A traditional Chinese medicine composition for repairing and / or activating residual ovarian function of aging ovary, a preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for repairing and / or activating residual ovary function of an aging ovary, a preparation method and application thereof, and relates to the technical field of biotechnology.The traditional Chinese medicine composition is composed of the following components: Astragalus membranaceus, Angelica sinensis, Ophiopogon japonicus, Polygonatum odoratum, Semen cuscutae, Morinda officinalis, Rubus chingii Hu, Dioscorea panthaica and Mulberry leaf.The application also provides application of the traditional Chinese medicine composition in repairing and / or activating residual ovary function of an aging ovary and improving quality of aging oocytes.The traditional Chinese medicine composition can increase the volume and weight of the aging ovary, increase the number of follicles at all levels in the aging ovary, reduce granulosa cell apoptosis in the ovary, reduce abnormal rate of oocytes, oxidative stress and DNA damage of oocytes, and promote and maintain development and maturation of residual follicles.The traditional Chinese medicine composition has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine biotechnology, specifically to a traditional Chinese medicine composition for repairing and / or activating residual ovarian function in aging ovaries, its preparation method, and its application. Background Technology

[0002] Ovarian aging is the process of declining ovarian reserve, mainly manifested by a decrease in the number of follicles and the quality of oocytes, until ovarian function fails, leading to decreased female fertility, menstrual disorders, and menopause. Ovarian aging is divided into age-related physiological ovarian aging and premature ovarian failure caused by pathological factors (premature ovarian insufficiency, i.e., premature ovarian failure). Ovarian aging is influenced by multiple factors such as age, genetic factors, immunity, iatrogenic factors, infection, and environment, and its occurrence, development, and clinical manifestations vary from person to person.

[0003] The basic structural and functional unit of the ovary—the follicle—is composed of an oocyte and surrounding granulosa cells. The primary oocyte, surrounded by a single layer of flattened granulosa cells, forms the primordial follicle, and the pool of primordial follicles established at this stage constitutes the ovarian reserve. During puberty, regulated by the gonadal axis, some of the primordial follicles are activated and enter a growth phase, successively developing into primary follicles, secondary follicles, preantral follicles, antral follicles, and finally ovulation. The vast majority of follicles in the ovary fail to mature; they cease growth and atresia at different stages of follicular development, leading to ovarian dysfunction. When follicular development ceases in the ovary, a woman enters menopause.

[0004] Ovarian function decline is a gradual process, and treatment strategies for ovarian aging need to focus on two key aspects: 1. In the mid-to-late stages of ovarian decline, elevated gonadotropins or an unfavorable ovarian microenvironment can lead to increased follicular atresia. Repairing aging ovarian function can reduce the rate of follicular atresia. 2. In the terminal stage of ovarian decline, a certain number of residual follicles still exist in the atrophied ovaries, retaining some potential for development and even fertilization. However, over time, due to a lack of developmental potential, these residual follicles will be permanently depleted. Therefore, finding effective methods to repair ovarian function, stimulate the development of residual follicles, and delay ovarian function decline is a pressing clinical challenge in the treatment of ovarian aging. In recent years, innovative technologies such as in vitro follicle activation (IVA), stem cell transplantation, and platelet-rich plasma (PRP) injection have been used to activate residual follicles. However, the research conclusions are mostly based on basic research and a limited number of small-sample observational clinical studies, and reliable safety and efficacy data are still lacking.

[0005] Traditional Chinese medicine (TCM) holds an important position in the field of anti-aging research. However, current TCM treatments for ovarian aging mainly focus on improving low estrogen-related symptoms caused by ovarian aging, or are used in combination with hormone replacement therapy to improve sex hormone levels. Research on drugs that protect / activate residual ovarian function is lacking. The TCM composition of this invention uses Astragalus membranaceus and Angelica sinensis as the principal herbs, Ophiopogon japonicus and Cuscuta chinensis as the assistant herbs, Rubus idaeus, Dioscorea opposita, Morinda officinalis, and Polygonatum odoratum as the adjuvant herbs, and Morus alba leaf as the guiding herb. This composition has the effects of tonifying qi and blood, promoting body fluid production and replenishing essence. This TCM composition, which can effectively delay ovarian aging, is of great significance for clinical practice. Summary of the Invention

[0006] The purpose of this invention is to provide a traditional Chinese medicine composition for delaying ovarian aging, its preparation method, and its application.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides a traditional Chinese medicine composition for repairing and / or activating residual ovarian function in aging ovaries, the traditional Chinese medicine composition being composed of the following components: Astragalus membranaceus, Angelica sinensis, Ophiopogon japonicus, Polygonatum odoratum, Cuscuta chinensis, Morinda officinalis, Rubus idaeus, Dioscorea opposita, and Morus alba leaves.

[0009] Furthermore, the traditional Chinese medicine composition comprises the following components by weight: 20-50 parts Astragalus membranaceus, 5-20 parts Angelica sinensis, 5-20 parts Ophiopogon japonicus, 5-20 parts Polygonatum odoratum, 10-30 parts Cuscuta chinensis, 5-20 parts Morinda officinalis, 5-20 parts Rubus idaeus, 5-20 parts Dioscorea opposita and 5-20 parts Morus alba.

[0010] Furthermore, the traditional Chinese medicine composition comprises the following components by weight: 20-40 parts Astragalus membranaceus, 5-15 parts Angelica sinensis, 5-15 parts Ophiopogon japonicus, 5-15 parts Polygonatum odoratum, 15-30 parts Cuscuta chinensis, 5-15 parts Morinda officinalis, 5-15 parts Rubus idaeus, 5-15 parts Dioscorea opposita and 5-15 parts Morus alba.

[0011] Furthermore, the traditional Chinese medicine composition comprises the following components by weight: 30 parts Astragalus membranaceus, 10 parts Angelica sinensis, 10 parts Ophiopogon japonicus, 10 parts Polygonatum odoratum, 20 parts Cuscuta chinensis, 10 parts Morinda officinalis, 10 parts Rubus idaeus, 10 parts Dioscorea opposita and 10 parts Morus alba.

[0012] In another aspect, the present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition, comprising the following steps:

[0013] (1) Take parts by weight of Astragalus membranaceus, Angelica sinensis, Ophiopogon japonicus, Polygonatum odoratum, Cuscuta chinensis, Morinda officinalis, Rubus idaeus, Dioscorea opposita and Morus alba leaves, and extract by reflux with water;

[0014] (2) Concentrate the extracted medicinal liquid to obtain a traditional Chinese medicine composition.

[0015] Specifically, the amount of water added is 8-15 times the total mass of the raw materials in the traditional Chinese medicine composition;

[0016] Furthermore, the amount of water added is 12 times the total mass of the raw materials of the traditional Chinese medicine composition.

[0017] Specifically, adding water also includes the step of soaking the medicinal herbs.

[0018] Furthermore, the soaking time is 20-60 minutes; even further, the soaking time is 30 minutes.

[0019] Specifically, the reflux extraction temperature is 70-90℃.

[0020] Specifically, the reflux extraction time is 60-150 min; further, the reflux extraction time is 90 min.

[0021] Specifically, the reflux extraction is performed 1-3 times, and more specifically, the reflux extraction is performed 2 times.

[0022] Specifically, the concentration refers to concentrating the drug solution to a final concentration of 1-2 g / ml;

[0023] Furthermore, the concentration refers to concentrating the drug solution to a final concentration of 1.2 g / ml.

[0024] In another aspect, the present invention provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of a drug for delaying ovarian aging.

[0025] Specifically, the ovarian aging mentioned includes physiological ovarian aging and pathological ovarian aging.

[0026] Furthermore, the ovarian aging described herein refers to physiological ovarian aging.

[0027] Furthermore, the pathological ovarian aging mentioned above includes ovarian aging caused by ovarian tumors, ovarian aging caused by drugs, and ovarian aging caused by iatrogenic factors.

[0028] Specifically, the drug may also include a pharmaceutically acceptable carrier.

[0029] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0030] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginate, gelatin, gum arabic, glyceryl monostearate, sodium glycolate starch, guar gum, glycerol, and propylene glycol.

[0031] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0032] Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglycerol-3 polyricinoleate.

[0033] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginate, cellulose ether, and carboxymethyl chitosan.

[0034] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0035] Specifically, the adhesive is selected from at least one of ethanol, starch paste, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinylpyrrolidone, gum arabic, gelatin, and alginic acid.

[0036] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0037] Specifically, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearate fumarate, and poloxamer.

[0038] Specifically, the pH adjuster is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0039] Specifically, the cryoprotectant is selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextrose, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, or formamide.

[0040] Specifically, the flavoring agent is selected from at least one of sweet orange flavoring, vanilla flavoring, strawberry flavoring, milk flavoring, banana flavoring, and cherry flavoring.

[0041] Specifically, the filler is selected from at least one of mannitol, xylitol, sorbitol, maltose, microcrystalline cellulose, glucose, lactose, sucrose, dextrin, starch, sodium alginate, and sodium bicarbonate.

[0042] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0043] In another aspect, the present invention provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of a medicine for repairing and / or activating residual ovarian function in aging ovaries.

[0044] Specifically, the drug may also include a pharmaceutically acceptable carrier.

[0045] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0046] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0047] The beneficial effects of this invention are as follows:

[0048] The traditional Chinese medicine composition provided by this invention can increase ovarian volume and weight, increase follicles at all stages in the ovary, specifically significantly increasing the number of primordial follicles, antral follicles, mature follicles and corpus luteum, reducing granulosa cell apoptosis in the ovary, better maintaining follicle development, increasing the number of follicles, reducing oocyte abnormality rate and oocyte DNA damage. The traditional Chinese medicine composition of this invention plays an important role in repairing and / or activating residual ovarian function in aging ovaries. Attached Figure Description

[0049] Figure 1 The average body weight of mice in each group.

[0050] Figure 2 The mean ovarian volume of mice in each group was **p<0.01 compared with the young control group and #p<0.05 compared with the aged control group.

[0051] Figure 3 The mean ovarian weight of mice in each group was ***p<0.001 compared with the young control group and #p<0.05 compared with the aged control group.

[0052] Figure 4 Pathological images of ovarian tissue from each group of mice.

[0053] Figure 5 The number of primordial follicles in each group of mice was ***p<0.001 compared with the young control group and #p<0.05 compared with the aged control group.

[0054] Figure 6 The initial number of follicles in each group of mice was compared with that of the young control group. ***p<0.001.

[0055] Figure 7 The number of secondary follicles in each group of mice was compared with that in the young control group, ***p<0.001.

[0056] Figure 8 The number of antral vesicles in each group of mice was ***p<0.001 compared with the young control group; and #p<0.05 and ##p<0.01 compared with the aged control group.

[0057] Figure 9 The number of corpora lutea in each group of mice was ***p<0.001 compared with the young control group and ##p<0.01 compared with the aged control group.

[0058] Figure 10 The mean number of mature follicles in each group of mice was ***p<0.001 compared with the young control group; and #p<0.05 and ##p<0.01 compared with the aged control group.

[0059] Figure 11 The image shows the results of ovarian apoptosis detection in mice of each group. In the image, the red arrows represent apoptotic granulosa cells.

[0060] Figure 12 Representative images of the ovaries, fallopian tubes, and number of oocytes retrieved from each group of mice are shown. Red arrows indicate MII stage oocytes in the ampulla of Vater.

[0061] Figure 13 The number of GV-stage oocytes in each group of mice was ***p<0.001 compared with the young control group and #p<0.05 compared with the aged control group.

[0062] Figure 14 The number of MII stage oocytes in each group of mice was ***p<0.001 compared with the young control group and #p<0.05 compared with the aged control group.

[0063] Figure 15The oocyte fragmentation rate in each group of mice was ***p<0.001 compared with the young control group; and ##p<0.01 and ###p<0.001 compared with the aged control group.

[0064] Figure 16 The germinal vesicle rupture rate of mouse oocytes in each group was ***p<0.001 compared with the young control group.

[0065] Figure 17 The oocyte polar body expulsion rate was calculated for each group of mice. Compared with the young control group, **p < 0.01, and compared with the aged control group, #p < 0.05.

[0066] Figure 18 The image shows the results of immunofluorescence γ-H2AX staining of mouse oocytes in each group.

[0067] Figure 19 The statistical results for γ-H2AX are shown in the figure.

[0068] Figure 20 A representative diagram of ROS in oocytes from each group.

[0069] Figure 21 The ROS statistics of oocytes in each group are shown. Compared with the young control group, ***p<0.001, and compared with the elderly control group, #p<0.05.

[0070] Figure 22 The graph shows the detection of mitochondrial membrane potential JC-1 in oocytes of each group.

[0071] Figure 23 The statistical graph shows the JC-1 fluorescence values ​​of mitochondrial membrane potential in oocytes of each group. Compared with the young control group, ***p<0.001, and compared with the elderly control group, #p<0.05.

[0072] Figure 24 The number of GV-stage oocytes in each group of mice in Example 2 was ***p<0.001 compared with the young control group and #p<0.05 compared with the aged control group.

[0073] Figure 25 The number of MII oocytes in each group of mice in Example 2 was compared with that of the young control group (***p<0.001) and the elderly control group (#p<0.05).

[0074] Figure 26 The images show the pathological images of ovarian tissue from each group of mice in Comparative Example 1.

[0075] Figure 27For the statistical count of follicles at all levels in each group of mice in Comparative Example 1, compared with the young control group, **p < 0.01, ***p < 0.001; compared with the old control group, #p < 0.05.

[0076] Figure 28 For the number of GV-stage oocytes in each group of mice in Comparative Example 2, compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05, and compared with the low-dose traditional Chinese medicine group, #p < 0.05.

[0077] Figure 29 For the number of MII-stage oocytes in each group of mice in Comparative Example 2, compared with the young control group, ***p < 0.001; compared with the old control group, ##p < 0.01, and compared with the low-dose traditional Chinese medicine group, #p < 0.05. Specific implementation manners

[0078] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further clarified below in conjunction with specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention and not all. Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the used operation methods are all conventional operation methods, the used equipment is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0079] Example 1

[0080] 1. Experimental animals

[0081] The animals used in this study were SPF-level ICR female mice provided by the Guangdong Provincial Medical Experimental Animal Center, with the license number: SCXK (Guangdong) 2019-0035, and the animal certificate number NO. 430727240100049428. The animal experiment protocol passed the ethical review of the Animal Ethics Committee of the Guangdong Provincial Traditional Chinese Medicine Experimental Animal Center. The whole experiment was completed in the Experimental Animal Center of Guangdong Provincial Hospital of Traditional Chinese Medicine, and the experimental animal use license: SYXK (Guangdong) 2018-0094. The animals were quarantined and adaptively fed for one week before the experiment. The mice had free access to water and food, the experimental environment was clean and quiet, and the basic welfare of the experimental animals was reliably guaranteed.

[0082] Astragalus membranaceus 30g, Angelica sinensis 10g, Polygonatum odoratum 10g, Morinda officinalis 10g, Rubus idaeus 10g, Dioscorea opposita 10g, Morus alba leaf 10g

[0083] 2. Standardized preparation of the compound

[0084] The prescription consists of 30g Astragalus membranaceus, 10g Angelica sinensis, 10g Ophiopogon japonicus, 10g Polygonatum odoratum, 20g Cuscuta chinensis, 10g Morinda officinalis, 10g Rubus idaeus, 10g Dioscorea opposita, and 10g Morus alba leaves. These ingredients are placed in a 5L round-bottom flask according to the above proportions, with water added in a volume 12 times the volume of the herbs. The herbs are soaked for 30 minutes, then a reflux condenser is installed. A standard temperature-controlled electric heating mantle is used for heating. After the water boils, heating continues for 90 minutes. The decoction is collected, and the process is repeated once. The second decoction is collected, and the two decoctions are combined. The decoction is then concentrated to a final concentration of 0.91g / ml using a rotary evaporator.

[0085] 3. Mouse grouping and administration

[0086] Forty-five 12-month-old female ICR mice were randomly divided into three groups: an aged control group, an aged + low-dose traditional Chinese medicine group, and an aged + equivalent dose traditional Chinese medicine group (n=15 in each group). Another 15 2-month-old female ICR mice were used as normal controls.

[0087] Calculation of final concentration of traditional Chinese medicine: Based on the clinical daily dosage of 120g for a 60kg adult female, the conversion factor between mice and humans is 9.1. Therefore, the equivalent drug mass "m" for mice should be: 160g / 60kg*9.1≈18.2g / kg, with the low dose being 9.1g / kg. The gavage volume V for mice is calculated at 10ml / kg (i.e., 0.01ml / g). The low dose is administered once daily by gavage, while the equivalent dose of traditional Chinese medicine is administered twice daily, once in the morning and once in the afternoon. The traditional Chinese medicine administration lasts for 30 days. Elderly and young controls are given the same amount of drinking water.

[0088] 4. Detection Indicators and Methods

[0089] 4.1 Weight recording: Mouse weight changes were recorded weekly during the dosing period.

[0090] 4.2 Ovarian pathological examination and follicle counting

[0091] Twenty-four hours after the last administration, five mice from each group were sacrificed, blood was collected, and ovaries were harvested. The ovaries were weighed and their length and diameter were measured with calipers. The ovaries were then fixed in 4% paraformaldehyde. After paraffin embedding, the tissues were serially sectioned, and the sections were stained with hematoxylin and eosin (HE). Ovarian pathological changes and follicular development were evaluated under a microscope. In addition, one ovarian slide from each group was stained with TUNEL to assess ovarian cell apoptosis.

[0092] HE staining includes the following steps:

[0093] (1) Take out the ovarian tissue that has been soaked in 4% paraformaldehyde solution overnight, peel off the surrounding adipose tissue, and place it in an embedding box;

[0094] (2) The embedded ovarian tissue was placed in an automatic dehydrator overnight for dehydration. The dehydration program was set as follows: ① Slow washing with running water for 12 hours; ② Dehydration in ethanol of varying concentrations: the dehydration time was 30 minutes for each concentration; the ethanol concentrations were 50%, 60%, 70%, 80%, 90%, 95%, 95%, 100%, and 100% respectively; ③ Clearing in xylene: xylene and 100% ethanol solution were mixed at a ratio of 1:1, and the ovarian tissue was immersed in the mixture for 10 minutes, and then immersed in xylene twice, each time for 5 minutes; ④ Paraffin immersion: the tissue was immersed in paraffin twice, each time for 60 minutes.

[0095] (3) After dehydration, the ovarian tissue was removed and completely embedded in paraffin.

[0096] (4) After waiting for the paraffin to solidify and form a wax block, use a microtome to slice the ovarian tissue. The slice thickness is 5μm. Slice continuously, and take one slice every 9 slices for HE staining.

[0097] (5) HE staining was performed using a fully automated staining and mounting workstation. The staining program was set as follows: ① Drying, 20 min; ② Soaking in xylene twice, 10 min each time; ③ Soaking in gradient concentrations of ethanol: 100% ethanol twice, 2 min each time; 95% ethanol twice, 2 min each time; 80% ethanol once, 1 min; 70% ethanol once, 1 min; ④ Washing with water, 2 min; ⑤ Soaking in hematoxylin solution for 8 min, then soaking again for 10 min to stain cell nuclei; ⑥ Water Wash twice to remove excess staining solution, each wash lasting 1 min; ⑦ Differentiate using 0.5% hydrochloric acid alcohol for 10 s; ⑧ Wash with water for 10 s; ⑨ Soak in eosin staining solution for 2 min to stain the cytoplasm; ⑩ Wash with water for 1 min; ⑪ Dehydrate by soaking in a gradient of ethanol concentrations: 80% ethanol for 5 s; 90% ethanol for 5 s; 95% ethanol for 1 min; 100% ethanol for 3 soaks, with durations of 2 min, 2 min, and 3 min respectively; ⑫ Clear: Soak twice in xylene, each soak lasting 2 min;

[0098] (6) After staining, mount the slide with neutral resin.

[0099] TUNEL staining includes the following steps:

[0100] 1. Fixation: After rinsing the ovarian tissue with physiological saline, fix it with 4% paraformaldehyde fixative for 30-50 minutes.

[0101] 2. Washing: Rinse three times with running water for five minutes each time to remove any residual fixative.

[0102] 3. Dehydration: The ovarian tissue is dehydrated in 70%, 80%, and 90% ethanol solutions for 30 minutes each, and then placed in 95% and 100% ethanol solutions twice for 20 minutes each time.

[0103] 4. Clearing and Paraffin Transdermal Treatment: Treat the tissue with a mixture of equal parts pure alcohol and xylene for 15 minutes, followed by xylene I and II for 15 minutes each until clear. Then, treat with a mixture of equal parts xylene and paraffin for 15 minutes, followed by paraffin I and II for 50-60 minutes each for paraffin transdermal treatment. The transdermal treatment is performed in a constant temperature chamber, maintaining the temperature at approximately 55-60℃.

[0104] 5. Embedding: The prepared ovarian tissue is embedded in paraffin.

[0105] 6. Sectioning: Fix the embedded paraffin block on the microtome, adjust it to the required thickness (4-6μm), and cut out tissue sections.

[0106] 7. Slides and mounting: Place the cut tissue slices in heated water to flatten them, then mount them onto glass slides and dry them in a 45°C constant temperature oven.

[0107] 8. Dewaxing and hydration: Paraffin sections are dewaxed with xylene I and II for 5 minutes each, then immersed in 100%, 95%, 90%, 80%, and 70% alcohol solutions for 3-5 minutes each, and then immersed in distilled water for 3 minutes.

[0108] 9. Antigen retrieval: Treat the slides with proteinase K working solution and incubate at 37°C for 20 minutes to increase cell membrane permeability.

[0109] 10. TUNEL reaction: Prepare TdT enzyme reaction solution. For each sample, add 1.0 μl of biotin-11-deoxyuridine triphosphate (biotin-11-dUTP) and 4.0 μl of terminal deoxyribonucleotidyl transferase (TdT Enzyme) to 45 μl of equilibration buffer, and incubate at 37°C in the dark for 60 minutes.

[0110] 11. Washing: Wash 3 times with PBS, 5 minutes each time.

[0111] 12. Labeling: Use TRITC-labeled streptavidin (TRITC) labeling solution and incubate at 37°C in the dark for 30 minutes.

[0112] 13. DAPI staining: Counterstain cell nuclei with DAPI (DNA blue fluorescent dye, Hoechst) staining solution and react at room temperature in the dark for 15 minutes.

[0113] 14. Observation: Observe under a fluorescence microscope, with an excitation wavelength of 543 nm and an emission wavelength of 571 nm.

[0114] 4.3 GV stage oocyte retrieval and in vitro maturation culture (IVM) of immature oocytes (5 oocytes per group)

[0115] Following the last administration of medication, mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG). Forty-eight hours later, the mice were euthanized by cervical dislocation, and both ovaries were dissected. In M2 culture medium (preheated to 37°C for 20 min using 1.5 ml EP tubes), the ovaries were repeatedly punctured with a 1 ml syringe needle or insulin needle until a cotton-like structure was formed. Immature oocytes (cocci) resembling a fried egg structure (granulosa cells tightly surrounding the oocyte without diffusion) were examined at the bottom of the dish. Then, morphologically normal GV-stage oocytes were collected using a pipette under a stereomicroscope. GV-stage oocytes were placed in M16 culture drops (culture medium drops prepared in advance, covered with paraffin oil, and pre-equilibrated in an incubator for 2 h) and cultured in a 37°C, 5% CO2 incubator. Germinal follicle rupture (GVBD) and first polar body expulsion (PB1) were observed and statistically analyzed at 2 h and 14 h of in vitro culture, respectively.

[0116] 4.4 Collection of MII stage oocytes (5 oocytes per group)

[0117] Female mice were injected with pregnant mare serum gonadotropin (MPG) for 48 hours, followed by an injection of 5 IU of human chorionic gonadotropin (hCG). Thirteen hours after hCG injection, MI-stage oocytes were collected, and both fallopian tubes were removed and transferred to M2 medium. Under a stereomicroscope, the ampulla of the fallopian tube was gently punctured with a syringe needle. The release of cumulus-oocyte complexes (COCs) was observed. These COCs were then transferred to M16 medium containing 1% hyaluronidase and incubated for 5-6 minutes (or up to 10 minutes) in an incubator. After the flocculent material loosened and the oocytes were exposed, the oocytes were collected under an inverted microscope using a pipette, and photographs were taken.

[0118] 4.5 Detection of mitochondrial membrane potential in oocytes (JC-1)

[0119] According to the instructions (purchased from Beyotime Biotechnology Co., Ltd., product number C2005), add the JC-1 probe to M16 medium (final concentration 10uM), mix thoroughly, place droplets in a sterile culture dish, and pre-equilibrate for 1 hour in a 37℃, 5% CO2 incubator; transfer the washed MII stage oocytes to droplets containing JC-1 dye, and incubate at 37℃ for 1 hour; transfer the oocytes from the M2 medium droplets containing JC-1 dye to clean M2 medium droplets, and wash 3 times; transfer the washed oocytes to fresh M2 medium droplets, observe under an inverted fluorescence microscope, and take pictures at the same fluorescence intensity for statistical analysis.

[0120] 4.6 Detection of reactive oxygen species (ROS) in oocytes

[0121] Take 1 μL of DCFH-DA probe (S0035S, Beyotime), add it to 1 mL of M2 medium, mix thoroughly by pipetting, and place it in a sterile culture dish. Incubate at 37°C in a 5% CO2 incubator for 1 hour to pre-equilibrate. Transfer the cleaned MII stage oocytes to the droplet containing the ROS probe and incubate at 37°C for 1 hour.

[0122] Oocytes were transferred from ROS-stained M2 culture medium droplets to clean M2 culture medium droplets and washed three times. After washing, the oocytes were transferred to fresh M2 culture medium droplets and observed under an inverted fluorescence microscope. Images were taken at the same fluorescence intensity for statistical analysis.

[0123] 4.7 Detection of DNA Damage by Immunofluorescence Staining of Oocytes

[0124] Reagent preparation: Fixative: 4% paraformaldehyde; Permeabilization solution: prepared with PBS, with the addition of 0.3% Triton X-100.

[0125] Washing buffer: Prepared with PBS, with the addition of 0.1% Tween-20 and 0.01% Triton X-100; Blocking buffer: Prepared with the washing buffer, containing 2% BSA.

[0126] Oocytes were fixed with 4% PFA at room temperature for 30 min, and then washed once with elution buffer (5 min).

[0127] Permeabilization: Permeabilize the membrane with permeabilization solution at room temperature for 20 minutes, then wash once with elution solution (5 minutes each time).

[0128] Sealing: Seal with sealing solution at room temperature for 60 minutes (2% BSA cleaning solution).

[0129] Primary antibody incubation: Oocytes were transferred to primary antibody dilution buffer (p-H2A·X, 9718, 1:200 dilution, CST) and incubated overnight at 4°C in a humidified chamber. The next day, oocytes were washed twice with washing buffer for 5 min each time. Oocytes were then transferred to secondary antibody dilution buffer (goat anti-rabbit IgG H&L (Alexa Fluor® 647, 1:500 dilution, ab150079, abcam) and incubated at 37°C for 1 h in a humidified chamber. After incubation, oocytes were washed twice with washing buffer for 5 min each time.

[0130] Oocytes were transferred to DAPI working solution and stained at room temperature for 5-10 min. Oocytes were then transferred to washing solution and washed twice, 5 min each time.

[0131] Mounting: Select a suitable glass slide and add 5 µL of DABCO anti-quenching solution to the corresponding center position on the slide. Then transfer the oocytes to the center of the DABCO solution, maintaining a suitable distance between the oocytes for easy confocal imaging. Cover with a coverslip and gently press the slide under a dissecting microscope using a pipette tip until the oocytes are 1.3 times their normal size. Mount the slide with colorless nail polish.

[0132] Experimental results:

[0133] 1. Analysis of body weight records for each group showed that older mice experienced weight gain, while the two dosage groups of traditional Chinese medicine did not affect mouse body weight. Figure 1 ).

[0134] 2. By statistically analyzing the ovarian volume and weight of mice ( Figures 2-3 It can be seen that aging leads to ovarian atrophy in mice, manifested in a reduction in volume and weight (p<0.01, p<0.001). Compared with the elderly control group, both low dose and equivalent dose of traditional Chinese medicine can increase ovarian volume and weight, with the equivalent dose of traditional Chinese medicine showing a statistically significant difference (p<0.05).

[0135] 3. Unilateral ovaries were harvested from four groups of mice, stained with hematoxylin and eosin (HE), and observed under a microscope. Pathological changes in the ovarian tissue of each group were compared, and photographs were taken and recorded. Figure 4 As shown in the figure. Through comparison, it can be clearly seen that: different levels of follicle development can be seen in the young control group, while compared with the young control group, the ovarian tissue of the old control group mice shows many ovarian cavities, very few follicles, and occasionally small antral follicles under development; while the number of follicles in the ovaries of the low-dose group and the equivalent dose group of traditional Chinese medicine is also less than that of the young control group, but more follicles under development can be observed than those of the old control group, and mature antral follicles can be seen.

[0136] 5. Mouse Follicle Grading and Counting: The number of developing follicles in the mouse ovary was used as the main efficacy indicator. This invention graded and counted them as follows: primordial follicles, primary follicles, secondary follicles, small antral follicles, large antral follicles, and mature follicles. Follicles in the transitional stage were included in the next stage. The statistical results are shown below. Figures 5-10 As shown: Compared with the young control group, the number of follicles at all stages of the ovary in the elderly control group was significantly reduced (p<0.001); the intervention of traditional Chinese medicine increased the number of follicles at all stages of the ovary in aged mice, with a significant increase in the number of primordial follicles, antral follicles, mature follicles and corpus luteum. When comparing the two traditional Chinese medicine groups, the equivalent dose of traditional Chinese medicine showed a more prominent effect in maintaining follicle development.

[0137] 6. Ovarian Apoptosis Detection: The number of follicles in the ovary is constant. The role of traditional Chinese medicine in maintaining follicle development does not induce the production of new follicles. It is speculated that its effect is mainly achieved by reducing follicular atresia or reducing follicular apoptosis during development. Ovarian apoptosis was detected using TUNEL assay. Figure 11 The results showed that aging caused an increase in ovarian granulosa cell apoptosis, which showed a stronger apoptotic fluorescence signal. Compared with the elderly control group, both dose groups of traditional Chinese medicine could reduce granulosa cell apoptosis in the ovary, thereby maintaining follicle development and increasing the number of follicles.

[0138] 7. To further investigate the protective effect of traditional Chinese medicine on follicular development in aging mice, mice were injected with pregnant mare serum gonadotropin and human chorionic gonadotropin to induce ovulation and retrieve oocytes. The number of oocytes was counted to assess their developmental potential. The results are as follows: Figures 12-16 As shown: Ovulation induction revealed a significant decrease in the number of oocytes in both the GV and MII phases in aged mice (p < 0.001), and an increased rate of oocyte abnormalities. After in vitro culture, their developmental potential was reduced, manifested by a significant decrease in the GVBD rate (GVBD rate = number of oocytes with GVBD observed under a microscope / total number of cultured GV phase oocytes) (p < 0.001). Compared with the aged control group, the number of oocytes retrieved by mice increased after treatment with traditional Chinese medicine, with the equivalent dose group showing better efficacy. Furthermore, the oocyte abnormality rate was significantly reduced in the traditional Chinese medicine group.

[0139] The polar body extrusion rate of oocytes is one of the important indicators for assessing their developmental potential. The polar body extrusion rate reflects the degree of maturation of oocytes during meiosis, and the extrusion of the first polar body (PB1) is usually used as a marker of oocyte nuclear maturation. By performing in vitro maturation culture on GV-stage oocytes obtained from different groups, the number of polar bodies extruded in each group was counted at half-hour intervals during the 10-14 h of culture, and the polar body extrusion rate (polar body extruded oocytes / GVBD oocyte count %) was calculated for each group. Figure 17As shown, the expulsion rate of the first polar body of oocytes in the young control group was 84%. Compared with the young control group, the expulsion rate of the polar body in the elderly control group was significantly lower (p < 0.01). Compared with the elderly control group, the expulsion rate of the polar body in the low dose and the clinically equivalent dose of the composition of the present invention was significantly increased (p < 0.05).

[0140] 8. After ovarian aging, the efficiency of DNA damage repair in oocytes decreases, leading to DNA damage accumulation and a decline in oocyte quality. We then assessed the DNA damage status of oocytes using immunofluorescence γ-H2AX staining: the results are as follows. Figures 18-19 As shown, the fluorescence of γ-H2AX, a key indicator of DNA damage in senescent oocytes, increased (p<0.05), while the DNA damage in oocytes of the traditional Chinese medicine group decreased compared with the elderly control group.

[0141] In addition, with age, the production of intracellular reactive oxygen species (ROS) increases, which can lead to mitochondrial dysfunction and thus affect mitochondrial membrane potential.

[0142] Next, we evaluated the levels and distribution of ROS and JC-1 in oocytes using DCFH-DA and JC-1 probes. The results showed that compared to the young group, older oocytes were more susceptible to oxidative damage, as evidenced by increased ROS fluorescence intensity (p < 0.001). However, compared to the older control group, the ROS fluorescence intensity values ​​of oocytes in both treatment groups were significantly reduced (p < 0.05). Figures 20-21 ).

[0143] The stability of mitochondrial membrane potential is directly related to cellular energy supply and survival. A decrease in mitochondrial membrane potential usually indicates that the cell may be in the early stages of apoptosis. JC-1 is a widely used fluorescent probe for measuring mitochondrial membrane potential. It has unique characteristics: under high membrane potential, JC-1 molecules aggregate in mitochondria, exhibiting red fluorescence; while under low membrane potential, JC-1 exists in monomeric form, emitting green fluorescence. Therefore, by detecting the ratio of red to green fluorescence, researchers can quantitatively analyze changes in intracellular mitochondrial membrane potential, as shown in the results. Figures 22-23 As shown, compared with young oocytes, the proportion of red / green fluorescence in aged oocytes was significantly reduced (p < 0.001), while compared with aged oocytes, oocytes treated with traditional Chinese medicine showed an increased proportion of red / green fluorescence, with a significant difference in the equivalent dose group of traditional Chinese medicine (p < 0.05).

[0144] Example 2

[0145] To investigate the effect of different dosage ratios on efficacy, this section compares the efficacy of three different traditional Chinese medicine compositions. Composition 1: 20 parts Astragalus membranaceus, 5 parts Angelica sinensis, 5 parts Ophiopogon japonicus, 5 parts Polygonatum odoratum, 10 parts Cuscuta chinensis, 5 parts Morinda officinalis, 5 parts Rubus idaeus, 5 parts Dioscorea opposita, and 5 parts Morus alba leaves; Composition 2: 50 parts Astragalus membranaceus, 20 parts Angelica sinensis, 20 parts Ophiopogon japonicus, 20 parts Polygonatum odoratum, 30 parts Cuscuta chinensis, 20 parts Morinda officinalis, 20 parts Rubus idaeus, 20 parts Dioscorea opposita, and 20 parts Morus alba leaves; Composition 3: the equivalent dosage of the traditional Chinese medicine in Example 1, 30g Astragalus membranaceus, 10g Angelica sinensis, 10g Ophiopogon japonicus, 10g Polygonatum odoratum, 20g Cuscuta chinensis, 10g Morinda officinalis, 10g Rubus idaeus, 10g Dioscorea opposita, and 10g Morus alba leaves.

[0146] The animal experiment was designed the same as in Example 1, and was divided into an aged control group, an aged + formulation 1 group, an aged + formulation 2 group, and an aged + formulation 3 group (n=10 per group). Ten 2-month-old female ICR mice were used as normal controls.

[0147] Based on the drug dosage conversion in Example 1, the dosages for mice in formulations 1-3 were 9.8 g / kg, 33.3 g / kg, and 18.2 g / kg, respectively. The gavage volume V for mice was calculated at 10 ml / kg (i.e., 0.01 ml / g); gavage was administered once daily. The traditional Chinese medicine was administered for a total of 30 days, and elderly and young controls were given the same amount of drinking water.

[0148] This section mainly uses ovarian weight, ovarian volume, and the number of oocytes retrieved as the main efficacy indicators for component comparison. The experimental results are shown in the table below: The results show that compared with the young control group, the elderly control group had ovarian atrophy, which was manifested in a significant decrease in ovarian weight and ovarian volume (p<0.01, consistent with the results of Example 1). Compared with the elderly control group, the ovarian weight and ovarian volume of mice in formulations 1-3 were significantly increased (p<0.05).

[0149] Table 1 Comparison of ovarian weight and volume in each group of mice ( )

[0150]

[0151] Note: Compared with the young control group, **p<0.01; compared with the elderly control group, #p<0.05.

[0152] Following the method in Example 1, oocytes in the GV and MII stages of each group were obtained and counted. The results are as follows: Figures 24-25 As shown: Compared with the elderly control group, formulations 1-3 all increased the number of oocytes retrieved in mice during the GV and MII phases (p<0.05).

[0153] The above examples illustrate that the following components of the traditional Chinese medicine composition—20-50 parts Astragalus membranaceus, 5-20 parts Angelica sinensis, 5-20 parts Ophiopogon japonicus, 5-20 parts Polygonatum odoratum, 10-30 parts Cuscuta chinensis, 5-20 parts Morinda officinalis, 5-20 parts Rubus idaeus, 5-20 parts Dioscorea opposita, and 5-20 parts Morus alba—all have the ability to improve, repair, and / or activate residual ovarian function in aging ovaries. The specific therapeutic effects vary depending on the proportions of the composition.

[0154] Comparative Example 1

[0155] The only difference between Comparative Example 1 and Example 1 is the composition and content of the traditional Chinese medicine composition. The traditional Chinese medicine composition in Comparative Example 1 consisted of 50g Astragalus membranaceus, 10g Angelica sinensis, 10g Dioscorea opposita, 10g Rehmannia glutinosa, 10g Epimedium brevicornu, 10g Cuscuta chinensis, and 10g Adenophora stricta. The model construction process and dosage were the same as in Example 1. The dosage in Comparative Example 1 was approximately 110g / 60kg*9.1≈16.7g / kg. The gavage volume V for mice was calculated at 10ml / kg (i.e., 0.01ml / g). The test results are shown below:

[0156] Unilateral ovaries were harvested from mice in each group, stained with hematoxylin and eosin (HE), and observed under a microscope. Pathological changes in the ovarian tissue of each group were compared and counted, and photographs were taken and recorded. Figures 26-27 As shown: The young control group exhibited numerous developing follicles of different grades, while the aged control group showed very few developing follicles. Compared to the young control group, the aged control group showed significantly fewer developing follicles in its ovarian pathology. Further follicle counting in each group revealed that, compared to the aged control group, both Comparative Example 1 and the traditional Chinese medicine group showed an increase in the number of follicles at all grades. The number of primordial, primary, and secondary follicles in Comparative Example 1 was slightly higher than that in the traditional Chinese medicine group (increased by 26.8%, 28.7%, and 26.6%, respectively). In contrast, the number of antral follicles, mature follicles, and corpora lutea in the traditional Chinese medicine group increased by 63.3%, 121.7%, and 80.3%, respectively, compared to the comparative example. In conclusion, the ovarian pathology and follicle counting results demonstrate that the efficacy of the traditional Chinese medicine composition of this invention is significantly higher than that of Comparative Example 1.

[0157] Comparative Example 2

[0158] The only difference between Comparative Example 2 and Example 1 is the composition of the traditional Chinese medicine composition: Astragalus membranaceus 30g, Angelica sinensis 10g, Polygonatum odoratum 10g, Morinda officinalis 10g, Rubus idaeus 10g, Dioscorea opposita 10g, and Morus alba 10g. The model construction process and dosage were the same as in Example 1. The dosage in Comparative Example 2 was 90g / 60kg*9.1≈13.7g / kg, and the gavage volume V for mice was calculated as 10ml / kg (i.e., 0.01ml / g).

[0159] To investigate the protective effects of the formulations in Comparative Example 2 and Example 1 on follicular development in aging mice, mice were injected with pregnant mare serum gonadotropin and human chorionic gonadotropin to induce ovulation and retrieve oocytes. The number of oocytes obtained in each group was counted. The results are as follows: Figures 28-29 As shown: Compared with the young control group, the number of oocytes retrieved in both the GV and MII phases was significantly lower in the elderly control group (p < 0.001). Compared with the elderly control group, the herbal medicine group of this invention significantly increased the number of oocytes retrieved in both the GV and MII phases (p < 0.05, p < 0.01). Compared with the herbal medicine group of this invention, the number of oocytes retrieved in both the GV and MII phases of Comparative Example 2 was significantly lower (p < 0.05). The number of oocytes retrieved during ovulation induction in mice is extremely important for assessing ovarian function and reproductive capacity. The statistical results of the number of oocytes retrieved in the GV and MII phases indicate that the efficacy of the herbal medicine composition of this invention is significantly higher than that of Comparative Example 2.

[0160] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for repairing and / or activating residual ovarian function in aging ovaries, characterized in that, The traditional Chinese medicine composition consists of the following components: 20-50 parts Astragalus membranaceus, 5-20 parts Angelica sinensis, 5-20 parts Ophiopogon japonicus, 5-20 parts Polygonatum odoratum, 10-30 parts Cuscuta chinensis, 5-20 parts Morinda officinalis, 5-20 parts Rubus idaeus, 5-20 parts Dioscorea opposita and 5-20 parts Morus alba.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following components by weight: 20-40 parts Astragalus membranaceus, 5-15 parts Angelica sinensis, 5-15 parts Ophiopogon japonicus, 5-15 parts Polygonatum odoratum, 15-30 parts Cuscuta chinensis, 5-15 parts Morinda officinalis, 5-15 parts Rubus idaeus, 5-15 parts Dioscorea opposita and 5-15 parts Morus alba.

3. The traditional Chinese medicine composition according to claim 2, characterized in that, The traditional Chinese medicine composition consists of the following components by weight: 30 parts Astragalus membranaceus, 10 parts Angelica sinensis, 10 parts Ophiopogon japonicus, 10 parts Polygonatum odoratum, 20 parts Cuscuta chinensis, 10 parts Morinda officinalis, 10 parts Rubus idaeus, 10 parts Dioscorea opposita and 10 parts Morus alba.

4. The method for preparing the traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Take parts by weight of Astragalus membranaceus, Angelica sinensis, Ophiopogon japonicus, Polygonatum odoratum, Cuscuta chinensis, Morinda officinalis, Rubus idaeus, Dioscorea opposita and Morus alba leaves, and extract by reflux with water; (2) Concentrate the extracted medicinal liquid to obtain a traditional Chinese medicine composition.

5. The preparation method according to claim 4, characterized in that, The amount of water added is 8-15 times the total mass of the raw materials of the traditional Chinese medicine composition, the reflux extraction time is 60-150 min, and the reflux extraction is performed 1-3 times.

6. The use of the traditional Chinese medicine composition according to any one of claims 1-3 in the preparation of a drug for delaying ovarian aging.

7. The application according to claim 6, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

8. The application according to claim 6, characterized in that, The dosage form of the drug is a mixture, tablet, powder, oral liquid, capsule, granule, suspension, powder or pill.

9. The use of the traditional Chinese medicine composition according to any one of claims 1-3 in the preparation of a medicament for repairing and / or activating residual ovarian function in aging ovaries.

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