripeptide, 3 parts of γ-aminobutyric acid, 3 parts of red grape yeast complex, 2 parts of yeast extract, 6 parts of Sophora japonica flower extract, 2 parts of Camellia chrysantha, 3 parts of Curcuma longa extract, 2 parts of blueberry anthocyanins, 6 parts of Panax ginseng, 3 parts of Angelica sinensis extract, 3 parts of Poria cocos extract, and 0.3 parts of sodium hyaluronate.

[0056] The composition of this example also includes excipients, including microcrystalline cellulose, hydroxypropyl methylcellulose ethanol solution, magnesium stearate, sorbitol, and silicon dioxide. The amounts of each excipient are 10.7%, 3%, 2%, 9.5%, and 1.5% of the total mass of the composition (including excipients and active ingredients), respectively. The hydroxypropyl methylcellulose ethanol solution is prepared by mixing 95% aqueous ethanol with hydroxypropyl methylcellulose; the concentration of hydroxypropyl methylcellulose in the hydroxypropyl methylcellulose ethanol solution is 2 wt%, but this is not limited to this.

[0057] This embodiment provides a method for preparing a composition for alleviating ovarian dysfunction, comprising the following steps: (1) Weigh the above raw materials in proportion and set aside. Grind sorbitol through an 80-mesh sieve and set aside.

[0058] (2) Collagen tripeptide, yeast extract, Sophora japonica flower extract, Camellia chrysantha, turmeric extract, ginseng, angelica extract, Poria cocos extract, and microcrystalline cellulose were placed in a wet granulator and mixed for 3 minutes, then hydroxypropyl methylcellulose ethanol solution was added and the mixture was continued to mix for 3 minutes before discharging.

[0059] (3) The granulated material is passed through a 20-mesh rocking granulator, then placed in an oven to dry, passed through a 30-mesh sieve, and then mixed with the remaining materials γ-aminobutyric acid, red grape yeast compound powder, sodium hyaluronate, blueberry anthocyanin, magnesium stearate, sorbitol and silicon dioxide in a three-dimensional mixer and waited for tableting.

[0060] (4) The material from step (3) is tableted and coated, with the tablet weight being 1000 mg / tablet ± 5%.

[0061] Example 2 This example refers to the composition for alleviating ovarian dysfunction and its preparation method of Example 1, with the only difference being that the active ingredient composition is different.

[0062] The composition for alleviating ovarian dysfunction of this embodiment comprises the following active ingredients, measured by weight: 10 parts of collagen tripeptide, 1 part of γ-aminobutyric acid, 1 part of red grape yeast complex, 0.5 parts of yeast extract, 1 part of Sophora japonica flower extract, 0.5 parts of Camellia chrysantha, 0.5 parts of Curcuma longa extract, 0.1 parts of blueberry anthocyanins, 1 part of Panax ginseng, 1 part of Angelica sinensis extract, 1 part of Poria cocos extract, and 0.1 parts of sodium hyaluronate.

[0063] Example 3 This example refers to the composition for alleviating ovarian dysfunction and its preparation method of Example 1, with the only difference being that the active ingredient composition is different.

[0064] The composition for alleviating ovarian dysfunction of this embodiment comprises the following active ingredients, measured by weight: 50 parts of collagen tripeptide, 5 parts of gamma-aminobutyric acid, 6 parts of red grape yeast complex, 2 parts of yeast extract, 6 parts of Sophora japonica flower extract, 2 parts of Camellia chrysantha, 6 parts of turmeric extract, 2.7 parts of blueberry anthocyanins, 8 parts of ginseng, 6 parts of angelica extract, 6 parts of Poria cocos extract, and 0.3 parts of sodium hyaluronate.

[0065] Experimental Example 1 The physical and chemical properties of the compositions of Examples 1 to 3 of the present invention were characterized. The product characteristic indicators of the compositions of each embodiment met the following requirements: protein (g / 100g) ≥33, peptide content (g / 100g) ≥30, crude polysaccharide (g / 100g) ≥8, and γ-aminobutyric acid (g / 100g) ≥0.8.

[0066] Experimental Example 2 1. The alleviating effect of the composition on TP-induced KGN cell activity inhibition Human ovarian granulosa cells KGN were used as test cells and purchased from Shanghai Biopharmaceutical Co., Ltd. They were divided into control group, TP group, Example 1 group, Example 2 group, and Example 3 group. 100 nmol / L TP was added to the TP group, Example 1 group, Example 2 group, and Example 3 group for 12 hours, and an equal amount of complete culture medium was added to the control group as a control. Then, complete culture medium containing the respective compositions was added to the Example 1 group, Example 2 group, and Example 3 group, so that the final concentration of the corresponding composition in each group was 60 mg / L. After culturing for 48 hours, the cell viability was detected by CCK-8 method. The test results are as follows. Figure 1 As shown. (Compared with the control group, # p <0.05,## p <0.01,### p <0.005,#### p <0.001; compared with TP group, * p <0.05,** p <0.01,*** p <0.005,**** p <0.001; the same applies to the subsequent from Figure 1 As can be seen from the results, the relative proliferation activity of KGN cells induced by TP was significantly lower than that of the control group. After intervention with the compositions of each example, the relative proliferation activity of KGN cells was increased. Among them, the cell activity was significantly increased after intervention with the composition of Example 1, and was higher than that of Examples 2 and 3, indicating that the composition of Example 1 is more effective in alleviating the TP-induced inhibition of KGN cell activity than the compositions of Examples 2 and 3. Subsequent functional verification was further conducted using the composition of Example 1 as the test substance.

[0067] 2. Effect of the composition on E2 and P secretion levels in TP-treated KGN cells The composition of Example 1 was used as the test substance, and KGN cells were seeded in 6-well plates and divided into a control group, a TP group, and a composition group. 100 nmol / L TP was added to the TP group and the composition group for 12 hours, and the control group was treated with an equal amount of complete culture medium as a control. Complete culture medium containing the composition of Example 1 was then added to the composition group to a final concentration of 60 mg / L. After culturing for 48 hours, the cell supernatant was collected and tested according to the instructions of the detection kit. The test results are as follows: Figure 2 shown.

[0068] from Figure 2 It can be seen that the E2 and P levels of KGN cells were significantly reduced after TP treatment ( p <0.001), after the intervention of the combination, the levels of E2 and P were significantly improved ( p <0.001), indicating that the composition of Example 1 of the present invention can significantly enhance the E2 and P secretion levels of KGN cells treated with TP.

[0069] 3. The composition alleviates excessive autophagy in TP-treated KGN cells based on the PI3K / Akt / mTOR pathway KGN cells were seeded in 6-well plates and divided into a control group, a TP group, and a combination group. 100 nmol / L TP was added to the TP and combination groups for 12 hours, respectively. The control group received an equal amount of complete culture medium as a control. Complete culture medium containing the composition of Example 1 was then added to the combination group to a final concentration of 60 mg / L. After 48 hours of incubation, KGN cells were lysed in an ice bath using a lysis buffer to extract total protein, and protein concentration was determined by the BCA assay. Western blot analysis was performed to determine the expression of relevant proteins. Equal amounts of protein per lane were loaded onto SDS-PAGE gels, electrophoresed, and transferred to PVDF membranes. Blocked with 5% skim milk powder for 1 hour, primary antibodies against Beclin 1, LC3 II, LC3 I, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, and β-actin were added, and the cells were incubated overnight at 4°C. After washing, secondary antibodies were added and incubated at room temperature for 30 minutes. ECL reagent was used for development, and the optical density of the target bands was analyzed using the AlphaEaseFC software processing system. β-actin served as a control. At the same time, MDC staining was used to detect the level of cell autophagy. The test results are as follows Figure 3 and Figure 4 shown.

[0070] from Figure 3 It can be seen that the expression of LC3Ⅱ / LC3Ⅰ and Beclin1 proteins in KGN cells increased significantly after TP treatment ( p<0.01), which is consistent with the results of MDC staining. TP-induced KGN cell autophagy increased. After the intervention of the combination, the expression of LC3Ⅱ / LC3Ⅰ, Beclin1 protein and MDC level were significantly reduced ( p <0.05).

[0071] from Figure 4 It can be seen that the expression of p-PI3K / PI3K, p-Akt / Akt and p-mTOR / mTOR proteins in KGN cells was significantly decreased after TP treatment ( p <0.001), after the intervention of the combination, the expression of p-PI3K / PI3K, p-Akt / Akt and p-mTOR / mTOR proteins were significantly increased ( p <0.005), indicating that the composition can promote the expression of PI3K, Akt and mTOR protein phosphorylation.

[0072] The PI3K inhibitor LY294002 was further used to verify the effect of the composition on the PI3K / Akt / mTOR pathway of KGN cells. Referring to the composition group, the PI3K inhibitor LY294002 (final concentration 10 μmol / L) was added to the complete culture medium containing the composition of Example 1 (final concentration 60 mg / L). The test results are as follows: Figure 5 As shown ( Figure 5 Compared with the control group, # p <0.05,## p <0.01,### p <0.005,#### p <0.001; compared with TP group, * p <0.05,** p <0.01,*** p <0.005, **** p <0.001; compared with the TP+combination group, △ p <0.05, △△ p <0.01, △△△ p <0.005, △△△△ p <0.001). The results showed that the expression of p-PI3K / PI3K, p-Akt / Akt and p-mTOR / mTOR proteins was significantly reduced after the addition of LY294002 ( p <0.01), indicating that LY294002 weakened the promoting effect of the composition on the expression of p-PI3K, p-Akt, and p-mTOR proteins. This proves that the composition regulates the level of TP-induced autophagy in KGN cells by mediating the PI3K / Akt / mTOR pathway.

[0073] 4. Effect of the composition on the methylation level of KGN cells treated with TP KGN cells were seeded in 6-well plates and divided into a control group, a TP group, and a combination group. 100 nmol / L TP was added to the TP group and the combination group for 12 h, and the control group was treated with an equal amount of complete culture medium as a control. Complete culture medium containing the composition of Example 1 was then added to the combination group to a final concentration of 60 mg / L. After culturing for 48 h, total RNA from KGN cells was extracted using the TRIzol method and fluorescent quantitative PCR was used to analyze the relative expression levels of DNMT3a, DNMT3b, TET1, and TET2 mRNA. The PCR reaction system, primer sequences, and amplification lengths are shown in Tables 1 and 2, and the test results are shown in Tables 1 and 2. Figure 6 .

[0074] Table 1 PCR reaction system

[0075] Table 2 Primer sequences and amplification lengths

[0076] from Figure 6 As shown in the results, TP treatment of KGN cells significantly increased the mRNA expression of methylation-related genes DNMT3a and DNMT3b ( p <0.005), and the mRNA expression of demethylation genes TET1 and TET2 was extremely significantly decreased ( p <0.001), indicating that TP promotes DNA methylation in KGN cells. Intervention with the composition reduced the mRNA expression of DNMT3a and DNMT3b and enhanced the mRNA expression of TET1 and TET2. This suggests that the composition can reduce TP-induced DNA methylation levels in KGN cells, thereby maintaining cellular homeostasis.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for alleviating ovarian dysfunction based on cell autophagy and DNA methylation levels, characterized in that: The invention comprises the following components in parts by weight: 10-50 parts of collagen tripeptide, 1-5 parts of gamma-aminobutyric acid, 1-6 parts of red grape yeast complex, 0.5-2 parts of yeast extract, 1-6 parts of Sophora japonica flower extract, 0.5-2 parts of Camellia chrysantha, 0.5-6 parts of turmeric extract, 0.1-2.7 parts of blueberry anthocyanin, 1-8 parts of ginseng, 1-6 parts of angelica extract, 1-6 parts of Poria cocos extract and 0.1-0.3 parts of sodium hyaluronate.

2. The composition according to claim 1, characterized in that The composition has at least one of the following characteristics: (1) Protein content ≥33wt%; (2) Peptide content ≥30wt%; (3) Crude polysaccharide content ≥8wt%; (4) The content of γ-aminobutyric acid is ≥0.8wt%.

3. A reagent, characterized in that A composition for alleviating ovarian dysfunction according to any one of claims 1 to 2.

4. The reagent according to claim 3, characterized in that It also includes acceptable excipients for functional foods or health foods.

5. The reagent according to claim 3, characterized in that The agent includes at least one of granules, tablets, pills, syrups, capsules and oral liquids.

6. Use of the composition according to any one of claims 1 to 2 or the agent according to any one of claims 3 to 5 in the preparation of a preparation for preventing and / or alleviating ovarian dysfunction or ovarian damage.

7. The use according to claim 6, characterized in that The preparation is used for enhancing the activity of ovarian granulosa cells induced by triptolide.

8. The use according to claim 6, characterized in that The preparation is used to upregulate the hormone secretion level of ovarian granulosa cells induced by triptolide; Preferably, the hormones include E2 and P.

9. The use according to claim 6, characterized in that The preparation inhibits the excessive autophagy of ovarian granulosa cells induced by triptolide by mediating the PI3K / Akt / mTOR pathway; Preferably, the preparation is used to upregulate the relative expression levels of p-PI3K, p-Akt and p-mTOR proteins in ovarian granulosa cells induced by triptolide.

10. The use according to claim 6, characterized in that The preparation is used to reduce the methylation level of ovarian granulosa cells induced by triptolide; Preferably, the preparation is used to down-regulate the mRNA expression of DNMT3a and DNMT3b in ovarian granulosa cells induced by triptolide, and is used to up-regulate the mRNA expression of TET1 and TET2 in ovarian granulosa cells induced by triptolide.

Images