An S-estrol composition for delaying ovarian aging and its uses

Abstract

This invention discloses an S-estrol composition for delaying ovarian aging and its uses, relating to the fields of biomedicine and health products. The S-estrol composition for delaying ovarian aging comprises, by weight, the following components: 1-5 parts S-estrol, 2-8 parts soy isoflavones, 3-10 parts coenzyme Q10, 2-6 parts vitamin E, 4-12 parts vitamin C, 0.01-0.05 parts folic acid, 0.1-0.3 parts zinc source, 0.5-1.5 parts anti-aging protective agent, 50-80 parts carrier, and 10-15 parts purified water. This invention uses high-purity S-estrol as the core, combined with soy isoflavones, coenzyme Q10 and a variety of antioxidant nutrients in a scientific formulation to form a synergistic system for estrogen receptor signal regulation and antioxidant defense. By constructing a complex antioxidant network and introducing novel anti-aging protectants for synergistic effect, the activity of antioxidant enzymes tends to increase and the level of lipid peroxidation tends to decrease, thereby improving the oxidative stress state in ovarian cells as a whole.

Description

Technical Field

[0001] This invention relates to the field of biomedicine and health care products, specifically to an S-estrol composition for delaying ovarian aging and its uses. Background Technology

[0002] Ovarian aging is a significant marker of aging in the female reproductive system and overall body. Its functional decline can lead to increased apoptosis of ovarian granulosa cells and an imbalance in oxidative stress, resulting in estrogen imbalances, abnormal follicle development, and a range of menopausal symptoms such as hot flashes, insomnia, and osteoporosis, severely impacting women's physiological health and quality of life. Currently, interventions for ovarian aging are limited, with hormone replacement therapy being the most common clinical approach. However, this method carries potential risks such as breast hyperplasia and endometrial lesions, and its applicability is strictly limited. Natural plant active ingredients have become a research hotspot due to their high safety. While phytoestrogens such as soy isoflavones have been shown to have a certain protective effect on the ovaries, their bioavailability is low when used alone, their target sites are limited, and their effect on delaying ovarian aging is limited. Existing related compositions also suffer from problems such as unreasonable ingredient compatibility, poor stability of active ingredients, and weak targeting of ovarian tissue. Some formulations do not take into account the dual needs of inhibiting oxidative stress and regulating apoptosis, and lack strict control over the purity, enantiomeric ratio, and receptor affinity of key active ingredients such as S-estradiol, making it difficult to achieve efficient and safe intervention for ovarian aging. In addition, existing compositions are prone to oxidative degradation of active ingredients during preparation, leading to reduced product potency. At the same time, the lack of dedicated anti-aging protective agents for synergistic effect prevents the full synergistic effect of each component from being fully utilized. Therefore, developing a composition with scientifically formulated ingredients, high purity of active ingredients, good stability, multi-target inhibition of ovarian granulosa cell apoptosis, reduction of oxidative stress, and high safety has become a technical challenge that urgently needs to be solved in the field of delaying ovarian aging. Summary of the Invention

[0003] The purpose of this invention is to address the limitations of existing ovarian aging intervention methods and the problems of unreasonable ingredient compatibility, poor stability of active ingredients, and lack of strict control over key components in existing natural plant active ingredient compositions. This invention provides an S-estrol composition for delaying ovarian aging and its uses. It aims to safely and effectively delay ovarian function decline by inhibiting ovarian granulosa cell apoptosis through multiple targets and reducing ovarian oxidative stress. To achieve the above objectives, the technical solution adopted by the present invention is as follows: An S-estrol composition for delaying ovarian aging, comprising, by weight, the following components: 1-5 parts S-estrol, 2-8 parts soy isoflavones, 3-10 parts coenzyme Q10, 2-6 parts vitamin E, 4-12 parts vitamin C, 0.01-0.05 parts folic acid, 0.1-0.3 parts zinc source, 0.5-1.5 parts anti-aging protectant, 50-80 parts carrier, and 10-15 parts purified water; The anti-aging protective agent is a compound represented by Formula 1; Formula 1: ; In Formula 1, the substituent R1 is selected from: , , Any one of them; * indicates a connection point. Furthermore, the purity of the S-estrol is ≥98%, and the affinity of S-estrol for ERβ is 8 to 12 times that for ERα. Furthermore, the soy isoflavones have a purity of ≥95% and are composed of daidzein and genistein in a mass ratio of 2:1. Furthermore, the vitamin E is D-α-tocopherol with a purity ≥96%; the vitamin C has a purity ≥99%; and the folic acid has a purity ≥98%. Furthermore, the zinc source is at least one of zinc gluconate, zinc citrate, zinc lactate, and zinc glycinate. Furthermore, the carrier is selected from at least one of microcrystalline cellulose, lactose, starch, and mannitol. Furthermore, the anti-aging protective agent is any one of the following compounds: ; . A method for preparing an S-estrol composition for delaying ovarian aging includes the following steps: S1 Raw Material Pretreatment: Take the S-estradiol, soy isoflavones, coenzyme Q10, vitamin E, vitamin C, folic acid, zinc source and carrier, crush them separately, pass them through an 80-120 mesh sieve to remove impurities, and then place them in an environment with relative humidity ≤45% and temperature of 20-25℃ for later use. S2 Mixing: First, put the S-estradiol, soy isoflavones, coenzyme Q10, vitamin E, vitamin C, folic acid, zinc source, and anti-aging protectant into a mixer and mix at 150-200 r / min for 15-25 min to obtain a premix; then add the carrier to the premix and continue mixing at 120-160 r / min for 20-30 min to ensure uniform mixing and obtain a mixture; S3 Molding: Add the purified water to the mixture, granulate using a granulator, and pass the granules through a 40-60 mesh sieve to obtain granules; S4 Drying and Packaging: Place the granules in a drying oven and dry at 40-50℃ for 1-2 hours, controlling the moisture content to ≤5%; package in a light-proof and sealed manner to obtain an S-estradiol composition for delaying ovarian aging. Furthermore, in step S1, the S-estrol pulverization is carried out under nitrogen protection; after the carrier is pulverized, it needs to be dried at a temperature of 50-60℃ for 2-3 hours to ensure that the moisture content of the carrier is ≤3%. Furthermore, in step S2, nitrogen gas is introduced for protection during the mixing process, with a nitrogen flow rate of 0.5-1 L / min. The application of an S-estrol composition for delaying ovarian aging in the preparation of drugs / health products that inhibit ovarian granulosa cell apoptosis, reduce ovarian oxidative stress, or delay ovarian function decline. The S-estrol composition of this invention, through the scientific formulation of its components, precise control of key active ingredient indicators, and the addition of a dedicated anti-aging protective agent, synergistically addresses numerous technical problems of existing ovarian aging intervention methods from multiple dimensions. The core component, S-estrol, achieves high targeting affinity for ERβ with high purity and a high enantiomeric ratio. Combined with high-purity soy isoflavones, it overcomes the shortcomings of low bioavailability and single target of single plant estrogens. The two work synergistically on ovarian tissue, precisely regulating the estrogen receptor signaling pathway and effectively inhibiting ovarian granulosa cell apoptosis. Coenzyme Q10, D-α-tocopherol, and high-purity vitamin C form an antioxidant synergistic system, while zinc and folic acid assist in regulating ovarian cell metabolism. The combined action of these multiple components effectively reduces ovarian oxidative stress, solving the problem that existing formulations do not simultaneously address the dual needs of oxidative stress inhibition and apoptosis regulation. The addition of a specially structured anti-aging protective agent synergistically enhances the effects of each active ingredient, fully leveraging their respective strengths. The formulation process employs nitrogen protection during S-estrol pulverization and component mixing, along with carrier drying and strict temperature and humidity control, effectively addressing the issues of poor stability and easy oxidation degradation of active ingredients in existing compositions, leading to reduced product potency. Furthermore, the rational selection of the carrier and the granulation process design enhance the composition's formability and stability, resolving existing formulations' lack of strict control over key component purity and receptor affinity, unreasonable component compatibility, and weak targeting of ovarian tissue. Ultimately, this achieves multi-target, safe, and efficient delaying of ovarian function decline while mitigating the potential risks of hormone replacement therapy, providing a safer and more effective solution for ovarian aging intervention. Compared with the prior art, the beneficial effects of the present invention are: 1. More comprehensive synergistic regulatory effect of multiple targets: This invention uses high-purity S-estrol as the core, combined with soy isoflavones, coenzyme Q10 and a variety of antioxidant nutrients to form a synergistic system of estrogen receptor signal regulation and antioxidant defense. It shows an overall improvement trend in inhibiting ovarian granulosa cell apoptosis and reducing oxidative stress, overcoming the problem of single target and limited intervention effect of existing single plant estrogens. 2. Significantly enhanced antioxidant and cell protection capabilities: By constructing a complex antioxidant network and introducing novel anti-aging protectants for synergistic effects, the activity of antioxidant enzymes tends to increase and the level of lipid peroxidation tends to decrease, thereby improving the overall oxidative stress state in ovarian cells and making up for the shortcomings of existing formulas that do not take into account both apoptosis regulation and oxidative damage mechanisms. 3. Improved stability and safety: This invention strictly controls the purity, receptor affinity, and preparation process of key active ingredients, and uses nitrogen protection and low-humidity treatment during the pulverization and mixing stages, which significantly reduces the oxidative degradation trend of active ingredients and improves product stability and consistency. Detailed Implementation

[0004] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Preparation Example 1 Preparation of anti-aging protective agent 1: first step: ; Under a nitrogen atmosphere, 15 mL of concentrated sulfuric acid was added to the reaction system and cooled in an ice-water bath. A mixture of 5.00 g of compound 1 and 3.07 g of compound 2 was slowly added dropwise through a constant-pressure dropping funnel. After the addition was complete, the reaction system was stirred in the ice bath for 30 minutes, then the ice bath was removed, and the reaction system was allowed to warm naturally to room temperature. The reaction was then stirred for another 4 hours. After the reaction was complete, 100 mL of an ice-water mixture was prepared. While stirring, the reaction mixture was slowly poured into the ice-water mixture, and a large amount of grayish-white solid precipitated immediately. The slurry was stirred for another 30 minutes and filtered under reduced pressure through a Buchner funnel. The filter cake was washed with copious amounts of cold deionized water until the filtrate was neutral. The filter cake was transferred to an Erlenmeyer flask, and anhydrous ethanol was added for recrystallization. The mixture was heated to reflux until the solid was completely dissolved, then slowly cooled to room temperature and placed in a freezer overnight. The precipitated needle-like crystals were collected by filtration, washed with a small amount of cold ethanol, and dried in a vacuum drying oven at 45°C to constant weight to obtain 4.28 g of intermediate 1. Step Two: ; Under a nitrogen atmosphere, 4.28 g of intermediate 1 and 70 mL of DMF were added to the reaction system. After stirring until the solid was completely dissolved, the reaction system was placed in an ice-water bath and cooled to 0 °C. Subsequently, 9.55 mL of N,N-diisopropylethylamine was slowly added through a syringe. After stirring for 5 minutes, 8.34 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate was added. The reaction mixture was activated and stirred at 0 °C for 15 minutes. Then, 3.24 g of compound 3 was added. The ice bath was removed, and the reaction system was allowed to rise naturally to room temperature. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was slowly poured into 500 mL of a vigorously stirred ice-water mixture. 1 N HCl was added dropwise to the aqueous phase to adjust the pH to about 3-4. Stirring was continued for 30 minutes. The mixture was then filtered under reduced pressure. The filter cake was washed successively with water and cold diethyl ether. The filter cake was transferred to a vacuum drying oven and dried at 50 °C for 4 hours to obtain the crude product. The crude product was dissolved in a hot DMSO / EtOH mixed solvent of 1:5, and slowly cooled to crystallize. The crystals were filtered and dried under vacuum to obtain 5.34 g of anti-aging protective agent 1. Structural assessment: Mass spectrometry (MS) of intermediate 1 (M+1): 235; Mass spectrometry (MS) of anti-aging protective agent 1 (M+1): 378; NMR of anti-aging protectant 1: 1 H NMR(Chloroform-d)δ9.56(s,1H),8.12-8.01(m,2H),7.75-7.64(m,2H),7.4 7(s,1H),6.89(d,1H),6.77(dd,1H),6.38(t,1H),3.98(d,2H),3.80(s,3H). Preparation Example 2 Preparation of anti-aging protective agent 2: Following the preparation method in Preparation Example 1, compound 3 was replaced with compound 4, and the rest remained the same as in Preparation Example 1. Structure of compound 4: Anti-aging protective agent 2 structure: Mass spectrometry (MS) of anti-aging protectant 2 (M+1): 378. Preparation Example 3 Preparation of anti-aging protective agent 3: Following the preparation method in Preparation Example 1, compound 3 was replaced with compound 5, and the rest remained the same as in Preparation Example 1. Structure of compound 5: Anti-aging protective agent 3 structure: Mass spectrometry (MS) of anti-aging protectant 3 (M+1): 392. Example 1 Preparation of an S-estrol composition for delaying ovarian aging: 1. Raw material components: S-Estrol: 3 parts, purity ≥98%; Soy isoflavones: 5 parts, purity ≥95%, composed of daidzein and genistein in a mass ratio of 2:1; Coenzyme Q10: 6 servings; Vitamin E: 4 parts, D-α-tocopherol, purity ≥96%; Vitamin C: 8 portions, purity ≥ 99%; Folic acid: 0.03 parts, purity ≥98%; Zinc source: 0.2 parts, zinc gluconate; Anti-aging protective agent: 1 part, which is the anti-aging protective agent 1 prepared in Preparation Example 1; Carrier: 65 parts, composed of microcrystalline cellulose and mannitol in a mass ratio of 1:1; Purified water: 12 parts. 2. Preparation method: S1 Raw Material Pretreatment: Take the S-estradiol, soy isoflavones, coenzyme Q10, vitamin E, vitamin C, folic acid, zinc source and carrier, and pulverize them respectively. The pulverization of S-estradiol is carried out under nitrogen protection. All raw materials are sieved through a 100-mesh sieve after pulverization to remove impurities. Place the carrier in a drying oven and dry it at 55℃ for 2.5h, controlling the moisture content of the carrier to ≤3%. Place all treated raw materials in a clean environment with relative humidity ≤45% and temperature of 22℃ for later use. S2 Mixing: First, pretreated S-estradiol, soy isoflavones, coenzyme Q10, vitamin E, vitamin C, folic acid, zinc source, and anti-aging protectant are placed in a three-dimensional mixer. During the mixing process, nitrogen gas is introduced for protection at a flow rate of 0.8 L / min, and the mixture is stirred at 180 r / min for 20 min to obtain a premix. Then, the pretreated carrier is added to the premix, and the mixture is stirred at 140 r / min for 25 min under the same nitrogen protection conditions to ensure that all components are mixed evenly to obtain a mixture. S3 Molding: Add the purified water to the mixture, granulate using a wet granulator, and pass the granules through a 50-mesh sieve to obtain uniform granules; S4 Drying and Packaging: The granules are placed in a hot air circulating drying oven and dried at 45°C for 1.5 hours. The moisture content of the granules is tested and controlled to be ≤5%. The dried granules are then packaged in an aluminum-plastic composite film for light protection and sealing to obtain an S-estradiol composition for delaying ovarian aging. Example 2-Example 3 The preparation of an S-estrol composition for delaying ovarian aging is carried out by referring to the preparation method in Example 1, except that the anti-aging protective agent is replaced with anti-aging protective agent 2-anti-aging protective agent 3 prepared in Preparation Examples 2-3, and the rest is the same as in Example 1. Comparative Example 1 The preparation of an S-estrol composition for delaying ovarian aging is carried out by referring to the preparation method in Example 1, except that the anti-aging protective agent is replaced with spermidine, and the rest is the same as in Example 1. Comparative Example 2 The preparation of an S-estrol composition for delaying ovarian aging is carried out by referring to the preparation method in Example 1, except that the anti-aging protective agent is replaced with pyrroloquinoline quinone, and the rest is the same as in Example 1. Comparative Example 3 The preparation of an S-estrol composition for delaying ovarian aging is carried out according to the preparation method in Example 1, without the addition of the anti-aging protective agent, and otherwise remains the same as in Example 1. Comparative Example 4 The preparation of an S-estrol composition for delaying ovarian aging was carried out according to the preparation method in Example 1, except that coenzyme Q10 was not added, and the rest remained the same as in Example 1. Performance testing I. Ovarian granulosa cell apoptosis and oxidative stress inhibition test: (I) Culture and modeling of ovarian granulosa cells: 1. Ovarian tissue from SD rats was collected, and primary ovarian granulosa cells were obtained by mechanical separation and trypsin digestion. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin antibiotics in a constant temperature incubator at 37°C and 5% CO2. 2. An ovarian granulosa cell oxidative stress injury model was established using H2O2 induction: Cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 1×10⁻⁶. 5 The cells were inoculated at a concentration of 1 / mL into a culture plate and cultured for 24 h. Then, H2O2 with a final concentration of 200 μmol / L was added and cultured for another 24 h to construct an aging damage model. (II) Experimental Grouping: Blank control group: No modeling was performed, only an equal volume of culture medium was added; Model control group: H2O2 modeling was performed, and an equal volume of sterile physiological saline was added; Example group: H2O2 modeling was performed, and the composition solutions of Examples 1-3 were added respectively; Comparative group: H2O2 modeling was performed, and the composition solutions of Comparative Examples 1-4 were added respectively. (III) Experimental Procedure: 1. Detection of ovarian granulosa cell apoptosis rate: After culturing cells for 48 h following drug administration, cells were collected by trypsin digestion, washed twice with pre-cooled PBS, centrifuged at 800 rpm for 5 min, and the supernatant was discarded. Cells were resuspended in 500 μL binding buffer according to the Annexin V-FITC / PI kit instructions, followed by the addition of 5 μL Annexin V-FITC and 5 μL PI, and gently mixed. Cells were incubated at room temperature in the dark for 15 min. Immediately afterward, cell apoptosis was detected by flow cytometry, and the proportions of early and late apoptotic cells were recorded. The total apoptosis rate was calculated as: total apoptosis rate = early apoptosis rate + late apoptosis rate. The results are shown in Table 1. 2. Detection of oxidative stress-related indicators: After culturing cells for 48 hours following drug administration, cell supernatant was collected, and cells were lysed with cell lysis buffer to extract cell homogenate. Following the instructions of the SOD, MDA, and GSH-Px kits, the SOD activity, MDA content, and GSH-Px activity in the cell supernatant and homogenate were detected using an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Table 1. Results interpretation: SOD and GSH-Px are antioxidant enzymes, and higher activity indicates stronger antioxidant capacity; MDA is a lipid peroxidation product, and lower content indicates less oxidative damage. Table 1. Performance Test Results Total apoptosis rate (%) SOD activity (U / mg prot) GSH-Px activity (U / mg prot) MDA content (nmol / mg prot) Example 1 12.56 187.25 125.05 3.21 Example 2 13.11 176.98 121.64 3.39 Example 3 11.86 195.42 129.52 2.98 Comparative Example 1 28.64 126.36 78.56 6.85 Comparative Example 2 26.39 132.51 82.35 6.49 Comparative Example 3 35.98 96.28 56.39 9.16 Comparative Example 4 30.15 106.56 69.87 8.21 Blank control group 8.65 206.37 137.64 2.84 Model control group 42.51 85.16 47.65 10.27 Table 1 shows that the example group (the composition containing the anti-aging protective agent of the present invention) was significantly superior to the comparative groups in inhibiting ovarian granulosa cell apoptosis, with a significantly reduced total apoptosis rate. Regarding oxidative stress indicators, the example group exhibited a stronger ability to enhance antioxidant enzyme activity, while the level of lipid peroxidation products was effectively controlled, resulting in outstanding overall antioxidant protection. In contrast, the comparative groups without the anti-aging protective agent or with other anti-aging ingredients, as well as the comparative group lacking coenzyme Q10, showed significantly weaker effects in inhibiting apoptosis and reducing oxidative stress. Overall, the composition of the present invention, through the synergistic combination of its components, especially the addition of the dedicated anti-aging protective agent, effectively inhibits ovarian granulosa cell apoptosis and significantly reduces oxidative stress, with performance approaching that of the blank control group. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An S-estrol composition for delaying ovarian aging, characterized in that, By weight, it consists of the following components: 1-5 parts S-estradiol, 2-8 parts soy isoflavones, 3-10 parts coenzyme Q10, 2-6 parts vitamin E, 4-12 parts vitamin C, 0.01-0.05 parts folic acid, 0.1-0.3 parts zinc source, 0.5-1.5 parts anti-aging protectant, 50-80 parts carrier, and 10-15 parts purified water; The anti-aging protective agent is a compound represented by Formula 1; Formula 1: ; In Formula 1, the substituent R1 is selected from: , , Any one of them; * indicates a connection point.

2. The S-estrol composition for delaying ovarian aging according to claim 1, characterized in that, The purity of the S-estrol is ≥98%.

3. The S-estrol composition for delaying ovarian aging according to claim 1, characterized in that, The soy isoflavones have a purity of ≥95% and are composed of daidzein and genistein in a mass ratio of 2:

1.

4. The S-estrol composition for delaying ovarian aging according to claim 1, characterized in that, The vitamin E is D-α-tocopherol with a purity ≥96%; the vitamin C has a purity ≥99%; and the folic acid has a purity ≥98%.

5. An S-estrol composition for delaying ovarian aging according to claim 1, characterized in that, The zinc source is at least one of zinc gluconate, zinc citrate, zinc lactate, and zinc glycinate.

6. An S-estrol composition for delaying ovarian aging according to claim 1, characterized in that, The carrier is selected from at least one of microcrystalline cellulose, lactose, starch, and mannitol.

7. A method for preparing an S-estrol composition for delaying ovarian aging according to any one of claims 1-6, characterized in that, Includes the following steps: S1 Raw Material Pretreatment: Take the S-estradiol, soy isoflavones, coenzyme Q10, vitamin E, vitamin C, folic acid, zinc source and carrier, crush them separately, pass them through an 80-120 mesh sieve to remove impurities, and then place them in an environment with relative humidity ≤45% and temperature of 20-25℃ for later use. S2 Mixing: First, put the S-estradiol, soy isoflavones, coenzyme Q10, vitamin E, vitamin C, folic acid, zinc source, and anti-aging protectant into a mixer and mix at 150-200 r / min for 15-25 min to obtain a premix; then add the carrier to the premix and continue mixing at 120-160 r / min for 20-30 min to ensure uniform mixing and obtain a mixture; S3 Molding: Add the purified water to the mixture, granulate using a granulator, and pass the granules through a 40-60 mesh sieve to obtain granules; S4 Drying and Packaging: Place the granules in a drying oven and dry at 40-50℃ for 1-2 hours, controlling the moisture content to ≤5%; By protecting it from light and sealing it in a sealed container, an S-estrol composition for delaying ovarian aging is obtained.

8. A method for preparing an S-estrol composition for delaying ovarian aging according to claim 7, characterized in that, In step S1, the S-estrol pulverization is carried out under nitrogen protection; after pulverization, the carrier needs to be dried at a temperature of 50-60℃ for 2-3 hours to ensure that the moisture content of the carrier is ≤3%.

9. A method for preparing an S-estrol composition for delaying ovarian aging according to claim 7, characterized in that, In step S2, nitrogen gas is introduced for protection during the mixing process, and the nitrogen flow rate is 0.5-1 L / min.

10. The use of an S-estrol composition for delaying ovarian aging according to any one of claims 1-6 in the preparation of a medicine / health product for inhibiting ovarian granulosa cell apoptosis, reducing ovarian oxidative stress, or delaying ovarian function decline.

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