Use of epicatechin in the preparation of a product for the treatment of premature ovarian insufficiency

Abstract

The application belongs to the technical field of medicines, and particularly relates to application of eriodictyol in preparation of products for treating early ovarian insufficiency. The application first finds that eriodictyol can simultaneously control four paths of an estrogen path, anti-oxidative ferroptosis, a CD38 / IL-17 inflammation axis and intestinal flora, breaks the efficacy limitation of traditional drugs which can only act on a single path, and realizes the effect of reversing pathological damage of POI from cells, tissues, endocrine to intestinal microecology multi-level. Moreover, as a natural flavone, eriodictyol has no carcinogenic risk, and its safety is better than that of hormone replacement therapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of sennaol in the preparation of products for the treatment of early-onset ovarian insufficiency. Background Technology

[0002] Premature ovarian insufficiency (POI) is a reproductive endocrine disorder characterized by premature follicle depletion, insufficient estrogen secretion, elevated gonadotropins, and decreased fertility. It is also accompanied by ovarian granulosa cell aging, excessive oxidative stress, chronic low-grade inflammation, and an imbalance of granulosa cell ferroptosis. It can also lead to secondary complications such as osteoporosis, ovarian fibrosis, and uterine atrophy. In recent years, due to environmental factors, stress, and irregular lifestyles, the incidence of this disease has been rising annually, with a global prevalence of approximately 1% to 3%.

[0003] Currently, the mainstream clinical treatment for POI is hormone replacement therapy, but long-term use of sex hormones increases the risk of breast and uterine lesions. In addition, non-hormonal antioxidants such as coenzyme Q10 and melatonin are commonly used to treat POI. However, coenzyme Q10 and melatonin only have single antioxidant effects and cannot simultaneously improve systemic inflammation, ovarian ferroptosis, and endocrine disorders, resulting in limited overall efficacy. Therefore, there is an urgent need in this field for a treatment regimen for POI that is effective and has fewer side effects.

[0004] Eucommia ulmoides is a traditional Chinese medicine used to tonify the liver and kidneys and stabilize pregnancy. Eri is a dihydroflavonoid monomer that can be isolated from Eucommia ulmoides leaves. Current research has only confirmed that Eri has in vitro anti-inflammatory and neuroprotective effects, but there is no existing technology to study the application of Eri in the treatment of POI. Summary of the Invention

[0005] To address the lack of effective and low-toxicity technical solutions for treating POI in existing technologies, this invention provides the application of sennaol in the preparation of products for treating early-onset ovarian insufficiency, specifically including the following technical solutions: The use of sennaol in the preparation of products for the prevention and / or treatment of premature ovarian insufficiency.

[0006] Preferably, the raw materials of the product include sennaol and excipients.

[0007] Preferably, the content of sennaol in the product is 30wt%~40wt%.

[0008] Preferably, the dosage form of the product includes an oral formulation.

[0009] Preferably, the oral preparation includes any one of tablets, capsules, granules, oral liquids, and suspensions.

[0010] Preferably, the application includes any one or more of the following: 1) Repair ovarian endocrine function; 2) Promotes estrogen secretion; 3) Inhibits granulocyte senescence; 4) Relieves inflammation; 5) Blocking ferrodeogenesis; 6) Repair the gut microbiota; 7) Improves osteoporosis; 8) Improve reproductive function.

[0011] Preferably, the product includes pharmaceuticals or health products.

[0012] Preferably, the health supplement includes health supplements that help regulate gut microbiota and / or health supplements that help with antioxidant activity.

[0013] The present invention also provides a medicament for treating early-onset infertility, the medicament comprising a functional component and excipients; the functional component comprising sennaol.

[0014] The present invention also provides the use of the drug as described above in the treatment of early-onset ovarian dysfunction complications, which include any one or more of uterine atrophy, ovarian fibrosis, osteoporosis, and inflammation.

[0015] The beneficial effects of this invention are as follows: This invention provides the application of sennarol in the preparation of products for the prevention and / or treatment of early-onset ovarian insufficiency. This invention is the first to discover that sennarol can simultaneously regulate four major pathways: the estrogen pathway, anti-ferrooxidative death, the CD38 / IL-17 inflammatory axis, and the gut microbiota, overcoming the limitations of traditional drugs that can only act on a single pathway. It achieves a multi-level reversal of POI pathological damage from the cellular, tissue, endocrine, and gut microbiota levels. Furthermore, as a natural flavonoid, sennarol has no carcinogenic risk, and its safety profile is superior to hormone replacement therapy.

[0016] This invention, when applied to POI-induced mice, not only repaired ovarian function and restored fertility, but also simultaneously improved POI-induced uterine atrophy, trabecular bone damage, and osteoporosis. It can treat both the primary disease and its complications simultaneously.

[0017] In animal experiments, this invention discovered that a dose of sennaol is only 2-10 mg / kg to achieve 300 mg / kg NMN ( β (Nicotinamide mononucleotide) has the same therapeutic effect, but requires a lower dosage and has a lower cost.

[0018] This invention validates the therapeutic efficacy of Eri through both in vitro cell experiments and in vivo animal experiments. The KGN cell model confirmed its anti-granulosa cell senescence effect; the D-galactose-induced POI mouse model demonstrated that Eri significantly affected the estrous cycle, follicle count, hormone levels, fertility, histopathology, protein molecules, and gut microbiota in the POI model. This invention provides a complete validation of Eri's pharmacological efficacy. Furthermore, molecular docking further corroborates the reliability of target binding. This invention provides a pathway for developing oral natural drugs using Eri for the prevention of POI in high-risk populations, intervention for mild POI, and improvement of ovarian reserve before assisted reproductive technology. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 Complete process flow diagram for extracting sennaol from Eucommia ulmoides leaves; Figure 2 The network pharmacology prediction of Eri targets is shown in Figure A; where A is the Venn diagram of common targets of Eri-POI and B is the network diagram of core targets of PPI. Figure 3 The results show the viability of D-gal-induced KGN cells under various treatments; Figure 4 SA-β-Gal senescence staining results of D-gal-induced KGN cells under various treatments; where A~F are blank group, D-gal model group (200 mg / L), NMN group (40 μM), and Eri low, medium and high dose groups (10 μM, 20 μM, 40 μM), respectively; magnification ×100; Figure 5 The images show the statistical results of changes in the ovaries and uterus of mice in each treatment group; the first row of images shows the gross appearance of the ovaries of mice in each group, and the second row of images shows the gross appearance of the uterus of mice in each group. Figure 6 A bar chart showing the ovarian index of mice in each treatment group; Figure 7 HE pathological sections of mouse ovaries from different treatments; Figure 8 HE pathological staining images of mouse uteruses from different treatments; Figure 9 HE pathological staining images of mouse bone tissue from various treatments; Figure 10 The number of mature follicles and atretic follicles in mice under each treatment was counted; the left figure shows the number of mature follicles, and the right figure shows the number of atretic follicles. Figure 11The counts of primordial follicles and growing follicles in mice under each treatment were statistically analyzed; the left figure shows the number of primordial follicles, and the right figure shows the number of growing follicles. Figure 12 The figures show the statistical results of estrous cycle changes in mice in each treatment group; the upper figure shows the statistical results of estrous cycles in the blank control, Jupiter group, and NMN group; the lower figure shows the statistical results of estrous cycles in low, medium, and high concentration Eri treatments. Figure 13 The statistical results of the cycle disorder rate in mice in each treatment group; Figure 14 The results show the hormone expression levels in the serum of mice under each treatment. The first row, from left to right, shows the FSH hormone expression level, LH hormone expression level, and the ratio of FSH to LH expression levels. The second row, from left to right, shows the E2, P4, and AMH hormone level results. Figure 15 The images show immunofluorescence staining results of ovarian tissue. The top image shows the staining results of Merge, Enlargement, ERα, ERβ, CD38, and DAPI in ovarian tissue. Images A through C show the statistical results of the relative fluorescence intensity of ERα, ERβ, and CD38, respectively. Figure 16 The images show Western blotting protein bands; the left side shows the protein bands of ERα, ERβ, CD38, and IL-17 in each treatment group; the right side shows the protein bands of Keap1, Nrf2, HO-1, and GPX4 in each treatment group. Figure 17 The gray-scale quantitative statistical charts of ERα and ERβ are shown; the left chart shows the gray-scale quantitative values ​​of ERα, and the right chart shows the gray-scale quantitative values ​​of ERβ. Figure 18 Statistical charts for CD38 and IL-17 grayscale quantification; the left chart shows the grayscale quantification of CD38, and the right chart shows the grayscale quantification of IL-17. Figure 19 The graphs show the quantitative grayscale values ​​of Keap1 and Nrf2; the left graph shows the quantitative grayscale values ​​of Keap1, and the right graph shows the quantitative grayscale values ​​of Nrf2. Figure 20 Quantitative statistical charts of grayscale values ​​for HO-1 and GPX4 are provided; the left chart shows the quantitative grayscale values ​​of HO-1, and the right chart shows the quantitative grayscale values ​​of GPX4. Figure 21 A schematic diagram showing the mouse birthing situation in each treatment group; Figure 22 The results show the pregnancy rate of mice in each group. Figure 23 The results show the litter size of mice in each group. Figure 24 The survival rate of the pups in each group of mice is statistically analyzed. Figure 25 This is a three-dimensional diagram illustrating the molecular docking and binding mode of sennaol and ERα. Figure 26 This is a three-dimensional diagram illustrating the molecular docking binding mode of sennaol and ERβ. Figure 27 This is a three-dimensional diagram illustrating the molecular docking binding mode of sennaol and Keap1. Figure 28 This is a three-dimensional diagram illustrating the molecular docking binding mode of sennaol and Nrf2. Figure 29 This is a three-dimensional diagram illustrating the molecular docking binding mode of sennaol and HO-1. Figure 30 This is a three-dimensional diagram illustrating the molecular docking binding mode of sennaol and GPX4. Figure 31 The results show the statistical results of intestinal function in mice under each treatment; the left figure shows the sparse curves of each treatment group; the right figure shows the OTU petal diagram of the intestinal flora of mice under each treatment. Figure 32 The results show the intestinal function statistics of mice in each treatment group; the left side shows the number of OTUs in each treatment group, and the right side shows the Shannon index in each treatment group. Figure 33 The results show the intestinal function statistics of mice in each treatment group; the left panel shows the Simpson index of mice in each treatment group; the right panel shows the Chao1 index of mice in each treatment group. Figure 34 The table shows the statistical results of intestinal function in mice under each treatment; the left panel shows the ACE index of mice in each treatment group; the right panel shows the β diversity analysis results of mice in each treatment group. Figure 35 The relative abundance of phylum-level bacteria in the gut of mice in each treatment group; Figure 36 The relative abundance of bacteria at the family level in the gut of mice in each treatment group; Figure 37 The relative abundance of mesoclass bacteria in the gut of mice in each treatment group; Figure 38 The relative abundance of Bacteroidetes and Firmicutes in the intestines of mice in each treatment group; Figure 39 The relative abundance of Bacteroidetes / Firmicutes and the relative abundance of Proteobacteria in the intestines of mice in each treatment group; Figure 40The relative abundance of Lachnospiraceae and Ruminococcaceae in the intestines of mice in each treatment group; Figure 41 The relative abundance of Muribauculaceae and Clostridia in the intestines of mice in each treatment group; Figure 42 The relative abundance of Bacteroidia and Gammaproteobacteria in the intestines of mice in each treatment group; Figure 43 The LEfSe analysis results for each treatment group; Figure 44 A comprehensive pathway diagram illustrating the mechanism by which sennaol enhances the synergistic effect of multiple targets of POI. Detailed Implementation

[0021] This invention provides the use of sennaol in the preparation of products for the prevention and / or treatment of early-onset ovarian insufficiency.

[0022] In one embodiment, the CAS number of the sennaroin described in this invention is 552-58-9. In one embodiment, the raw materials of the product include sennaroin and excipients. In one embodiment, the content of sennaroin in the product is 30wt%~40wt%. In an optional embodiment, the content of sennaroin in the product can be any one or any midpoint between two values ​​from 30wt%, 31wt%, 32wt%, 33wt%, 34wt%, 34.75wt%, 35wt%, 36wt%, 37wt%, 38wt%, 39wt%, and 40wt%. In a specific embodiment, when the product is a capsule, the raw materials are 100 mg of 98% pure sennaroin, 80 mg of lactose, 100 mg of starch, and 2 mg of magnesium stearate, wherein the content of pure sennaroin is 34.75wt%. In one embodiment, the dosage form of the product includes an oral preparation. In one embodiment, the oral preparation includes any one of tablets, capsules, granules, oral liquids, and suspensions. In one embodiment, the product includes pharmaceuticals or health supplements. In another embodiment, the health supplements include those that help regulate gut microbiota and / or those that contribute to antioxidant activity.

[0023] Eri (Eri) is a natural, low-toxicity flavonoid compound widely found in various medicinal plants. In this invention, erythrophenol was extracted from Eucommia ulmoides leaves, which have the advantages of readily available raw materials and low preparation costs. This invention utilized erythrophenol for in vitro cell experiments and in vivo animal experiments, verifying the therapeutic effect of Eri from two aspects. The KGN cell model confirmed that Eri has anti-granulosa cell senescence properties; the D-galactose-induced POI mouse model confirmed that Eri significantly affected the estrous cycle, follicle count, hormone levels, fertility, histopathology, protein molecules, and gut microbiota in the POI model. This invention provides a complete verification of the efficacy of Eri. Furthermore, molecular docking was used to corroborate the reliability of target binding. This invention provides a solid theoretical basis and data support for the application of erythrophenol in the treatment of early-onset ovarian insufficiency. In this invention, erythrophenol improves the synergistic mechanism of POI targeting multiple targets, such as... Figure 44 As shown.

[0024] In one embodiment, the premature ovarian insufficiency includes D-galactose-induced premature ovarian insufficiency. In one embodiment, the application includes any one or more of the following: 1) repairing ovarian endocrine function; 2) promoting estrogen secretion; 3) inhibiting granulosa cell senescence; 4) alleviating inflammation; 5) blocking ferroptosis; 6) repairing gut microbiota; 7) improving osteoporosis; 8) improving fertility. In one embodiment, repairing ovarian endocrine function includes improving uterine atrophy and ovarian fibrosis. In one embodiment, promoting estrogen secretion includes upregulating the expression of ERα and ERβ proteins in the ovary and uterus, activating estrogen signaling pathways. In one embodiment, the granulosa cells include ovarian cells. In a specific embodiment, this invention demonstrates that sennaol can inhibit D-galactose-induced granulosa cell senescence through D-galactose-induced granulosa cell senescence. In one embodiment, alleviating inflammation includes reducing the expression of CD38 and IL-17, reducing chronic inflammation. In one embodiment, the inflammation includes local and systemic inflammation of the ovary. As one implementation, blocking ferroptosis includes downregulating Keap1, promoting Nrf2 nuclear translocation, and increasing the expression levels of HO-1 and GPX4 to inhibit ferroptosis. As one implementation, repairing the gut microbiota includes increasing gut microbiota diversity, increasing the abundance of beneficial bacteria, and inhibiting one or more of harmful pro-inflammatory bacteria. As one implementation, improving osteoporosis includes improving osteoporosis caused by early-onset ovarian insufficiency. As one implementation, improving osteoporosis includes reducing trabecular bone fracture, increasing bone mass, and repairing bone microstructure, one or more of these. As one implementation, improving fertility includes increasing the conception rate in patients with early-onset ovarian insufficiency. As one implementation, when the subject of improving fertility is an animal, the indicators of improvement also include litter size and / or offspring survival rate.

[0025] The present invention also provides a medicament for treating early-onset infertility, the medicament comprising a functional component and excipients; the functional component comprising sennaol.

[0026] In one embodiment, the excipients include pharmaceutically acceptable excipients. In one embodiment, the excipients include any one or more of sodium carboxymethyl cellulose, starch, lactose, mannitol, and magnesium stearate. In one embodiment, the in vivo animal dose of the drug, based on the dosage of sennaol, is 2–10 mg / kg / day, and the in vitro cell administration concentration is 10–40 μM.

[0027] The present invention also provides a method for preparing the drug as described above, comprising: mixing functional components and excipients to obtain the drug.

[0028] In one embodiment, when the drug is an oral capsule, the preparation steps involve pulverizing each raw material through an 80-mesh sieve, mixing the sieved raw materials evenly using an equal-volume incremental addition method to obtain a medicinal powder. The medicinal powder is then filled into hollow hard capsules, each capsule containing 100 mg of sennaol.

[0029] To further illustrate the present invention, the application of the sennaol provided by the present invention in the preparation of products for treating early-onset ovarian insufficiency is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1: Preparation of Eucommia ulmoides leaf senna monomer This embodiment utilizes Eucommia ulmoides leaves to extract sennaol, and the extraction process flow diagram is shown below. Figure 1 As shown, the specific extraction steps are as follows: The dried Eucommia ulmoides leaves were pulverized to obtain Eucommia ulmoides leaf powder. The Eucommia ulmoides leaf powder and 80% ethanol were mixed at a mass-volume ratio of 1 g: 10 mL to obtain a mixture. The mixture was extracted twice by reflux extraction, 2 h each time. The extracts from the two extractions were combined and concentrated under reduced pressure in a rotary evaporator at 45℃ and 0.08 MPa to obtain an extract. The extract and water were mixed at a mass-volume ratio of 1 g: 15 mL to obtain a suspension; after defatting with petroleum ether, the suspension was extracted with ethyl acetate at a volume ratio of 1:1, and the ethyl acetate phase was collected. The ethyl acetate phase was loaded onto a silica gel column and eluted with petroleum ether-ethyl acetate at a volume ratio of 8:1 under normal pressure and room temperature to collect the sennaol fraction. The sennaol fraction was then eluted with dichloromethane-methanol at a volume ratio of 20:1, and the concentrate was used to obtain fraction Fr.3. Subsequently, Fr.3D was purified by elution with a mixture of petroleum ether and ethyl acetate at a volume ratio of 1:1. A preparative C18 column (250×20mm, 10μm) was used for isocratic elution with acetonitrile-0.1% formic acid water (30:70, V / V) as the mobile phase, with a detection wavelength of 290nm, a flow rate of 15mL / min, and a column temperature of 30℃. The Fr.3D fraction was dissolved in methanol, filtered through a 0.45μm filter membrane, and injected. The chromatographic peak fraction corresponding to sennaol was collected, concentrated under reduced pressure, and then lyophilized to obtain the sennaol monomer.

[0031] In this embodiment, the components of the purified sennaol were identified by ESI-MS, 1H-NMR, and 13C-NMR spectroscopy. The identification results showed that the product was sennaol, and the purity was ≥98.0% as detected by HPLC. This product was used as a sennaol sample for future use.

[0032] Example 2: Network pharmacology prediction of potential targets of Eri for POI diseases This embodiment uses network pharmacology to screen potential targets for Eri intervention in POIs. The specific steps are as follows: The SMILES structure of Eri was submitted to SwissTargetPrediction, PharmMapper, and the BATMAN-TCM online platform to predict potential targets for Eri. Simultaneously, in the GeneCards and OMIM databases, POI disease-related targets were searched using "Premature Ovarian Failure" and "POI" as search terms, with a relevance score ≥1.0 as the inclusion criterion. All obtained compound and disease targets were submitted to the UniProt database, limited to the human species (Homo sapiens), and uniformly converted to standard official gene names, removing duplicate targets. The intersection of the Eri predicted target set and the POI disease target set was taken to obtain the Eri-POI common target; the Venn diagram of this common target is shown below. Figure 2 A.

[0033] The obtained Eri-POI common targets were imported into the STRING database to construct a protein-protein interaction (PPI) network. Interaction confidence was set >0.4 to filter out protein interaction data. The interaction relationship file exported from the STRING database was imported into Cytoscape 3.9.1 software for network visualization. The cytoNCA plugin was used to calculate the degree centrality, betweenness centrality, and proximity centrality topological parameters of each node in the network. Nodes with all three topological parameters (degree centrality, betweenness centrality, and proximity centrality) higher than their respective medians were selected as potential core targets for Eri intervention in POIs. The resulting PPI core target network diagram is shown below. Figure 2 B.

[0034] Depend on Figure 2 As can be seen, this embodiment uses network pharmacology analysis to screen out potential common and core targets of Eri in treating POI, providing candidate targets for subsequent cell experiments to verify activity and molecular mechanism of action.

[0035] Example 3 In vitro cell experiments In this embodiment, the senna sample prepared in Example 1 was used for in vitro cell experiments to verify the protective effect of senna on D-galactose (D-gal)-induced senescence of KGN ovarian granulosa cells.

[0036] This embodiment uses human ovarian granulosa cells (KGN) for experiments. KGN cells were cultured in DMEM / F12 complete medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. The culture conditions were 37°C and 5% CO2. After reaching the logarithmic growth phase, the medium was discarded, and the cells were digested with 1 mL of trypsin for 1 min. Digestion was terminated with 1 mL of DMEM / F12 complete medium, followed by centrifugation. The cells were resuspended and seeded at 8000 cells / well in 96-well plates for adherent culture at 37°C. After cell adhesion, the cells were grouped and treated.

[0037] The grouping is as follows, with 5 duplicate holes in each group: Blank control group: Discard the original culture medium and replace it with DMEM / F12 complete culture medium; D-gal model group: Add D-gal to a final concentration of 200 mg / L and incubate for 24 h to complete cell senescence modeling. After modeling, discard the original culture medium and replace it with DMEM / F12 complete culture medium. NMN positive group: D-gal was added to a final concentration of 200 mg / L and incubated for 24 h to complete the cell senescence model. After the model was completed, the original culture medium was discarded and replaced with DMEM / F12 complete culture medium. At the same time, NMN was added to a final concentration of 40 μM and the intervention lasted for 24 h. Low-dose Eri group: D-gal was added to a final concentration of 200 mg / L and incubated for 24 h to complete the cell senescence model. After the model was established, the original culture medium was discarded, blank culture medium was added, and Eri was added to a final concentration of 10 μM. The intervention lasted for 24 h. Medium-dose Eri group: D-gal was added to the cell senescence model for 24 h and incubated for 200 mg / L. After the model was established, the original culture medium was discarded and blank culture medium was added. At the same time, Eri was added to the cell senescence model for 24 h. High-dose Eri group: D-gal was added to the cell senescence model for 24 h. After the model was established, the original culture medium was discarded, blank culture medium was added, and Eri was added to the cell senescence model for 24 h.

[0038] Immediately after each treatment intervention was completed, the cells in each treatment group were analyzed using the following methods: 1. MTT assay for cell viability: 20 μL of 5 mg / mL MTT working solution was added to each well, and the cells were incubated at 37°C in the dark for 4 h. After incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well. The mixture was stirred at room temperature until dissolved and crystallized. The absorbance of each group was then measured at 490 nm, and cell viability was calculated using the following formula: Cell viability = (absorbance value of experimental group / absorbance value of blank control group) × 100%.

[0039] 2.SA- β -Gal staining for senescent cells: Cells in each treatment group were washed once with PBS, then fixed with 150 μL of fixative for 15 min. After fixation, 150 μL of SA- β -Gal staining was performed and incubated at 37°C in the dark for 12 hours. After incubation, the staining solution was discarded, and cell morphology was observed under a microscope. For each treatment, five fields of view were randomly selected under the microscope, and the total number of cells and the number of blue-stained senescent cells in the five fields of view were counted. The percentage of senescent positive cells was calculated using the following formula: Percentage of senescent positive cells (%) = Blue-stained senescent cells / Total number of cells × 100%.

[0040] Cell viability assay results as follows Figure 3 As shown, the staining results of senescent cells are as follows: Figure 4 As shown in the figure. It can be seen that, compared to the blank control, the cell viability of the D-gal model group (Model) was reduced (##, P<0.01), the number of senescent cells increased significantly, and the proportion of senescent cells increased significantly; cell viability rebounded dose-dependently after intervention with 10, 20, and 40 μM Eri, while the proportion of senescent positive cells decreased synchronously. Among them, 40 μM Eri had the most significant effect on improving senescent cells (*, P <0.05), can significantly reverse D-gal-induced ovarian granulosa cell senescence, with efficacy comparable to the positive control drug NMN.

[0041] Example 4: In vivo animal experiments In this embodiment, the sennaol sample prepared in Example 1 was used to conduct in vivo animal experiments to verify the improvement effect of sennaol on the D-galactose-induced POI mouse model.

[0042] In this embodiment, 60 SPF-grade ICR female mice aged 6-8 weeks were randomly divided into 6 groups of 10 mice each. The grouping and treatment of each group are shown below: Control group: 0.5% CMC-Na was administered by gavage at a dose of 10 mL / (kg·d) daily for 6 weeks.

[0043] Model group: D-gal 500 mg / kg was subcutaneously injected into the back of mice daily for 6 consecutive weeks; NMN 300 mg / kg positive group: D-gal 500 mg / kg was injected subcutaneously into the back of mice daily. NMN was dissolved in 0.5% CMC-Na and then administered by gavage at a dose of 300 mg / kg NMN daily for 6 consecutive weeks. Low-dose Eri group: D-gal 500 mg / kg was injected subcutaneously into the back of mice daily. Eri was dissolved in 0.5% CMC-Na and then administered by gavage at a dose of 2 mg / kg Eri daily for 6 consecutive weeks. Medium-dose Eri group: D-gal 500 mg / kg was injected subcutaneously into the back of mice daily. Eri was dissolved in 0.5% CMC-Na and then administered by gavage at a dose of 5 mg / kg Eri daily for 6 consecutive weeks. High-dose Eri group: D-gal 500 mg / kg was injected subcutaneously into the back of mice daily. Eri was dissolved in 0.5% CMC-Na and then administered by gavage at a dose of 10 mg / kg Eri daily for 6 consecutive weeks.

[0044] (1) Ovarian index / body weight: The body weight of mice in each group was recorded daily during the experimental period to calculate the ovarian index; The estrous cycle was observed by microscopic examination of vaginal smears daily for 14 consecutive days, and the percentage of mice with cycle disorders was counted. After the intervention, the mice were sacrificed, and both ovaries were completely removed, weighed, and the ovarian index was calculated. Ovarian index = ovarian wet weight (mg) / mouse body weight (g) × 100%; Statistical results are as follows Figures 5-6 and 12~13 shown by Figures 5-6 As shown in Figures 12-13, the ovaries of mice in the model group were shrunken, the ovarian index was reduced, and the estrous cycle disorder rate was 80%. Compared with the model group, high doses of Eri significantly increased the ovarian index of mice and reduced the cycle disorder rate to 25%, with no significant difference in effect from the positive control drug NMN.

[0045] (2) Ovarian / uterine pathological HE staining: After the intervention, bilateral ovarian and uterine tissues were completely harvested from the mice and fixed in 4% paraformaldehyde. The fixed tissues were then dehydrated with graded ethanol, embedded in paraffin, and 4 μm thick paraffin sections were prepared, followed by hematoxylin-eosin (HE) staining. The dehydration-embedding-staining procedures were performed according to standard paraffin sectioning procedures in this field. The pathological morphology of follicles at various stages, endometrium, and stroma in the sections was observed using an optical microscope. The staining results are shown in [Figure number missing]. Figures 7-8 And 10~11.

[0046] Depend on Figures 7-8 As seen in Figures 10-11, the model group showed a decrease in follicles at all levels, an increase in atretic follicles, endometrial atrophy, and stromal inflammatory infiltration; after Eri intervention, the number of primordial / growing / mature follicles significantly increased, uterine structure recovered, and fibrosis was reduced.

[0047] (3) Bone histopathology: After the intervention, femurs were removed from each group of mice. The femur samples were decalcified using a 10% EDTA-Na2 (ethylenediaminetetraacetic acid disodium salt) decalcification solution until a pin could be inserted into the bone cortex without resistance, resulting in decalcified femurs. The decalcified femurs were then embedded in paraffin and sectioned. After hematoxylin and eosin (HE) staining, the continuity, number, and spacing of trabeculae were observed under a microscope to assess the degree of bone microstructural damage. The results are as follows: Figure 9 As shown.

[0048] Depend on Figure 9 It is evident that the model group mice exhibited femoral trabecular fracture and reduced bone mass; Eri dose-dependently repaired bone microstructure, indicating that Eri can be used to improve POI-induced secondary osteoporosis.

[0049] (4) Serum hormone ELISA: After the intervention, mice were anesthetized before sacrifice, and whole blood was collected. Whole blood samples were centrifuged at 3000 rpm for 15 min, and serum was separated and collected. The following ELISA kits were used: mouse estradiol (E2) ELISA kit (Catalog No.: JL11790-48T), mouse follicle-stimulating hormone (FSH) ELISA kit (Catalog No.: JL10239-48T), progesterone (P4, PROG) ELISA kit (Catalog No.: JL54600-48T), and human luteinizing hormone (LH) ELISA kit (Catalog No.: JL12917-48T) from Jianglai Biotechnology; and the anti-Müllerian hormone (AMH) kit (Catalog No.: H324-1-2) from Nanjing Jiancheng Bioengineering Institute.

[0050] Strictly follow the instructions for each kit, measure the OD value of each well using an ELISA reader, and calculate the serum levels of E2, P4, AMH, FSH, and LH in the sample by fitting a standard curve based on the kit's accompanying standards. The test results are shown below. Figure 14 .

[0051] Depend on Figure 14 As can be seen, compared with the blank group, the serum levels of E2 and AMH in the model group mice were decreased, while the levels of FSH and LH were increased, and P4 showed no significant change. After administration of Eri, the serum levels of E2 and AMH in mice were significantly upregulated, and the levels of gonadotropins were decreased, indicating that Eri can be used to balance the endocrine levels of POI mice.

[0052] (5) Evaluation of fertility function: After the modeling and drug administration were completed, female mice in each group were mated with healthy male mice at a ratio of 2:1. The pregnancy rate and litter size of the female mice were recorded. The survival rate of the offspring was observed for 21 consecutive days, and the pregnancy rate, litter size, and pup survival rate were statistically analyzed. The results are as follows: Figures 21-24 As shown.

[0053] Depend on Figures 21-24 As can be seen, the conception rate in the model group was almost zero, and compared with the control group, the number of pups and the survival rate of offspring in the model group were significantly reduced. The conception rate, litter size, and offspring survival rate of mice in the medium- and high-dose Eri groups were significantly increased, indicating that medium- and high-dose Eri can improve the reproductive function of POI mice; (6) Molecular mechanism of immunofluorescence + Western blotting: Western Blot Procedure: Ovarian and uterine tissues were accurately weighed and lysed using RIPA lysis buffer containing protease and phosphatase inhibitors. The tissues were thoroughly ground at low temperature, and the supernatant was collected by centrifugation to obtain total protein. Protein concentration was determined using the BCA method, and the loading amount for each group was standardized. Protein samples were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane using wet transfer. The membrane was blocked with 5% skim milk at room temperature for 1.5 h. Then, ERα (1:1000), ERβ (1:1000), Keap1 (1:1000), Nrf2 (1:1000), HO-1 (1:1000), GPX4 (1:1000), CD38 (1:1000), IL-17 (1:1000), and an internal control were added. β Incubate Acatin (1:1000) primary antibody overnight at 4 ℃; after thorough washing with TBST, add HRP-labeled secondary antibody (1:5000) and incubate at room temperature for 1 h; after washing again, use ECL luminescent reagent for imaging, and ImageJ software to quantitatively analyze the band gray level and calculate the relative expression level of the target protein.

[0054] Immunofluorescence procedure: Ovarian tissue sections were dewaxed, antigen-retrievaled, and permeabilized. Non-specific binding was blocked with 5% BSA. Primary antibody against the target protein was added and incubated overnight at 4°C. After washing with PBS, fluorescently labeled secondary antibody was added and incubated at room temperature in the dark. Nuclear staining was performed with DAPI, and the sections were mounted with anti-fluorescence quenching mounting medium. Fluorescence images were acquired using a laser confocal microscope to observe protein localization. Image J analysis was used to analyze the average fluorescence intensity of the sections. Results are shown below. Figures 15-20 As shown.

[0055] Depend on Figures 15-20 It is evident that Eri can improve the ovarian microenvironment through multiple molecular mechanisms. On one hand, Eri can upregulate the expression of ERα and ERβ proteins in the ovary and uterus, activating the estrogen signaling pathway. On the other hand, Eri can also regulate the Keap1 / Nrf2 / GPX4 axis, downregulating Keap1, promoting Nrf2 nuclear translocation, increasing the expression levels of HO-1 and GPX4, and inhibiting ferroptosis. Furthermore, Eri can significantly reduce the expression of CD38 and IL-17, effectively alleviating local chronic inflammation in the ovary. Eri achieves its therapeutic effect on POI through the combined action of multiple pathways.

[0056] Example 5: Molecular docking verification of target binding ability Crystal structures of Erα (ID: 1X7R), ERβ (ID: 1X7J), Keap1 (ID: 6HWS), Nrf2 (ID: 7K2L), HO-1 (ID: 5BTQ), and GPX4 (ID: 2HGS) proteins were downloaded from the PDB database. The three-dimensional structure of Eri was generated and optimized using Chem 3D. Using the CB-Dock2 platform, Eri was molecularly docked with Erα, ERβ, Keap1, Nrf2, HO-1, and GPX4 proteins, respectively. The optimal binding conformation was selected based on the Vina score to obtain the docking complexes. The docking complexes were imported into PyMOL software, and the ligands and proteins were separated. The interaction patterns between Eri and key residues of the target proteins were analyzed based on visibility distance and hydrophobic contact, and binding pattern diagrams were exported. The results are as follows: Figures 25-30 As shown.

[0057] Depend on Figures 25-30 The binding sites and amino acid residue binding of Eri to each target protein are shown below, along with the binding energies (kcal / mol): ERα (-8.2), ERβ (-9.5), Keap1 (-9.6), Nrf2 (-9.7), HO-1 (-8.0), and GPX4 (-9.0). It is evident that the target binding energies of Eri to the key residues of all target proteins are <-5 kcal / mol, demonstrating that Eri can stably bind to the core target proteins and can directly act on the target sites to exert its therapeutic effect.

[0058] Example 6: Gut Microbiota 16S rRNA Sequencing Experiment Thirty-six cecal contents were collected from mice in each group. Microbial DNA was extracted from the cecal contents using a DNA extraction kit. The microbial DNA was then subjected to agarose gel electrophoresis and quality control using NanoDrop2000. Using the quality-controlled DNA as a template, the V3-V4 hypervariable region of 16S rRNA was amplified. The amplified products were then sequenced using the Illumina NovaSeq PE250 platform. After quality control filtering using FASTP software, the sequencing data were clustered using the UPARSE algorithm to generate OTUs. Further analysis of bacterial diversity, species abundance, and differential bacterial counts was performed based on the OTU abundance matrix. The results are shown below. Figures 31-43 As shown.

[0059] Depend on Figures 31-43It is evident that Eri has a significant regulatory effect on the gut microbiota of POI mice. α-Diversity: Analysis showed that the Shannon index increased after Eri administration, indicating a restoration of gut microbiota diversity. Regarding microbiota composition, compared to the model group, the Eri-treated group not only reversed the decreasing trend of the Bacteroidetes / Firmwallis ratio but also upregulated α-diversity. Muribaculaceae , Lachnospiraceae The relative abundance of beneficial bacteria such as NK4A136 was reduced, while the enrichment of pro-inflammatory Proteobacteria was inhibited.

[0060] PCoA and LEfSe analyses further confirmed that after Eri intervention, the gut microbiota structure of POI mice approached that of the normal control group. This demonstrates that Eri can reduce harmful pro-inflammatory bacteria, regulate the gut microbiota, and systematically alleviate systemic inflammation through the gut-ovarian axis, thereby synergistically improving the POI effect.

[0061] Example 7: A Sacred Herbol Oral Capsule The ingredients are: 100 mg of 98% pure sennaol, 80 mg of lactose, 100 mg of starch and 2 mg of magnesium stearate.

[0062] Preparation steps: Grind all raw materials into powder and pass them through an 80-mesh sieve. Mix the sieved raw materials evenly using an equal-volume incremental addition method to obtain a medicinal powder. Fill the medicinal powder into hollow hard capsules, with each capsule containing 100 mg of sennaol.

[0063] The method of use for the oral capsules is as follows: adults take 1 capsule twice a day, suitable for long-term oral intervention in people with early-onset ovarian insufficiency.

[0064] In summary, this invention provides the application of sennarol in the preparation of products for treating early-onset ovarian insufficiency. This invention is the first to discover that sennarol can simultaneously regulate four major pathways: the estrogen pathway, anti-ferrooxidative death, the CD38 / IL-17 inflammatory axis, and the gut microbiota, overcoming the limitations of traditional drugs that can only act on a single pathway. It achieves a multi-level reversal of POI pathological damage from the cellular, tissue, endocrine, and gut microbiota levels. Furthermore, as a natural flavonoid, sennarol has no carcinogenic risk, and its safety profile is superior to hormone replacement therapy.

[0065] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of sennaol in the preparation of products for the prevention and / or treatment of premature ovarian insufficiency.

2. The application according to claim 1, characterized in that, The raw materials for the product include sennaol and excipients.

3. The application according to claim 1, characterized in that, The product contains 30wt% to 40wt% of sennaol.

4. The application according to claim 1, characterized in that, The dosage form of the product includes oral formulations.

5. The application according to claim 1, characterized in that, The oral preparations include any one of tablets, capsules, granules, oral liquids, and suspensions.

6. The application according to claim 1, characterized in that, The application includes any one or more of the following: 1) Repair ovarian endocrine function; 2) Promotes estrogen secretion; 3) Inhibits granulocyte senescence; 4) Relieves inflammation; 5) Blocking ferrodeogenesis; 6) Repair the gut microbiota; 7) Improves osteoporosis; 8) Improve reproductive function.

7. The application according to claim 1, characterized in that, The products include pharmaceuticals or health supplements.

8. The application according to claim 7, characterized in that, The health supplements include those that help regulate gut microbiota and / or those that help with antioxidant effects.

9. A drug for treating early-onset ovarian insufficiency, characterized in that, The drug includes functional ingredients and excipients; the functional ingredients include sennaol.

10. The use of the drug according to claim 9 in the treatment of premature ovarian dysfunction complications, characterized in that, The early-onset ovarian dysfunction complications include any one or more of the following: uterine atrophy, ovarian fibrosis, osteoporosis, and inflammation.

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