Composition for improving ovarian function as well as preparation method and application thereof

Abstract

The invention provides a composition for improving an ovarian function as well as a preparation method and application thereof, and relates to the technical field of compositions. The composition is prepared from urolithin and S-equol. The mass ratio of the urolithin to the S-equol in the composition is (1-30): 1. The composition can significantly improve the ovarian function and repair ovarian structure damage, and through the synergistic effect of urolithin and S-equol, the composition can significantly improve the survival rate of zebra fish oocyte, effectively increase the ovary weight index, reverse cisplatin-induced ovarian atrophy, and inhibit activation of apoptosis pathway key molecule Caspase-3. And the composition can improve the fertility, reduce the problem of poor development of the offspring, and realize dual improvement of the fertility and the health of the offspring.

Description

Technical Field

[0001] This invention belongs to the field of composition technology, specifically relating to a composition for improving ovarian function, its preparation method, and its application. Background Technology

[0002] The ovary is the core organ of the female reproductive system, possessing both reproductive functions (producing oocytes and ovulating) and endocrine functions (secreting estrogen, progesterone, etc.). Its functional status directly affects a woman's fertility and physical and mental health. Currently, with increasing social pressure, delayed childbearing age, exposure to environmental toxins, and the widespread use of medical interventions such as radiotherapy and chemotherapy for tumors, the incidence of ovarian dysfunction is rising year by year, mainly manifesting in various forms such as premature ovarian failure, premature ovarian insufficiency, and age-related decline in ovarian function.

[0003] Premature ovarian failure (POF) refers to the decline of ovarian function in women before the age of 40, characterized by amenorrhea, elevated gonadotropin levels, and decreased estrogen levels, accompanied by loss of fertility, and is showing a trend towards affecting younger women. Age-related ovarian function decline is characterized by a decrease in the number and quality of oocytes, a reduced fertilization rate, and an increased risk of embryo implantation failure, becoming one of the important causes of female infertility. Furthermore, chemotherapy drugs such as cisplatin are widely used in clinical cancer treatment, but these drugs have clear ovarian toxicity, directly damaging ovarian granulosa cells and oocytes, inducing apoptosis, and leading to a significantly increased incidence of post-chemotherapy POI, seriously affecting the subsequent fertility needs and quality of life of young cancer patients.

[0004] Ovarian dysfunction not only leads to loss of fertility but also triggers a series of endocrine-related complications, such as osteoporosis, cardiovascular disease, mood disorders, and reproductive system atrophy, severely reducing patients' quality of life and imposing a heavy burden on families and society. Therefore, developing safe and effective interventions to improve ovarian function and enhance fertility has become a crucial issue urgently needing to be addressed in the field of reproductive health.

[0005] Currently, clinical interventions for ovarian dysfunction mainly include hormone replacement therapy (HRT), assisted reproductive technology (ART), and drug intervention. However, all of these have significant limitations. Hormone replacement therapy primarily alleviates low estrogen symptoms by supplementing estrogen and progesterone, but it cannot repair ovarian function itself. Furthermore, long-term use may increase the risk of malignant tumors such as breast cancer and endometrial cancer, making it unsuitable for patients with fertility needs and limiting its clinical application. Assisted reproductive technology, such as in vitro fertilization-embryo transfer (IVF-ET), is currently the main method for treating infertility. However, for patients with insufficient oocyte quantity or extremely poor quality, the success rate is extremely low, and it cannot address the fundamental problem of ovarian function decline. It also has drawbacks such as long treatment cycles, high costs, and the risk of multiple pregnancies. Commonly used ovulation-inducing drugs (such as clomiphene and letrozole) can only induce ovulation in the short term, cannot improve ovarian reserve function, and may cause serious side effects such as ovarian hyperstimulation syndrome (OHSS).

[0006] Therefore, there is an urgent need to provide a composition that can effectively repair ovarian structural damage, improve oocyte quality, and improve reproductive outcomes, thus providing a new solution for the treatment of ovarian dysfunction and the improvement of fertility. Summary of the Invention

[0007] To address the above problems, the present invention provides a composition for improving ovarian function, its preparation method, and its application.

[0008] In a first aspect, the present invention provides a composition for improving ovarian function, the composition comprising urolithin and S-estrol.

[0009] Specifically, the mass ratio of urolithiasis to S-estradiol in the composition is (1-30):1; According to some embodiments of the present invention, the mass ratio of urolithin to S-estrol in the composition can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. According to some embodiments of the present invention, the mass ratio of urolithin to S-estrol in the composition can be 1:1, 8:1, or 30:1; Preferably, the mass ratio of urolithiasis to S-estradiol in the composition is 8:1.

[0010] Secondly, the present invention provides a method for preparing the above composition, which is to mix urolithin and S-estrol.

[0011] Thirdly, the present invention provides the use of the above-described composition in the preparation of products that improve ovarian function.

[0012] Specifically, the product has at least one of the following functions: (1) Improve oocyte survival rate; (2) Repairing ovarian function damage; (3) Inhibit the activation of Caspase-3, a key molecule in the apoptosis pathway, and reduce excessive apoptosis of oocytes.

[0013] Fourthly, the present invention provides the use of the above-described composition in the preparation of products that improve ovarian function and enhance fertility.

[0014] Specifically, the product has at least one of the following functions: (1) Increase fertilization rate; (2) Increase the hatching rate; (3) Increase survival rate; (4) Reversing fertility decline; (5) Improve the developmental problems of offspring.

[0015] Specifically, the product in question is a medicine.

[0016] Furthermore, the drug includes excipients.

[0017] Furthermore, the excipients are selected from one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0018] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginate, gelatin, gum arabic, glyceryl monostearate, sodium glycolate starch, guar gum, glycerol, and propylene glycol.

[0019] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0020] Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglycerol-3 polyricinoleate.

[0021] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginate, cellulose ether, and carboxymethyl chitosan.

[0022] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0023] Specifically, the adhesive is selected from at least one of ethanol, starch paste, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinylpyrrolidone, gum arabic, gelatin, and alginic acid.

[0024] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0025] Specifically, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearate fumarate, and poloxamer.

[0026] Specifically, the pH adjuster is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0027] Specifically, the cryoprotectant is selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextrose, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, or formamide.

[0028] Specifically, the flavoring agent is selected from at least one of sweet orange flavoring, vanilla flavoring, strawberry flavoring, milk flavoring, banana flavoring, and cherry flavoring.

[0029] Specifically, the filler is selected from at least one of mannitol, xylitol, sorbitol, maltose, microcrystalline cellulose, glucose, lactose, sucrose, dextrin, starch, sodium alginate, and sodium bicarbonate.

[0030] Specifically, the dosage form of the drug, depending on the route of administration, includes, but is not limited to, gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0031] Furthermore, the gastrointestinal dosage forms include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0032] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to: injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0033] Furthermore, the injectable dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0034] Furthermore, the respiratory drug delivery formulations include, but are not limited to, sprays, aerosols, and powder inhalers.

[0035] Furthermore, the skin delivery dosage forms include, but are not limited to, lotions, ointments, topical solutions, plasters, pastes, and patches.

[0036] Furthermore, the mucosal drug delivery dosage forms include, but are not limited to, eye drops, nasal drops, ophthalmic ointments, sublingual tablets, and patches.

[0037] Furthermore, the cavity drug delivery dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pellets.

[0038] The beneficial effects of this invention are as follows: (1) The composition of the present invention can significantly improve ovarian function and repair ovarian structural damage. Through the synergistic effect of urolithin and S-estrol, the composition of the present invention can significantly improve the survival rate of zebrafish oocytes, effectively increase the ovarian weight index, reverse cisplatin-induced ovarian atrophy, inhibit the activation of Caspase-3, a key molecule in the apoptosis pathway, significantly reduce the positive area of ​​Caspase-3 immunohistochemical staining in ovarian tissue, reduce excessive apoptosis of ovarian granulosa cells and oocytes, repair the structural integrity of ovarian tissue, and restore normal physiological function of the ovary.

[0039] (2) The composition of the present invention can enhance fertility, increase the fertilization rate, hatching rate and 3-day survival rate of zebrafish. In the cisplatin-induced POI mouse model, the composition can significantly enhance the reproductive capacity of the model mice and effectively reverse the problems of reduced litter size and poor offspring development caused by POI, thus achieving a dual improvement in fertility and offspring health. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0041] Source of experimental materials: Urolithiasis precipitate was purchased from Denuobangshang Company, product number 1143-70-0; S-Estrol was purchased from Shanghai Ergothioneine Biotechnology Group Co., Ltd., product number 531-95-3.

[0042] Example 1 Preparation of the composition The composition comprises 1 part urolithin and 1 part S-estrol.

[0043] The composition is prepared by mixing urolithin and S-estradiol.

[0044] Example 2 Preparation of the composition The composition comprises 8 parts urolithin and 1 part S-estradiol.

[0045] The composition was prepared using the same method as in Example 1.

[0046] Example 3 Preparation of the composition The composition comprises 30 parts of urolithin and 1 part of S-estrol.

[0047] The composition was prepared using the same method as in Example 1.

[0048] Comparative Example 1 The composition contains only 8 parts of urolithiasis.

[0049] Comparative Example 2 The composition contains only 1 part of S-estrol.

[0050] Comparative Example 3 The composition contains 40 parts urolithin and 1 part S-estrol in a mass ratio of 40:1.

[0051] Comparative Example 4 The composition comprises 8 parts coenzyme Q10 and 1 part S-estrol.

[0052] The composition was prepared using the same method as in Example 1.

[0053] Experimental Example 1: Composition Improves Ovarian Function 1. Zebrafish Experiment Experimental methods: Female zebrafish aged 15 months, weighing 0.4-0.6g, were acclimatized for 1 week (water temperature 26±1℃, light-dark cycle 12h:12h, pH 7.0-7.5, dissolved oxygen ≥6mg / L).

[0054] Experimental Groups: Control group: Standard aquaculture water; Example 1 group: S-estrol 9.5 μg / L, urolithiasis 9.5 μg / L; Example 2 group: S-estrol 9.5 μg / L, urolithiasis 76 μg / L; Example 3 group: S-estrol 9.5 μg / L, urolithiasis 285 μg / L; Comparative Example 1: Urolithiasis 76 μg / L; Comparative Example 2: S-estrol 9.5 μg / L; Comparative Example 3: S-estrol 9.5 μg / L, urolithiasis 95 μg / L; Comparative Example 4: S-estrol 9.5 μg / L, Coenzyme Q10 76 μg / L; Each group has 3 parallels, with 10 zebrafish in each parallel and an exposure volume of 10L.

[0055] The zebrafish were continuously exposed for 4 weeks, during which the rearing conditions were the same as the adaptive rearing conditions. The condition of the zebrafish was observed daily, and dead fish were removed in a timely manner.

[0056] After exposure, zebrafish were anesthetized, their bodies were rinsed with sterile saline, and both ovaries were separated under a microscope. Adipose tissue and connective tissue were removed, and the fish were rinsed three times with saline. The surface moisture was then dried, and oocyte viability was tested.

[0057] Oocyte viability assay: (1) Take a portion of ovarian tissue, gently pipette the oocytes in Hank's solution to disperse them, filter to remove tissue debris, and adjust the cell concentration to 1×10⁻⁶. 4 cells / mL; (2) Add FDA working solution (final concentration 5 μg / mL) and PI working solution (final concentration 2 μg / mL), and incubate at 37°C in the dark for 15 min; (3) Observe under a fluorescence microscope (excitation wavelength 488nm / FDA, 535nm / PI), count 1000 oocytes, live cells: FDA positive (green fluorescence), PI negative; dead cells: PI positive (red fluorescence), FDA negative.

[0058] Calculate oocyte survival rate = (number of live cells / total number of cells) × 100%.

[0059] Table 1 Oocyte survival rate

[0060] 2. Mouse experiments Experimental methods: SPF-grade ICR female mice, 5-6 weeks old, were acclimatized for one week and kept in a 12-day light / dark cycle. They were randomly divided into 9 groups of 6 mice each, as follows: Control group: given normal saline; Model group: cisplatin 1.5 mg / kg was administered; Example 1 group: Intraperitoneal injection of cisplatin 1.5 mg / kg + gavage administration of Example 1 group (S-estrol 0.1 mg / ml, urolithiasis 0.1 mg / ml) 0.1 ml; Example 2 group: Intraperitoneal injection of cisplatin 1.5mg / kg + gavage administration of Example 2 group (S-estrol 0.1mg / ml, urolithiasis 0.8mg / ml) 0.1ml; Example 3 group: Intraperitoneal injection of cisplatin 1.5mg / kg + gavage administration of Example 3 group (S-estrol 0.1mg / ml, urolithiasis 3mg / ml) 0.1ml; Comparative Example 1: Intraperitoneal injection of cisplatin 1.5 mg / kg + gavage administration of 0.1 ml of urolithiasis (0.8 mg / ml); Comparative Example 2: Intraperitoneal injection of cisplatin 1.5 mg / kg + oral gavage of 0.1 ml of S-estrol (0.1 mg / ml); Comparative group 3: Intraperitoneal injection of cisplatin 1.5 mg / kg + gavage administration of comparative group 3 (S-estrol 0.1 mg / ml, urolithiasis 4 mg / ml) 0.1 ml; Comparative Group 4: Intraperitoneal injection of cisplatin 1.5 mg / kg + oral gavage of comparative group 4 (S-estrol 0.1 mg / ml, coenzyme Q10 0.8 mg / ml) 0.1 ml.

[0061] The experiment lasted for 4 weeks. For the first two weeks, cisplatin was injected intraperitoneally once a week to establish the model. After the model was established, the drug was administered by gavage daily for 2 weeks. The weight of the mice was recorded daily.

[0062] After the experiment, mice were anesthetized with sodium pentobarbital. The ovary and surrounding adipose tissue were completely removed from the mouse's abdominal cavity. The ovary and surrounding adipose tissue were completely separated under a stereomicroscope. Excess blood and fat on the ovary were washed away in physiological saline. After the water was absorbed by filter paper, the ovary was weighed.

[0063] Ovarian weight index = ovarian weight / body weight.

[0064] Immunohistochemistry: (1) The baking, dewaxing, and dehydration processes are carried out sequentially; (2) Rinsing: After rinsing the dehydrated and dewaxed ovarian tissue under tap water for 2 minutes, place it in PBS buffer. Prevent detachment during the operation. (3) Antigen retrieval: Place the ovarian tissue slices in the antigen retrieval solution. The amount of retrieval solution should be sufficient to cover the tissue slices after antigen retrieval to avoid drying out the slices. Place the slides immersed in the antigen retrieval solution in a microwave oven and heat on high for 5 minutes, then on low for 15 minutes. After heating, remove the slides and allow them to cool naturally at room temperature. (4) Washing: After cooling, remove the slide from the antigen retrieval solution and wash it with PBS buffer on a shaker for 5 min*3 times. (5) Blocking endogenous peroxidase: Drop reagent 1 (endogenous peroxidase blocker) onto the slice to cover the tissue and incubate at room temperature for 10 min; (6) Washing: Shake the glass slide slightly to dry it, and wash it on a decolorizing shaker for 5 minutes*3 times; (7) Blocking: Add reagent 2 (working solution of normal goat serum for blocking) to the ovarian tissue section, ensuring complete coverage of the tissue, incubate at room temperature for 10 min, pour off the serum, do not wash; (8) Incubation of primary antibody: Add an appropriate amount of primary antibody dilution solution to the ovarian tissue section, ensuring complete coverage of the tissue, and then place the section in a humidified box and place it in a refrigerator at 4°C overnight; (9) Rewarming: The humidification box was taken out of the refrigerator at 4℃ and allowed to warm to room temperature for 30 minutes; (10) Washing: Shake the glass slide slightly to dry it, discard the primary antibody dilution solution, and wash it with PBS buffer on a shaker for 5 min*3 times. (11) Incubation of secondary antibody: Add 3 drops of reagent to the slide and incubate at room temperature for 10 min; (12) Washing: Shake the glass slide slightly to dry it, immerse it in PBS buffer, and wash it on a shaker for 5 min * 3 times; (13) Add reagent 4: Add reagent 4 (horseradish enzyme labeled streptavidin working solution) to the slide, ensuring that the working solution can completely cover the tissue, and incubate at room temperature for 10 min; (14) Washing: Shake the glass slide slightly to dry it, and wash it with PBS buffer on a decolorizing shaker for 5 min * 3 times; (15) Color development: Observe the tissue with added DAB color development solution under a microscope, and stop the reaction with tap water when appropriate; (16) Hematoxylin staining: After the staining is completed, rinse the slide in tap water for 2 minutes and then soak it in the prepared hematoxylin staining tank for 20 seconds. (17) Rinsing: Rinse the hematoxylin-stained sections under tap water for 2 minutes; (18) Differentiation: Immerse ovarian tissue slices in 1% hydrochloric acid alcohol for 6 seconds, then rinse under running tap water for 2 minutes; (19) Blueing: Immerse the ovarian slices in a saturated lithium carbonate solution for 5 seconds, then rinse with tap water for 2 minutes; (20) Dehydration: Soak in alcohol of 100%, 95%, 80% and 75% concentration gradients for 2 min in sequence to dehydrate; (21) Clearing: Soak the tissue sections in xylene I and xylene II for 15 min each; (22) Mounting: In a fume hood, drop neutral resin onto the ovarian slices on the slide, cover with a coverslip, and handle carefully to prevent air bubbles from forming. After mounting, place the slide in a fume hood to air dry. (23) Taking pictures: Review the slides under a microscope and take pictures.

[0065] Experimental results: Table 2 Ovarian weight index

[0066] Note: Compared with the control group, *** p <0.001, compared to the model group, # p <0.05, ## p <0.01.

[0067] Table 3. Area of ​​Caspase-3 immunohistochemical staining positive (%)

[0068] Note: Compared with the control group, *** p <0.001, compared to the model group, # p <0.05, ## p <0.01.

[0069] Experimental Example 2: Composition Improves Fertility 1. Zebrafish Experiment Experimental methods: Female zebrafish aged 15 months, weighing 0.5-0.8g (male zebrafish should be matched with females in weight), should be acclimatized for 1 week (water temperature 26±1℃, light-dark cycle 12h:12h, pH 7.0-7.5, dissolved oxygen ≥6mg / L) to ensure normal reproductive capacity.

[0070] Experimental Groups: Control group: Standard aquaculture water; Example 1 group: S-estrol 9.5 μg / L, urolithiasis 9.5 μg / L; Example 2 group: S-estrol 9.5 μg / L, urolithiasis 76 μg / L; Example 3 group: S-estrol 9.5 μg / L, urolithiasis 285 μg / L; Comparative Example 1: Urolithiasis 76 μg / L; Comparative Example 2: S-estrol 9.5 μg / L; Comparative Example 3: S-estrol 9.5 μg / L, urolithiasis 380 μg / L; Comparative Example 4: S-estrol 9.5 μg / L, Coenzyme Q10 76 μg / L; The zebrafish were continuously exposed for 4 weeks, during which the rearing conditions were the same as those for acclimatization. The condition of the zebrafish was observed daily, and dead fish were removed in a timely manner.

[0071] Twelve female zebrafish were selected for each group and paired with healthy male zebrafish in a 1:1 ratio (12 pairs / group). On the last day at 18:00, the paired parent fish were placed in a breeding tank containing the corresponding exposure concentration, maintaining a light-dark cycle of 14:10h and a water temperature of 26±1℃. The lighting was turned on at 8:00 the next day, and the zebrafish naturally laid eggs. Eggs were collected until 10:00.

[0072] Statistics on egg production and fertilization rate: Collect all eggs from the breeding tank and transfer them to a grid-lined culture dish. Count the total number of eggs laid under a stereomicroscope (record the number of eggs laid by each pair of parent fish separately). Four hours after spawning, the morphology of the eggs was observed under a microscope: fertilized eggs appeared transparent with visible yolk granules; unfertilized eggs appeared cloudy with no yolk aggregation. The number of fertilized eggs was counted, and the fertilization rate was calculated. Fertilization rate (%) = (Number of fertilized eggs / Total number of eggs laid) × 100%.

[0073] Hatching rate statistics: Each group of fertilized eggs was randomly divided into 3 replicates (≥100 eggs per replicate), transferred to petri dishes, and incubated at a constant temperature of 28℃, with half of the culture medium replaced daily. Three days after spawning, the number of hatched fry was observed and counted, and the hatching rate was calculated. Hatching rate (%) = (Number of fry hatched in 72 hours / Number of fertilized eggs) × 100%.

[0074] Embryo survival rate at 3 days: After hatching, continue culturing until 72 hours after spawning, count the number of surviving fry (active and without deformities), and calculate the survival rate: Embryo survival rate at 3 days (%) = (Number of surviving larvae at 72 hours / Number of fertilized eggs) × 100%.

[0075] Experimental results: Table 4 Fertilization rate (%)

[0076] Table 5 Hatching Rate (%)

[0077] Table 6. Embryo survival rate at 3 days (%)

[0078] 2. Mouse experiments Experimental methods: SPF-grade ICR female mice, 5-6 weeks old, were acclimatized for one week and kept in a 12-mouse light / dark cycle. They were randomly divided into 9 groups of 10 mice each, as follows: Control group: given normal saline; Model group: cisplatin 1.5 mg / kg was administered; Example 1 group: Intraperitoneal injection of cisplatin 1.5 mg / kg + gavage administration of Example 1 group (S-estrol 0.1 mg / ml, urolithiasis 0.1 mg / ml) 0.1 ml; Example 2 group: Intraperitoneal injection of cisplatin 1.5mg / kg + gavage administration of Example 2 group (S-estrol 0.1mg / ml, urolithiasis 0.8mg / ml) 0.1ml; Example 3 group: Intraperitoneal injection of cisplatin 1.5mg / kg + gavage administration of Example 3 group (S-estrol 0.1mg / ml, urolithiasis 3mg / ml) 0.1ml; Comparative Example 1: Intraperitoneal injection of cisplatin 1.5 mg / kg + gavage administration of 0.1 ml of urolithiasis (0.8 mg / ml); Comparative Example 2: Intraperitoneal injection of cisplatin 1.5 mg / kg + oral gavage of 0.1 ml of S-estrol (0.1 mg / ml); Comparative group 3: Intraperitoneal injection of cisplatin 1.5 mg / kg + gavage administration of comparative group 3 (S-estrol 0.1 mg / ml, urolithiasis 4 mg / ml) 0.1 ml; Comparative Group 4: Intraperitoneal injection of cisplatin 1.5 mg / kg + oral gavage of comparative group 4 (S-estrol 0.1 mg / ml, coenzyme Q10 0.8 mg / ml) 0.1 ml.

[0079] The experiment lasted for 4 weeks. For the first two weeks, cisplatin was injected intraperitoneally once a week to establish the model. After the model was established, the drug was administered by gavage daily for 2 weeks. The weight of the mice was recorded daily.

[0080] At the end of the fourth week, 8 female mice were selected from each group and housed together with healthy male mice in a 1:1 ratio, for a total of 8 pairs / group; Observe the vaginal plugs of mice daily (the presence of a vaginal plug indicates successful mating), and record the mating time; After the mice became pregnant (delivered on days 19-21 after mating), the number of pups in each litter was recorded (total number of pups = sum of the number of pups delivered by all mother mice in each group), and the weight of the pups was measured using an electronic scale on days 1, 3, and 7 after birth (5 pups were randomly selected from each litter for measurement, and the average was taken).

[0081] Table 7. Average number of pups per litter (pups / litter)

[0082] Table 8. Weight of cubs at 7 days after birth (g)

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for improving ovarian function, characterized in that, The composition consists of urolithin and S-estrol.

2. The composition according to claim 1, characterized in that, The mass ratio of urolithiasis to S-estradiol in the composition is (1-30):

1.

3. The composition according to claim 1, characterized in that, The mass ratio of urolithiasis to S-estradiol in the composition is 8:

1.

4. A method for preparing the composition according to any one of claims 1-3, characterized in that, The solution is obtained by mixing urolithin and S-estrol.

5. The use of the composition according to any one of claims 1-3 in the preparation of products that improve ovarian function.

6. The use of the composition according to any one of claims 1-3 in the preparation of products that improve ovarian function and enhance fertility.

7. The application according to any one of claims 5-6, characterized in that, The product in question is a medicine.

8. The application according to claim 7, characterized in that, The drug includes excipients.

9. The application according to claim 8, characterized in that, The excipients are selected from one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

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