Application of berberine in preparation of medicine for preventing and / or delaying ovarian physiological senescence

Abstract

The invention discloses application of berberine (hereinafter referred to as BBR) in preparation of a medicine for preventing and / or delaying ovarian physiological senescence, and belongs to the technical field of biological medicine. The invention aims to provide a medicine application scheme capable of effectively delaying ovarian senescence and improving ovarian functions. Animal experiment research finds that the BBR can significantly increase the number and quality of oocytes in physiologically aged mice and promote the increase of the levels of anti-mullerian hormone and estradiol, so that the ovarian endocrine function is improved. The invention has the following beneficial effects: BBR can improve ovarian function at animal level and delay ovarian physiologic aging, and provides a new idea and technical basis for development of related drugs.

Description

Technical Field

[0001] This invention belongs to the field of molecular biomedical technology, and in particular relates to the application of berberine in the preparation of drugs for preventing and / or delaying physiological ovarian aging. Background Technology

[0002] The ovary is a vital organ of the female reproductive system, responsible for ovulation and reproduction, and regulating the body's endocrine balance by secreting estrogen and progesterone. Ovarian function directly impacts a woman's fertility, endocrine levels, and overall health. With age, ovarian function gradually declines, manifested as a decrease in the number and quality of follicles, hormonal imbalances, and irregular ovulation cycles, ultimately leading to menopause—a process known as physiological ovarian aging.

[0003] The physiological aging mechanism of the ovary is complex, involving the combined effects of multiple factors such as follicle depletion, granulosa cell dysfunction, interstitial tissue fibrosis, mitochondrial dysfunction, hormonal feedback axis dysregulation, chronic inflammation, and enhanced oxidative stress. Especially during perimenopause, the levels of pro-inflammatory factors and reactive oxygen species (ROS) in ovarian tissue are significantly elevated, inducing local cellular stress responses and programmed cell death, accelerating the structural and functional degeneration of ovarian tissue.

[0004] Currently, hormone replacement therapy (HRT) is the primary clinical approach to alleviate ovarian aging or menopausal symptoms. This method relies on exogenous estrogen supplementation, which can improve symptoms in the short term, but long-term use may increase the risk of breast cancer, endometrial cancer, and cardiovascular disease. Therefore, there is an urgent need to develop safer, long-term-use natural drugs or small-molecule interventions to prevent and improve physiological ovarian aging.

[0005] Berberine (BBR) is a natural isoquinoline alkaloid found in traditional Chinese medicinal plants such as Coptis chinensis and Phellodendron amurense, exhibiting good oral safety and various pharmacological activities. Numerous studies have shown that berberine has significant effects in anti-inflammation, anti-oxidation, regulating energy metabolism, improving insulin resistance, anti-tumor activity, and delaying cellular senescence. Its mechanisms involve multiple inflammatory signaling pathways, including AMPK / mTOR, PI3K / AKT, Nrf2 / ARE, and NF-κB. However, there are currently no reports in the literature regarding the application of berberine in physiological ovarian aging. Summary of the Invention

[0006] To address the lack of safe and effective drugs for intervening in ovarian physiological aging in existing technologies, the purpose of this invention is to provide the application of berberine in the preparation of drugs for preventing and / or delaying ovarian physiological aging. Animal experiments have shown that berberine can significantly improve manifestations related to ovarian physiological aging, including increasing the number and quality of oocytes, promoting follicle development, and effectively improving endocrine disorders such as decreased estradiol (E2) and anti-Müllerian hormone (AMH) levels and increased follicle-stimulating hormone (FSH) levels. Further research results indicate that berberine can improve ovarian function and delay the degeneration of ovarian tissue structure and function. Therefore, the beneficial effects of this invention are: berberine, as a natural small molecule component, has good safety and a clear biological effect, and can serve as an effective active pharmaceutical ingredient for delaying ovarian physiological aging and improving ovarian function, providing new pharmacological ideas and application value for the prevention and treatment of ovarian aging.

[0007] To achieve the above objectives, this application provides the following technical solution: One object of the present invention is the use of berberine in the preparation of drugs for preventing and / or delaying physiological ovarian aging.

[0008] In some preferred embodiments of this, the physiological aging of the ovaries is manifested as estrous cycle disorder.

[0009] In some preferred embodiments of this aspect, the physiological aging of the ovaries is manifested by changes in serum sex hormones, specifically: a decrease in E2 and AMH levels, and an increase in FSH levels.

[0010] In some preferred embodiments of this aspect, the physiological aging of the ovary is characterized by a decrease in the number of growing follicles in the ovary.

[0011] In some preferred embodiments of this aspect, the physiological aging of the ovary is manifested as a decrease in the number of ovarian oocytes and a decline in the quality of ovarian oocytes; wherein, the evaluation indicators of ovarian oocyte quality are: the proportion of oocytes expelled from the first polar body and the proportion of fragmented oocytes.

[0012] In some preferred embodiments of this aspect, the physiological aging of the ovary is manifested as a decrease in the fertilization rate of ovarian oocytes and a decrease in embryonic developmental potential; wherein, the evaluation index of embryonic developmental potential is the blastocyst formation rate.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing and / or delaying physiological ovarian aging, the pharmaceutical composition comprising berberine and a pharmaceutically acceptable carrier or excipient.

[0014] In some preferred embodiments of this aspect, the carrier or excipient includes, but is not limited to, the following types: Diluents: such as starch, pregelatinized starch, lactose, dextrin, sucrose, microcrystalline cellulose (MCC), mannitol, sorbitol, and inorganic calcium salts (such as calcium hydrogen phosphate, calcium carbonate, and calcium sulfate), are used to increase drug volume, improve formability, or improve taste. Adhesives: such as pure water, ethanol, starch paste, hydroxypropyl methylcellulose (HPMC) or hydroxymethylcellulose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), gelatin, etc., are used to improve the adhesion between powders and enhance the mechanical strength of the formulation. Lubricants, such as magnesium stearate (MS), micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol (PEG), sodium lauryl sulfate or magnesium salt, are used to improve the flowability and release properties of formulations during tableting, capsule filling and other processes.

[0015] The specific types and amounts of the excipients can be optimized and adjusted according to the target formulation (such as tablets, capsules, granules, pellets, solutions, etc.), administration route (oral, transdermal, topical, etc.), and the physicochemical properties of berberine to achieve good pharmaceutical performance and biological stability.

[0016] In a preferred embodiment of this aspect, berberine (BBR) is the main active ingredient in the pharmaceutical composition, enabling it to contact ovarian tissue and thereby achieve a therapeutic or interventional effect. This pharmaceutical composition can be used to reduce or inhibit the activity of pro-inflammatory pathways in the ovary, improving ovarian physiological function decline caused by their abnormal activation.

[0017] Compared with the prior art, the present invention has the following advantages: This invention involves intervention with berberine (BBR) in a physiologically aging mouse model. Experimental results show that BBR significantly reduces the expression levels of pro-inflammatory pathways in oocytes and granulosa cells of the ovary, thereby alleviating local inflammation and cellular stress. This regulatory effect is further manifested in correcting estrous cycle disorders and improving sex hormone levels (including elevated estradiol E2 and anti-Müllerian hormone AMH), indicating a significant improvement in ovarian endocrine function. Furthermore, BBR intervention effectively increases ovarian reserve, specifically by increasing the number of primordial and growing follicles and reducing follicular atresia, suggesting a protective effect on follicle survival and development. Regarding oocyte quality, mice in the BBR-treated group exhibited higher oocyte structural integrity, more uniform mitochondrial distribution, and better spindle formation, demonstrating superior fertilization capacity and embryonic developmental potential.

[0018] This invention, through in-depth research, has discovered that berberine has a significant protective effect during ovarian aging, and its mechanism partly depends on the negative regulation of inflammatory signaling pathways. Therefore, berberine, as a reliable and low-toxicity natural active ingredient, not only has broad application prospects in anti-ovarian aging, but its molecular mechanism mediated by inflammatory pathways is also clearly defined, providing a scientific basis and technological innovation for drug development and patent protection.

[0019] This invention demonstrates that BBR has significant effects in delaying physiological ovarian aging, protecting ovarian function, and improving oocyte quality, with good safety profile and broad clinical application prospects and translational value. It fills a research gap in the targeted regulation of ovarian aging signaling pathways by natural products, with clear molecular mechanism support and good application and translational potential. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the drug administration experiment in mice. Specifically, 60 physiologically aging mice (aged 10 months) were divided into two groups: an aging control group (n=30) and an aging-treated BBR group (n=30), with an additional 30 young control mice. The aging BBR group received 300 mg / kg of BBR (dissolved in physiological saline) via gavage, while the aging and young control groups received an equal volume of physiological saline via gavage. Drug administration was performed every two days for one month, followed by subsequent experimental validation.

[0021] Figure 2 The results show the morphology, mass, and hormone levels of mouse ovaries. A shows the ovarian morphology (hematoxylin-eosin staining, scale bar 200 μm) of the three groups of mice after drug administration, with the upper left corner showing the bright field image of the ovaries (scale bar 100 μm); B shows the ovarian weight of the three groups of mice; C shows the estrous cycle ratio of the three groups of mice; D shows the statistical results of ovarian follicle growth in the three groups of mice; E shows the serum AMH hormone level of the three groups of mice; and F shows the serum E2 hormone level of the three groups of mice.

[0022] Figure 3 Bright field plots and statistical results of superovulation in three groups of mice.

[0023] Figure 4 Figure 1 shows the results of mitochondrial function staining in mouse oocytes after superovulation. Figure A shows the results and statistical graphs of Mito Tracker staining in mitochondria of the three groups of mouse oocytes (scale bar: 25 μm); Figure B shows the results and statistical graphs of MitoSox staining in mitochondria of the three groups of mouse oocytes (scale bar: 25 μm); Figure C shows the results and statistical graphs of JC-1 staining of mitochondrial membrane potential in the three groups of mouse oocytes (scale bar: 25 μm).

[0024] Figure 5 The results of Annexin V and ROS staining in mouse oocytes after superovulation are presented, along with statistical analysis. Figure A shows the Annexin V staining results and statistical graph (scale bar 25 μm) of mouse oocytes from the three groups; Figure B shows the ROS staining results and statistical graph (scale bar 25 μm) of mouse oocytes from the three groups.

[0025] Figure 6 Immunofluorescence results and statistical graphs of oocyte spindle fibers and chromosome alignment after superovulation in mice (scale bar: 25 μm).

[0026] Figure 7 Immunofluorescence results and statistical graphs of chromosome staining on oocyte slices after superovulation in mice (scale bar: 25 μm).

[0027] Figure 8 The results of in vitro fertilization culture of mice after superovulation and the statistical results of pregnancy rate of mice in cages were presented. Detailed Implementation

[0028] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] The experimental animals used in this embodiment were clean-grade C57BL / 6 female mice, purchased from Beijing Spaford Laboratory Animal Technology Co., Ltd. The specific experimental groups are as follows: Young control group: 8-week-old female mice; Aged control group: 10-month-old female mice; BBR intervention group: 10-month-old female mice were administered BBR by gavage; Male mice used for breeding: 3-month-old C57BL / 6 male mice.

[0030] The mouse housing environment was set as follows: temperature maintained at 22–25°C, relative humidity ≤50%, and a 12-hour light-dark cycle. Animals had free access to standard feed and water; both feed and water were sterile, and bedding was changed weekly. All animal experiments were ethically approved and conducted in accordance with the "Regulations on the Management of Laboratory Animals." Unless otherwise specified, the procedures and techniques used in the experiments were standard experimental techniques well-known to those skilled in the art, or performed according to the operating conditions recommended by the relevant reagent and equipment suppliers.

[0031] Example 1: BBR improves ovarian follicle reserve in mice Sixty physiologically aging mice (10 months old) were randomly divided into two groups: an aging control group (n=30) and an aging-treated BBR group (n=30), along with a young control group (n=30). The aging-treated BBR group received 300 mg / kg of BBR (dissolved in saline) via gavage, while the aging and young control groups received an equal volume of saline via gavage. Administration was performed every two days for one month, followed by subsequent experimental validation.

[0032] One month after gavage administration, vaginal exfoliated cells were collected from the young control group, aging control group, and BBR intervention group every morning for 30 consecutive days to prepare smears. These smears were then stained with hematoxylin and eosin to observe the estrous cycle. These 20 mice were used in subsequent experiments. Six mice were randomly selected and sacrificed. Ovaries from all three groups were collected, fixed in 4% formaldehyde, and prepared into 5μm paraffin sections. After HE staining, the morphology and count of growing follicles (primary follicles, secondary follicles, and antral follicles) were observed under a microscope. Serum was also collected, and AMH and E2 were detected by ELISA (Elabscience). The results are shown below. Figure 1 and Figure 2 .

[0033] from Figure 1 The specific experimental groups, as well as the experimental drug dosage and timing, can be seen from the data.

[0034] from Figure 2 China A and Figure 2 As can be seen from B, the ovarian volume of mice in the BBR intervention group was significantly larger than that in the aging group; from Figure 2 The results show that the rate of irregular estrous cycles in mice was significantly higher in the aging control group compared to the young control group; while the rate of regular estrous cycles was higher in the BBR intervention group than in the aging control group. Figure 2 As can be seen from the data, the number of growing follicles decreases during ovarian aging, and the BBR intervention group mice had more growing follicles in their ovaries than the aging control group mice. Figure 2 China E and Figure 2 The results showed that the serum levels of the hormones AMH and E2 in the BBR intervention group were significantly higher than those in the aging control group.

[0035] Figure 2 The results showed that BBR intervention could correct estrous cycle disorders and increase serum AMH and E2 hormone levels in aging mice; and improve ovarian follicle reserve in aging mice.

[0036] Example 2: BBR improves the number of superovulations in aging mice Six mice were randomly selected from each of the aging control group, BBR intervention group, and young control group in Example 1 to perform a superovulation experiment. The specific procedure was as follows: 10 IU of pregnant mare serum gonadotropin (PMSG) was injected into each of the three groups of mice. 48 hours later, 10 IU of human chorionic gonadotropin (hCG) was injected. 14 hours later, cumulus oocyte complexes (COCs) expelled into the ampulla of the fallopian tube were removed. The granulosa cells surrounding the oocytes were then removed using 0.1% hyaluronidase. The morphology of the oocytes was observed and counted under a microscope.

[0037] from Figure 3 It can be seen that the number of ovulations decreased in the aging control group compared with the young control group, while the number of ovulations in the BBR intervention group increased significantly compared with the aging control group.

[0038] Figure 3 The results showed that BBR intervention could improve the number of superovulations in aging mice.

[0039] Example 3: BBR intervention improves mitochondrial abnormalities in oocytes of aging mice Six mice from each of the groups in Example 1 were selected, and oocytes were obtained from the three groups of mice using the same superovulation method as in Example 2. The obtained oocytes were then subjected to the following operations: 1. Mito Tracker staining of oocytes: Mito-Tracker-Red was diluted to 200 nM using Opti-MEM medium. Oocytes were transferred into droplets and incubated at 37 °C in the dark for 30 min. After washing three times in PBS containing 0.1% BSA, the oocytes were transferred to a glass-bottomed confocal microscope dish and coated with paraffin oil. Images were observed and acquired under a confocal microscope within 1 hour.

[0040] 2. MitoSox staining of oocytes: MitoSox was diluted to 50 nM using Opti-MEM medium. Oocytes were transferred into droplets and incubated at 37 °C in the dark for 30 min. After washing three times in PBS containing 0.1% BSA, the oocytes were transferred to a glass-bottomed confocal microscope dish and coated with paraffin oil. Images were observed and acquired under a confocal microscope within 1 hour.

[0041] 3. Mitochondrial membrane potential staining of oocytes: The experiment was performed using a mitochondrial membrane potential detection kit. 1 μl of JC-10 (200x) probe was diluted in 160 μl of ultrapure water, and 40 μl of JC-1 staining buffer was added and mixed thoroughly to form a droplet. Oocytes were transferred into the droplet and incubated at 37 ℃ in the dark for 20 min. After washing three times with staining buffer (1x), the cells were transferred to a glass-bottomed confocal microscope dish and coated with paraffin oil. Images were observed and acquired under a confocal microscope within 1 hour.

[0042] from Figure 4 China A and Figure 4 As can be seen from B, compared with aging control mice, the abnormal aggregation and distribution of mitochondria in oocytes of aged mice treated with BBR were reduced; Figure 4 The results show that, compared with aging control mice, the mitochondrial membrane potential of oocytes in BBR-treated aged mice was significantly increased.

[0043] Figure 4 The results showed that BBR intervention could improve mitochondrial abnormalities in oocytes of aging mice.

[0044] Example 4: BBR intervention improves the quality of oocytes in aging mice Six mice from each of the groups in Example 1 were selected, and oocytes were obtained from the three groups of mice using the same superovulation method as in Example 2. The obtained oocytes were then subjected to the following operations: 1. Detection of oocyte apoptosis signals: Annexin V-mCherry cell apoptosis detection was performed. The assay kit was used for the experiment. Following the instructions, 5 μl of Annexin V-mCherry was added to 195 μl of binding buffer. Oocytes were transferred into freshly prepared Annexin V-mCherry binding buffer and stained at room temperature in the dark for 20 min. After washing three times in PBS containing 0.1% BSA, the cells were transferred to a glass-bottomed confocal microscope and coated with paraffin oil. Images were observed and acquired under a confocal microscope within 1 hour.

[0045] 2. Reactive oxygen species (ROS) staining of oocytes: 2,7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) was diluted to 10 μM using Opti-MEM medium. Oocytes were transferred into droplets and incubated at 37°C in the dark for 30 min. After washing three times in PBS containing 0.1% BSA, the oocytes were transferred to a glass-bottomed confocal microscope dish and coated with paraffin oil. Images were observed and acquired under a confocal microscope within 1 hour.

[0046] from Figure 5As can be seen from Figure A, compared with aging control mice, the level of reactive oxygen species in oocytes of aged mice treated with BBR was significantly reduced; Figure 5 The results from B indicate that, compared with aging control mice, the apoptosis signaling level of oocytes in BBR-treated aged mice was significantly reduced.

[0047] Figure 5 The results showed that BBR intervention could improve the quality of oocytes in aging mice.

[0048] Example 5: BBR intervention improves the quality of oocytes in aging mice, reduces the incidence of chromosomal ploidy abnormalities, abnormal spindle structures, and chromosomal misalignment. Six mice from each of the groups in Example 1 were selected, and oocytes were obtained from the three groups of mice using the same superovulation method as in Example 2. The obtained oocytes were then subjected to the following operations: 1. The obtained oocytes were fixed in paraformaldehyde for 30 min, then permeabilized with 0.5% Triton X-100 for 20 min, blocked with 1% BSA for 1 h, and incubated overnight at 4°C with 1:200 anti-α-tubulin monoclonal antibody. After washing three times with IVF solution, the cells were incubated with FITC-conjugated secondary antibody at room temperature for 1 h, and then the nuclei were stained with DAPI. The cells were then transferred into 10 μL of anti-quenching agent droplets and observed under a Zeiss LSM880 laser confocal microscope to observe the spindle morphology and chromosome arrangement.

[0049] 2. Removal of the zona pellucida: Collect oocytes and wash them three times in M2 processing solution. Then, transfer the oocytes to acidic stearic acid solution, minimizing the amount of M2 processing solution introduced. Next, observe under a stereomicroscope. When the zona pellucida is completely invisible, quickly aspirate the oocytes and transfer them back to the M2 processing solution to wash away the acidic stearic acid solution and prevent oocyte adhesion. Spreading: Use an adhesive slide for chromosome spreading. First, draw a rectangle approximately 1 cm × 0.5 cm on the reverse and front sides of the slide using a marker and a hydrophobic pen, respectively. Add approximately 5-10 μL of spreading solution to the hydrophobic pen-marked rectangle on the front side, and aspirate the supernatant after centrifugation to reduce impurities. Next, replace with a finer pipette (with an inner diameter not too different from the oocyte diameter) and aspirate 7-10 oocytes from which the zona pellucida has been removed. In the preparation solution, with the bottom of the pipette pressed firmly against the slide, gently slide it from left to right, ejecting the oocytes one by one in a straight line, maintaining a certain gap between adjacent oocytes. At this point, the oocytes can be observed exploding under a stereomicroscope, gradually becoming invisible or leaving only blurry traces. Then, allow them to air dry naturally on a flat surface at room temperature. In summary, from the time the oocytes explode until the preparation solution is completely dry, the movement of the droplet should be avoided as much as possible. On the one hand, during the ejection of oocytes, the pipette should be moved gently to the right. After the last oocyte in the pipette is ejected, the bottom of the pipette should continue to slide to the right with the bottom of the pipette pressed against the slide until it leaves the droplet. The speed should not be too fast or too slow throughout the process, and the movement should be kept as still as possible to avoid internal flow within the droplet. Nuclear staining and mounting: The nuclei of the oocytes are stained with DAPI for 15 minutes and placed in a humidified chamber at room temperature. The number of chromosomes is observed under a Zeiss LSM880 laser confocal microscope.

[0050] from Figure 6 It can be seen that, compared with aging control mice, the incidence of abnormal spindle structures in oocytes and chromosome misalignment was significantly reduced in aging mice treated with BBR.

[0051] from Figure 7 It can be seen that, compared with aging control mice, the oocyte chromosomal ploidy abnormalities in aging mice treated with BBR were significantly reduced.

[0052] Figure 6 and Figure 7 The results showed that BBR intervention could improve the quality of oocytes in aging mice.

[0053] Example 6: BBR intervention improves oocyte fertilization rate and enhances embryonic developmental potential in aging mice. The COCs obtained in Example 2 were fertilized in vitro with sperm from 3-month-old male mice that had undergone in vitro capacitation treatment in IVF droplets for 6 hours. The fertilized eggs were then transferred to KSOM culture droplets, washed, and transferred again to new KSOM droplets for further culture for 6 hours. Pronucleus formation was observed, and the blastocyst rate was recorded. A co-breeding experiment was conducted on the three groups of mice, and their pregnancy rates were calculated.

[0054] from Figure 8 As can be seen from A, compared with aging control mice, the blastocyst rate of in vitro fertilization development in BBR-treated aging mice was significantly increased; from Figure 8 The results show that, compared with aging control mice, the pregnancy rate of aging mice treated with BBR was significantly increased.

[0055] Figure 8 The results showed that BBR intervention could improve the fertilization rate of oocytes in aging mice, enhance embryonic developmental potential, and improve the fertility of aging mice.

[0056] It is understood that the above specific description of the present invention is only for illustrating the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention to achieve the same technical effect; as long as the use needs are met, they are all within the protection scope of the present invention.

Claims

1. Application of berberine in the preparation of drugs for preventing and / or delaying physiological ovarian aging.

2. The application according to claim 1, characterized in that, The physiological aging of the ovaries is manifested as estrous cycle disorder in animals.

3. The application according to claim 1, characterized in that, The physiological aging of the ovaries is characterized by changes in serum sex hormones, specifically: a decrease in estradiol and anti-Müllerian hormone levels, and an increase in follicle-stimulating hormone levels.

4. The application according to claim 1, characterized in that, The physiological aging of the ovaries is characterized by a decrease in the number of follicles growing in the ovaries.

5. The application according to claim 1, characterized in that, The physiological aging of the ovary is characterized by a decrease in the number of ovarian oocytes and a decline in the quality of ovarian oocytes; among which, the evaluation indicators of oocyte quality include the proportion of oocytes expelled from the first polar body and the proportion of fragmented oocytes.

6. The application according to claim 1, characterized in that, The physiological aging of the ovary is characterized by a decrease in oocyte fertilization rate and a decrease in embryonic developmental potential; among which, the evaluation indicators of embryonic developmental potential include blastocyst formation rate.

7. A pharmaceutical composition for preventing and / or delaying physiological ovarian aging, characterized in that, The pharmaceutical composition includes berberine and a pharmaceutically acceptable carrier or excipient.

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