Traditional Chinese medicine composition for repairing and / or activating residual ovarian function of senescence ovary and preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for repairing and / or activating residual ovarian functions of an aging ovary, and a preparation method and application thereof, and relates to the technical field of biology. The traditional Chinese medicine composition is prepared from the following components: radix astragali, radix angelicae sinensis, radix ophiopogonis, radix polygonati officinalis, semen cuscutae, radix morindae officinalis, fructus rubi, rhizoma dioscoreae and folium mori. The invention further provides application of the traditional Chinese medicine composition in repairing and / or activating the residual ovarian function of the senescent ovary and improving the quality of the senescent oocyte, the traditional Chinese medicine composition can increase the volume and weight of the senescent ovary, increase the number of follicles of all levels in the senescent ovary, reduce apoptosis of granular cells in the ovary and improve the survival rate of the senescent ovary. The abnormal rate of oocytes, oxidative stress of the oocytes and DNA damage are reduced, development and maturation of residual follicles are promoted and maintained, and the traditional Chinese medicine composition has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine biotechnology, and particularly relates to a traditional Chinese medicine composition for repairing and / or activating the residual ovarian function of senescent ovaries, a preparation method thereof, and an application thereof. Background Art

[0002] Ovarian senescence is a process of declining ovarian reserve capacity, mainly manifested by a decrease in the number of follicles and the quality of oocytes, until ovarian function fails, resulting in a decline in female fertility, menstrual disorders, and menopause. Ovarian senescence is divided into age-related physiological ovarian senescence and premature decline of ovarian function caused by pathological factors (premature ovarian insufficiency, i.e., premature ovarian failure, etc.). Ovarian senescence is affected by multiple factors such as age, genetic factors, immunity, iatrogenic factors, infection, and environment, and its occurrence, development, and clinical manifestations have individual differences.

[0003] The basic structural and functional unit of the ovary - the follicle is composed of an oocyte and the surrounding granulosa cells. The primary oocyte is surrounded by a single layer of flattened granulosa cells to form primordial follicles, and the established primordial follicle pool at this time is the ovarian reserve. During puberty, under the regulation of the gonadal axis, some primordial follicles are activated and enter the growth stage, and sequentially develop into primary follicles, secondary follicles, preantral follicles, antral follicles to ovulation. Most follicles in the ovary cannot develop to maturity, and they stop growing and atresia at different stages of follicle development, leading to ovarian function decline. When no follicle development occurs in the ovary, a woman enters menopause.

[0004] The decline of ovarian function is a gradual process. For the treatment strategy of ovarian senescence, two links need to be emphasized: 1. In the middle and late stages of ovarian decline, increased follicle atresia will occur due to elevated gonadotropins or a poor ovarian microenvironment. Repairing the function of senescent ovaries can reduce the atresia rate of follicles. 2. At the end stage of ovarian decline, there are still a certain number of residual follicles in the atrophic ovary, which retain a certain potential for development and even fertilization. However, over time, due to the lack of developmental potential, the residual follicles will be permanently exhausted. Therefore, finding effective methods to repair ovarian function, stimulate the development of residual follicles, and delay the decline of ovarian function is an urgent clinical challenge in the current treatment of ovarian senescence. In recent years, innovative technologies such as in vitro activation of follicles (IVA), stem cell transplantation, and platelet-rich plasma (PRP) injection have been used to activate residual follicles, but the research conclusions mostly come from basic research and a limited number of small-sample clinical observational studies, and there is still a lack of reliable safety and effectiveness data.

[0005] In the field of anti-aging research, traditional Chinese medicine occupies an important position. However, at present, the treatment of ovarian aging with traditional Chinese medicine mainly focuses on improving the low estrogen-related symptoms caused by ovarian aging, or using it in combination with hormone replacement therapy to improve sex hormone levels. There is a lack of research on drugs that protect / activate the remaining ovarian function. The monarch drugs of the traditional Chinese medicine composition of the present invention are Astragalus membranaceus and Angelica sinensis, the ministerial drugs are: Ophiopogon japonicus and Cuscuta chinensis, the adjuvant drugs are: Rubus idaeus, Dioscorea opposita, Morinda officinalis, and Polygonatum odoratum, and the guiding drug is: Mori Folium. It has the effects of replenishing qi and nourishing blood, promoting the production of body fluids and filling essence. The traditional Chinese medicine composition that can effectively delay ovarian aging is of great significance to clinical practice. Summary of the Invention

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

[0007] To achieve the above-mentioned invention purpose, the technical solution of the present invention is as follows: On the one hand, the present invention provides a traditional Chinese medicine composition for repairing and / or activating the remaining ovarian function of aging ovaries. The traditional Chinese medicine composition is composed of the following components: Astragalus membranaceus, Angelica sinensis, Ophiopogon japonicus, Polygonatum odoratum, Cuscuta chinensis, Morinda officinalis, Rubus idaeus, Dioscorea opposita and Mori Folium.

[0008] Further, the traditional Chinese medicine composition is composed of the following components according to weight parts: 20-50 parts of Astragalus membranaceus, 5-20 parts of Angelica sinensis, 5-20 parts of Ophiopogon japonicus, 5-20 parts of Polygonatum odoratum, 10-30 parts of Cuscuta chinensis, 5-20 parts of Morinda officinalis, 5-20 parts of Rubus idaeus, 5-20 parts of Dioscorea opposita and 5-20 parts of Mori Folium.

[0009] Still further, the traditional Chinese medicine composition is composed of the following components according to weight parts: 20-40 parts of Astragalus membranaceus, 5-15 parts of Angelica sinensis, 5-15 parts of Ophiopogon japonicus, 5-15 parts of Polygonatum odoratum, 15-30 parts of Cuscuta chinensis, 5-15 parts of Morinda officinalis, 5-15 parts of Rubus idaeus, 5-15 parts of Dioscorea opposita and 5-15 parts of Mori Folium.

[0010] Even further, the traditional Chinese medicine composition is composed of the following components according to weight parts: 30 parts of Astragalus membranaceus, 10 parts of Angelica sinensis, 10 parts of Ophiopogon japonicus, 10 parts of Polygonatum odoratum, 20 parts of Cuscuta chinensis, 10 parts of Morinda officinalis, 10 parts of Rubus idaeus, 10 parts of Dioscorea opposita and 10 parts of Mori Folium.

[0011] On the other hand, the present invention provides a preparation method of the above-mentioned traditional Chinese medicine composition, including the following steps: (1) Take Astragalus membranaceus, Angelica sinensis, Ophiopogon japonicus, Polygonatum odoratum, Cuscuta chinensis, Morinda officinalis, Rubus idaeus, Dioscorea opposita and Mori Folium in the weight parts, and carry out reflux extraction with water; (2) Concentrate the extracted medicinal liquid to obtain the traditional Chinese medicine composition.

[0012] Specifically, the amount of water added is 8-15 times the total mass of the raw materials of the traditional Chinese medicine composition; Further, the amount of water added is 12 times the total mass of the raw materials of the traditional Chinese medicine composition.

[0013] Specifically, after adding water, it further includes the step of soaking the medicinal materials.

[0014] Further, the soaking time is 20 - 60 min; still further, the soaking time is 30 min.

[0015] Specifically, the temperature for reflux extraction is 70 - 90 °C.

[0016] Specifically, the reflux extraction time is 60 - 150 min; further, the reflux extraction time is 90 min.

[0017] Specifically, the number of times of reflux extraction is 1 - 3 times, and further, the number of times of reflux extraction is 2 times.

[0018] Specifically, the concentration is to concentrate the medicinal liquid to a final concentration of 1 - 2 g / ml; Further, the concentration is to concentrate the medicinal liquid to a final concentration of 1.2 g / ml.

[0019] On the other hand, the present invention provides the application of the above traditional Chinese medicine composition in the preparation of a drug for delaying ovarian aging.

[0020] Specifically, the ovarian aging includes physiological ovarian aging and pathological ovarian aging.

[0021] Further, the ovarian aging is physiological ovarian aging.

[0022] Further, the pathological ovarian aging includes ovarian aging caused by ovarian tumors, ovarian aging caused by drugs, and ovarian aging caused by iatrogenic factors.

[0023] Specifically, the drug may further include a pharmaceutically acceptable carrier.

[0024] Further, the pharmaceutically acceptable carrier is selected from one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents, and fillers.

[0025] 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, alginic acid, gelatin, gum arabic, glyceryl monostearate, sodium starch glycolate, guar gum, glycerol, and propylene glycol.

[0026] Specifically, the buffering agent is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0027] 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 polyglyceryl-3 polyricinoleate.

[0028] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitin ester.

[0029] 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.

[0030] Specifically, the binder 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.

[0031] Specifically, the preservative is selected from at least one of methyl paraben, propyl paraben, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, parachlorometacresol, benzalkonium bromide, benzalkonium chloride, and ethyl paraben.

[0032] 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 stearyl fumarate, and poloxamer.

[0033] Specifically, the pH regulator is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0034] Specifically, the cryoprotectant is selected from at least one of sucrose, glucose, mannitol, fructose, trehalose, dextran, lactose, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, or formamide.

[0035] Specifically, the flavoring agent is selected from at least one of sweet orange flavor, vanilla flavor, strawberry flavor, milk flavor, banana flavor, and cherry flavor.

[0036] 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.

[0037] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, dripping pill, pill, buccal tablet, freeze-dried powder injection, gel, suppository or aerosol.

[0038] On the other hand, the present invention provides the use of the above-mentioned traditional Chinese medicine composition in the preparation of a drug for repairing and / or activating the residual ovarian function of senescent ovaries.

[0039] Specifically, the drug may further include a pharmaceutically acceptable carrier.

[0040] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH regulators, cryoprotectants, flavoring agents, and fillers.

[0041] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, dripping pill, pill, buccal tablet, freeze-dried powder injection, gel, suppository or aerosol.

[0042] The beneficial effects of the present invention are as follows: The traditional Chinese medicine composition provided by the present invention can increase the volume and weight of the ovaries, increase the follicles at all levels in the ovaries, specifically significantly increase the number of primordial follicles, antral follicles, mature follicles and corpora lutea, reduce the apoptosis of granulosa cells in the ovaries, can better maintain follicle development, increase the number of follicles, reduce the abnormal rate of eggs and DNA damage of oocytes. The traditional Chinese medicine composition of the present invention plays an important role in repairing and / or activating the residual ovarian function of senescent ovaries. Description of the Drawings

[0043] Figure 1 is the average body weight of each group of mice.

[0044] Figure 2 is the average ovarian volume of each group of mice. Compared with the young control group, **p < 0.01; compared with the old control group, #p < 0.05.

[0045] Figure 3 is the average ovarian weight of each group of mice. Compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05.

[0046] Figure 4 is the pathological section of ovarian tissue of each group of mice.

[0047] Figure 5 The number of primordial follicles in each group of mice. Compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05.

[0048] Figure 6 The number of primary follicles in each group of mice. Compared with the young control group, ***p < 0.001.

[0049] Figure 7 The number of secondary follicles in each group of mice. Compared with the young control group, ***p < 0.001.

[0050] Figure 8 The number of antral follicles in each group of mice. Compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05, ##p < 0.01.

[0051] Figure 9 The number of corpora lutea in each group of mice. Compared with the young control group, ***p < 0.001; compared with the old control group, ##p < 0.01.

[0052] Figure 10 The average number of mature follicles in each group of mice. Compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05, ##p < 0.01.

[0053] Figure 11 The results of ovarian apoptosis detection in each group of mice. In the figure, the red arrows represent apoptotic granulosa cells.

[0054] Figure 12 Representative pictures of the ovaries, oviducts and the number of retrieved oocytes in each group of mice. The red arrows indicate the presence of MII oocytes at the dilated ampulla.

[0055] Figure 13 The number of GV-stage oocytes in each group of mice. Compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05.

[0056] Figure 14 The number of MII-stage oocytes in each group of mice. Compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05.

[0057] Figure 15 The oocyte fragmentation rate in each group of mice. Compared with the young control group, ***p < 0.001; compared with the old control group, ##p < 0.01, p < 0.001.

[0058] Figure 16The germinal vesicle breakdown rate of mouse oocytes in each group. Compared with the young control group, ***p < 0.001.

[0059] Figure 17 The polar body extrusion rate of mouse oocytes in each group. Compared with the young control group, **p < 0.01, and compared with the old control group, #p < 0.05.

[0060] Figure 18 The immunofluorescence γ-H2AX staining results of mouse oocytes in each group.

[0061] Figure 19 The statistical results of γ-H2AX.

[0062] Figure 20 The representative graphs of ROS in oocytes of each group.

[0063] Figure 21 The statistical graphs of ROS in oocytes of each group. Compared with the young control group, ***p < 0.001, and compared with the old control group, #p < 0.05.

[0064] Figure 22 The detection graphs of mitochondrial membrane potential JC-1 in oocytes of each group.

[0065] Figure 23 The statistical graphs of the fluorescence values of mitochondrial membrane potential JC-1 in oocytes of each group. Compared with the young control group, ***p < 0.001, and compared with the old control group, #p < 0.05.

[0066] Figure 24 The number of GV-stage oocytes of mice in each group of Example 2. Compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05.

[0067] Figure 25 The number of MII-stage oocytes of mice in each group of Example 2. Compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05.

[0068] Figure 26 The pathological graphs of ovarian tissues of mice in each group in the experiment of Comparative Example 1.

[0069] Figure 27 The statistical count of follicles at all levels of mice in each group in the experiment of Comparative Example 1. Compared with the young control group, **p < 0.01, ***p < 0.001; compared with the old control group, #p < 0.05.

[0070] Figure 28For the number of GV-stage oocytes in each group of mice in the experimental control 2, compared with the young control group, ***p < 0.001; compared with the old control group, #p < 0.05, compared with the low-dose traditional Chinese medicine group, #p < 0.05.

[0071] Figure 29 For the number of MII-stage oocytes in each group of mice in the experimental control 2, compared with the young control group, ***p < 0.001; compared with the old control group, ##p < 0.01, compared with the low-dose traditional Chinese medicine group, #p < 0.05. Specific implementation mode

[0072] In order to make the technical means, creative features, achieved purposes and functions 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 mode, other embodiments obtained by those skilled in the art without creative labor all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operation methods used are all conventional operation methods, the equipment used is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0073] Example 1 1. Experimental animals The animals used in this study were SPF-grade ICR female mice provided by the Guangdong Provincial Center for Medical Laboratory Animals, 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 Laboratory 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. After the animals were quarantined and adaptively fed for one week, the experiment began. 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.

[0074] Astragalus membranaceus 30g, Angelica sinensis 10g, Polygonatum odoratum 10g, Morinda officinalis 10g, Rubus idaeus 10g, Dioscorea opposita 10g, Morus alba 10g 2. Standardized preparation of the compound The prescription composition is 30g of Astragalus membranaceus, 10g of Angelica sinensis, 10g of Ophiopogon japonicus, 10g of Polygonatum odoratum, 20g of Cuscuta chinensis, 10g of Morinda officinalis, 10g of Rubus idaeus, 10g of Dioscorea opposita, and 10g of Morus alba L. According to the above ratio, it is put into a 5L round-bottom flask, and the volume of water added is 12 times the volume of the medicinal materials; the medicinal materials are soaked for 30 minutes, a condensing reflux device is installed, and it is heated with an ordinary temperature-controlled electric heating mantle. After the water boils, continue to heat for 90 minutes, collect the medicinal liquid, repeat the decocting process once, collect the medicinal liquid obtained from the second decoction, combine the two medicinal liquids, and use a rotary evaporator to concentrate the medicinal liquid to a final concentration of 0.91g / ml.

[0075] 3. Grouping and administration of mice Forty-five 12-month-old female ICR mice were randomly divided into an old control group, an old + low-dose traditional Chinese medicine group, and an old + equivalent-dose traditional Chinese medicine group (15 mice in each group). Another 15 2-month-old female ICR mice were used as normal controls.

[0076] Calculation of the final concentration of traditional Chinese medicine: Converted according to the daily clinical dosage of 120g for an adult female weighing 60kg: The conversion coefficient between mice and humans is 9.1. Therefore, the equivalent drug mass "m" for mice should be: 160g / 60kg * 9.1 ≈ 18.2g / kg, and the low dose is 9.1g / kg; the gavage volume V for mice is calculated according to 10ml / kg (i.e., 0.01ml / g); the low dose is gavaged once a day, and the equivalent dose of traditional Chinese medicine is gavaged twice a day, once in the morning and once in the afternoon. The traditional Chinese medicine administration lasts for 30 days, and the old control group and the young control group are given an equal amount of drinking water.

[0077] 4. Detection indexes and methods 4.1 Body weight record: The body weight change of mice is detected once a week during the administration period.

[0078] 4.2 Ovarian pathological detection and follicle counting Twenty-four hours after the last administration, 5 mice were taken from each group, sacrificed, blood was taken, ovaries were taken, weighed and the length and width were measured with a vernier caliper. Then the ovaries were fixed in 4% paraformaldehyde for fixation. After the tissues were embedded in paraffin, serial sections were made. The sections were stained with HE and the ovarian pathological changes and follicle development were evaluated under a microscope; in addition, 1 white section was taken from the ovaries of each group for ovarian TUNEL staining to evaluate the apoptosis of ovarian cells.

[0079] The HE staining includes the following steps: (1) Take out the ovarian tissue that has been soaked in 4% paraformaldehyde solution overnight, strip the surrounding adipose tissue, and place it in an embedding cassette; (2) The embedded ovarian tissue was placed in an automatic dehydrator and dehydrated overnight. The dehydration program was set as follows: ① Slowly wash with running water for 12 h; ② Dehydrate in ethanol with gradient concentrations: The dehydration time was 30 min each; The ethanol concentrations were 50%, 60%, 70%, 80%, 90%, 95%, 95%, 100%, and 100% in sequence; ③ Clear in xylene: Mix xylene and 100% ethanol solution at a ratio of 1:1, immerse the ovarian tissue in it for 10 min, and then immerse it in xylene twice, with each immersion duration of 5 min; ④ Impregnate with wax: Immerse in paraffin twice, with each duration of 60 min; (3) After the dehydration was completed, the ovarian tissue was taken out and completely embedded with paraffin; (4) After waiting for the paraffin to solidify into a wax block, the ovarian tissue was sectioned using a microtome, with the section thickness of 5 μm, sectioned continuously, and every 9th section was taken for HE staining; (5) HE staining was performed using a fully automatic staining and coverslipping integrated workstation, and the staining program was set as follows: ① Dry for 20 min; ② Immerse in xylene twice, with each time for 10 min; ③ Immerse in ethanol with gradient concentrations: 100% ethanol twice, with each time for 2 min; 95% ethanol twice, with each time for 2 min; 80% ethanol once, with a duration of 1 min; 70% ethanol once, with a duration of 1 min; ④ Wash with water for 2 min; ⑤ Immerse in hematoxylin stain for 8 min and then immerse again for 10 min to stain the cell nuclei; ⑥ Wash with water twice to remove the excess stain, with each water wash duration of 1 min; ⑦ Differentiate with 0.5% hydrochloric acid alcohol for 10 s; ⑧ Wash with water for 10 s; ⑨ Immerse in eosin stain for 2 min to stain the cytoplasm; ⑩ Wash with water for 1 min; ⑪ Immerse in ethanol with gradient concentrations for dehydration: 80% ethanol for 5 s; 90% ethanol for 5 s; 95% ethanol for 1 min; Immerse in 100% ethanol three times, with the durations of 2 min, 2 min, and 3 min in sequence; ⑫ Clear: Immerse in xylene twice, with each duration of 2 min; (6) After the staining was completed, the sections were coverslipped with neutral balsam.

[0080] TUNEL staining includes the following steps: 1. Fixation: After rinsing the ovarian tissue with normal saline, it was fixed with 4% paraformaldehyde fixative for 30 - 50 minutes.

[0081] 2. Washing: Rinse with running water 3 times, 5 minutes each time, to remove the residual fixative.

[0082] 3. Dehydration: The ovarian tissue was dehydrated successively through 70%, 80%, 90% ethanol solutions for 30 minutes each, and then placed in 95%, 100% ethanol solutions twice, 20 minutes each time.

[0083] 4. Clearing and paraffin infiltration: Treat the tissue with an equal mixture of absolute alcohol and xylene for 15 minutes, and then use xylene I and xylene II for 15 minutes each until the tissue becomes clear. Then place it in an equal mixture of xylene and paraffin for 15 minutes, and then use paraffin I and paraffin II for paraffin infiltration for 50 - 60 minutes each. Paraffin infiltration is carried out in an incubator with the temperature maintained at about 55 - 60 °C.

[0084] 5. Embedding: Embed the treated ovarian tissue in paraffin.

[0085] 6. Sectioning: Fix the embedded paraffin block on a microtome, adjust it to the required thickness (4 - 6 μm), and cut thin tissue slices.

[0086] 7. Spreading and mounting: Place the cut thin tissue slices in heated water to flatten them, then mount them on glass slides and dry them in an incubator at 45 °C.

[0087] 8. Deparaffinization and hydration: Deparaffinize the paraffin sections with xylene I and xylene II for 5 minutes each, then place them in 100%, 95%, 90%, 80%, and 70% alcohol solutions for 3 - 5 minutes each, and then place them in distilled water for 3 minutes.

[0088] 9. Antigen retrieval: Treat the sections with proteinase K working solution and incubate at 37 °C for 20 minutes to increase cell membrane permeability.

[0089] 10. TUNEL reaction: Prepare the TdT enzyme reaction solution. The dosage for each sample is to add 1.0 μl of biotin - 11 - dUTP and 4.0 μl of terminal deoxynucleotidyl transferase (TdT Enzyme) to 45 μl of equilibration buffer, and incubate at 37 °C in the dark for 60 minutes.

[0090] 11. Washing: Wash with PBS 3 times, 5 minutes each time.

[0091] 12. Labeling: Use streptavidin - TRITC labeling solution and incubate at 37 °C in the dark for 30 minutes.

[0092] 13. DAPI staining: Counterstain the cell nuclei with DAPI (DNA blue fluorescent dye, Hoechst) staining solution and react at room temperature in the dark for 15 minutes.

[0093] 14. Observation: Observe under a fluorescence microscope with an excitation wavelength of 543 nm and an emission wavelength of 571 nm.

[0094] 4.3 Obtaining GV - stage oocytes and in vitro maturation culture (IVM) of immature oocytes (5 per group) After the last administration to the mice, 10 IU of pregnant mare serum gonadotropin (PMSG) was injected intraperitoneally. 48 hours later, the mice were sacrificed by cervical dislocation, and the bilateral ovaries were dissected. In M2 culture medium (the M2 culture medium was pre-warmed in a 1.5 ml EP tube at 37 °C for 20 min), the ovaries were repeatedly punctured with a 1 ml syringe needle or an insulin needle until they became cotton-like. Immature eggs coc (granulosa cells did not spread and tightly wrapped around the oocyte) similar to the structure of fried eggs were searched for at the bottom of the dish. Then, under a stereomicroscope, morphologically normal GV-stage oocytes were picked up with a mouth pipette. Among them, the GV-stage oocytes were placed in M16 culture drops (the culture drops were prepared in advance, covered with paraffin oil, and pre-equilibrated in an incubator for 2 h), and placed in an incubator at 37 °C, 5% CO 2 and cultured. At 2 h and 14 h of in vitro culture, the germinal vesicle breakdown (GVBD) and the extrusion of the first polar body (PB1) of oocytes in each group were observed and counted respectively.

[0095] 4.4 Collection of MII-stage oocytes (5 per group) 48 h after injecting pregnant mare serum gonadotropin into female mice, 5 IU of human chorionic gonadotropin (hCG) was injected. 13 hours after injecting human chorionic gonadotropin, MII-stage oocytes were collected. The bilateral oviducts were removed and transferred to M2 medium. Under a stereomicroscope, the ampulla of the oviduct was gently scratched with a syringe needle. When the cumulus-oocyte complexes (COCs) were observed to be released, they were transferred to M16 medium containing 1% hyaluronidase with a pipette and left standing in an incubator for 5 - 6 minutes (or up to 10 minutes). After the floccules became loose and the oocytes were exposed, the oocytes were collected with a pipette under an inverted microscope and photographed for record.

[0096] 4.5 Detection of mitochondrial membrane potential of oocytes (JC-1) According to the instruction manual (purchased from Beyotime Biotechnology Co., Ltd., product number C2005), the JC-1 probe was added to M16 culture medium (final concentration 10 uM). After thorough mixing, drops were made in a sterile culture dish and placed in an incubator at 37 °C, 5% CO 2 for pre-equilibration for 1 hour; the washed MII-stage oocytes were transferred to the droplet containing JC-1 dye and placed in an incubator at 37 °C for incubation for 1 hour; the oocytes were transferred from the M2 culture medium droplet containing JC-1 dye to a clean M2 culture medium droplet and washed 3 times; the washed oocytes were transferred to a new M2 culture medium droplet and observed under an inverted fluorescence microscope, and photographed and counted at the same fluorescence intensity.

[0097] 4.6 Detection of reactive oxygen species in oocytes (ROS) Take 1 μL of DCFH-DA probe (S0035S, Beyotime), add it to 1 mL of M2 medium, mix well by pipetting, then make drops in a sterile Petri dish and place it in an incubator at 37 °C with 5% CO 2 Pre-equilibrate for 1 hour; transfer the washed metaphase II oocytes into the droplet containing the ROS probe, place it in the incubator and incubate at 37 °C for 1 hour.

[0098] Transfer the oocytes from the M2 medium droplet containing the ROS stain to a clean M2 medium droplet and wash 3 times; transfer the washed oocytes to a new M2 medium droplet, place it under an inverted fluorescence microscope for observation, and take pictures at the same fluorescence intensity for statistics.

[0099] 4.7 Detection of DNA damage in oocytes by immunofluorescence staining Reagent preparation: Fixative: 4% paraformaldehyde; Permeabilization solution: Prepared with PBS, add 0.3% Triton X-100.

[0100] Washing solution: Prepared with PBS, add 0.1% Tween-20 and 0.01% Triton X-100; Blocking solution: Prepared with the washing solution, a washing solution containing 2% BSA.

[0101] Fix the oocytes with 4% PFA at room temperature for 30 min, and wash once with the eluent after fixation (5 min).

[0102] Permeabilize: Permeabilize with the permeabilization solution at room temperature for 20 min, then wash once with the eluent (5 min each time).

[0103] Block: Block with the blocking solution at room temperature for 60 min (washing solution containing 2% BSA).

[0104] Incubate with primary antibody: Transfer the oocytes to the primary antibody dilution solution (p-H2A·X, 9718, diluted 1:200, CST), and incubate overnight at 4 °C in a humidified chamber. The next day, transfer the oocytes to the washing solution and wash 2 times, 5 min each time. Transfer the oocytes to the secondary antibody dilution solution (Goat anti-rabbit IgG H&L (Alexa Fluor® 647, diluted 1:500, ab150079, abcam), and incubate at 37 °C for 1 h in a humidified chamber. After incubation, transfer the oocytes to the washing solution and wash 2 times, 5 min each time.

[0105] Transfer the oocytes to the DAPI working solution and stain at room temperature for 5 - 10 min. Transfer the oocytes to the washing solution and wash 2 times, 5 min each time.

[0106] Mounting: Select an appropriate glass slide, add 5 μL of DABCO anti-quenching liquid at the corresponding central position of the glass slide. Then transfer the oocytes to the center of the DABCO liquid, keeping an appropriate distance between the oocytes for convenient confocal imaging. Then cover with a coverslip and gently press the oocytes with a pipette tip under a dissecting microscope until the oocytes reach 1.3 times their normal size, and seal the slide with colorless nail polish.

[0107] Experimental results: 1. According to the analysis of the body weight records of each group, the body weight of the elderly mice increased, and the two Chinese medicine dosage groups did not affect the body weight of the mice ( Figure 1 ).

[0108] 2. By statistically analyzing the ovarian volume and ovarian weight of the mice ( Figures 2 - 3 ), it can be seen that aging led to ovarian atrophy in mice, manifested as a decrease in volume and weight (p < 0.01, p < 0.001). Compared with the elderly control group, both the low-dose Chinese medicine group and the equivalent-dose Chinese medicine group could increase the ovarian volume and weight, and the equivalent-dose Chinese medicine group showed a statistical difference (p < 0.05).

[0109] 3. Take the unilateral ovaries of the 4 groups of mice, perform HE staining and observe under the microscope, compare the pathological changes of the ovarian tissues of each group of mice, and take pictures and record as Figure 4 shown. By comparison, it can be intuitively found that different levels of developing follicles can be seen in the young control group. Compared with the young control group, many ovarian cavities can be seen in the ovarian tissues of the elderly control group, the number of developing follicles is extremely small, and occasionally small antral follicles in development can be seen; the number of follicles in the ovaries of the low-dose Chinese medicine group and the equivalent-dose group is also less than that of the young control group, but more developing follicles can be observed than in the elderly control group, and mature antral follicles can be seen.

[0110] 5. Grading and counting of mouse follicles: The number of developing follicles in the mouse ovaries was used as the main efficacy index, and the present invention performed grading and counting on it, which were respectively: primordial follicles, primary follicles, secondary follicles, small antral follicles, large antral follicles, mature follicles. When follicles in the transitional stage were found, they were all counted in the latter stage. The statistical results are as Figures 5 - 10 shown: Compared with the young control group, the number of follicles at all levels in the ovaries of the elderly control group decreased significantly (p < 0.001); Chinese medicine intervention increased the follicles at all levels in the ovaries of aging mice, among which the number of primordial follicles, antral follicles, mature follicles and corpora lutea increased significantly. Comparing the two Chinese medicine groups, the equivalent-dose Chinese medicine showed a more prominent role in maintaining follicle development.

[0111] 6. Detection of ovarian apoptosis: The number of follicles in the ovary is constant. The effect of traditional Chinese medicine in maintaining follicular development cannot cause the ovary to produce new follicles. It is speculated that its effect is mainly achieved by reducing follicular atresia or apoptosis during follicular development. The apoptosis situation of the ovary was detected by TUNEL: Figure 11 The results showed that aging caused an increase in apoptosis of ovarian granulosa cells, showing stronger apoptotic fluorescence signals. Compared with the old control group, both doses of traditional Chinese medicine could reduce the apoptosis of granulosa cells in the ovary, thereby maintaining follicular development and increasing the number of follicles.

[0112] 7. To further explore the effect of traditional Chinese medicine in maintaining follicular development in aging mice, pregnant mare serum gonadotropin and human chorionic gonadotropin were injected into the mice for superovulation and egg collection, and the number of oocytes was counted to evaluate their developmental potential. The results are as Figures 12 - 16 shown: Through superovulation, it was found that the number of GV-stage and MII-stage eggs in aging mice decreased significantly (p < 0.001), and the abnormality rate of eggs increased. After in vitro culture, their developmental potential decreased, manifested as a significant decrease in the GVBD rate (GVBD rate = the number of oocytes with GVBD monitored under the microscope / the number of all cultured GV-stage oocytes) (p < 0.001). Compared with the old control group, the number of eggs obtained by mice after the action of traditional Chinese medicine increased, and the curative effect of the equivalent-dose group of traditional Chinese medicine was better. In addition, the abnormality rate of eggs in the traditional Chinese medicine group also decreased significantly.

[0113] The polar body extrusion rate of oocytes is one of the important indicators for evaluating their developmental potential. The polar body extrusion rate reflects the maturity of oocyte meiosis. Usually, the extrusion of the first polar body (PB1) is used as a marker of oocyte nuclear maturity. By performing in vitro maturation culture on GV-stage oocytes obtained from different groups, during the 10 - 14 h of culture, the number of polar bodies extruded in each group was counted every half hour, and the polar body extrusion rate of each group was counted (the percentage of oocytes with extruded polar bodies / the number of GVBD oocytes), as Figure 17 shown. The first polar body extrusion rate of oocytes in the young control group was 84%. Compared with the young control group, the polar body extrusion rate in the old control group decreased significantly (p < 0.01). Compared with the old control group, the polar body extrusion rates of the low dose and clinical equivalent dose of the composition of the present invention increased significantly (p < 0.05).

[0114] 8. After ovarian aging, the efficiency of DNA damage repair in oocytes decreases, resulting in the accumulation of DNA damage and the decline of oocyte quality. Then, we evaluated the DNA damage situation of oocytes by immunofluorescence γ-H2AX staining of oocytes: The results are as Figures 18 - 19 shown. The fluorescence of γ-H2AX, a key indicator of DNA damage in aging oocytes, increased (p < 0.05). Compared with the old control group, the DNA damage of oocytes in the traditional Chinese medicine group decreased.

[0115] In addition, with the increase of age, the production of reactive oxygen species (ROS) in cells increases, and these ROS can lead to mitochondrial dysfunction, which in turn affects the mitochondrial membrane potential.

[0116] Then, we evaluated the levels and distributions of ROS and JC-1 in oocytes using DCFH-DA and JC-1 probes. The results showed that compared with the young group, the ROS fluorescence intensity in old oocytes was increased, indicating that old oocytes were more vulnerable to oxidative damage (p < 0.001). Compared with the old control group, the ROS fluorescence intensity values in the two dose groups of traditional Chinese medicine-treated oocytes were significantly decreased (p < 0.05, Figures 20 - 21 )

[0117] The stability of the mitochondrial membrane potential is directly related to the energy supply and survival status of cells. When the mitochondrial membrane potential decreases, it usually indicates that the cells may be in the early apoptosis stage. JC-1 is a fluorescent probe widely used to measure the mitochondrial membrane potential. It has unique properties: in the state of high membrane potential, JC-1 molecules aggregate in mitochondria and show red fluorescence; while in the state of low membrane potential, JC-1 exists in monomer form and emits green fluorescence. Therefore, by detecting the ratio of red / green fluorescence, researchers can quantitatively analyze the changes in the mitochondrial membrane potential in cells. The results are as Figures 22 - 23 shown. Compared with young oocytes, the ratio of red / green fluorescence in old oocytes was significantly decreased (p < 0.001). Compared with old oocytes, the ratio of red / green fluorescence in traditional Chinese medicine-intervened oocytes increased, and the difference in the equivalent dose group of traditional Chinese medicine was significant (p < 0.05).

[0118] Example 2 To investigate the effect of different dose ratios on the efficacy, this part of the example compared the curative effects of three traditional Chinese medicine compositions with different ratios. Prescription 1: 20 parts of Astragalus membranaceus, 5 parts of Angelica sinensis, 5 parts of Ophiopogon japonicus, 5 parts of Polygonatum odoratum, 10 parts of Cuscuta chinensis, 5 parts of Morinda officinalis, 5 parts of Rubus idaeus, 5 parts of Dioscorea opposita, and 5 parts of Morus alba; Prescription 2: 50 parts of Astragalus membranaceus, 20 parts of Angelica sinensis, 20 parts of Ophiopogon japonicus, 20 parts of Polygonatum odoratum, 30 parts of Cuscuta chinensis, 20 parts of Morinda officinalis, 20 parts of Rubus idaeus, 20 parts of Dioscorea opposita, and 20 parts of Morus alba; Prescription 3: the equivalent dose of traditional Chinese medicine in Example 1, 30 g of Astragalus membranaceus, 10 g of Angelica sinensis, 10 g of Ophiopogon japonicus, 10 g of Polygonatum odoratum, 20 g of Cuscuta chinensis, 10 g of Morinda officinalis, 10 g of Rubus idaeus, 10 g of Dioscorea opposita, and 10 g of Morus alba.

[0119] The animal experiment design was the same as that in Example 1, and it was divided into an old control group, an old + Prescription 1 group, an old + Prescription 2 group, and an old + Prescription 3 group (10 in each group). Another 10 2-month-old female ICR mice were used as normal controls.

[0120] According to the drug dose conversion in Example 1, the dosages for mice in Formulas 1-3 are 9.8 g / kg, 33.3 g / kg, and 18.2 g / kg respectively. The gavage volume V for mice is calculated as 10 ml / kg (i.e., 0.01 ml / g); gavage is performed once a day. The traditional Chinese medicine administration lasts for 30 days, and the elderly control group and the young control group are given an equal amount of drinking water.

[0121] In this part, the main efficacy indicators for component comparison are the ovarian weight, ovarian volume, and the number of oocytes obtained. The experimental results are statistically shown in the following table: The results show that compared with the young control group, the ovarian atrophy in the elderly control group is 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 Formulas 1-3 are significantly increased compared with the elderly control group (p < 0.05).

[0122] Table 1 Comparison of ovarian weight and volume of mice in each group ( )

[0123] Note: Compared with the young control group, **p < 0.01; compared with the elderly control group, #p < 0.05.

[0124] According to the method of Example 1, the oocytes in the GV stage and MII stage of each group were obtained and counted respectively. The results are as Figures 24 - 25 shown: Compared with the elderly control group, Formulas 1-3 all increased the number of oocytes obtained by mice in the GV stage and MII stage (p < 0.05).

[0125] The above examples show that within the following component ranges of the traditional Chinese medicine composition: 20-50 parts of Astragalus membranaceus, 5-20 parts of Angelica sinensis, 5-20 parts of Ophiopogon japonicus, 5-20 parts of Polygonatum odoratum, 10-30 parts of Cuscuta chinensis, 5-20 parts of Morinda officinalis, 5-20 parts of Rubus idaeus, 5-20 parts of Dioscorea opposita, and 5-20 parts of Morus alba, all have the effects of improving, repairing, and / or activating the residual ovarian function of the aging ovary. The specific efficacy varies with different compositions.

[0126] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is only in the components and contents of the traditional Chinese medicine composition. The traditional Chinese medicine composition in Comparative Example 1 is 50 g of Astragalus membranaceus, 10 g of Angelica sinensis, 10 g of Dioscorea opposita, 10 g of Rehmannia glutinosa, 10 g of Epimedium brevicornu, 10 g of Cuscuta chinensis, and 10 g of Adenophora stricta. The model construction process and the administration dose are the same as those in Example 1. The administration dose in Comparative Example 1 is 110 g / 60 kg * 9.1 ≈ 16.7 g / kg. The gavage volume V for mice is calculated as 10 ml / kg (i.e., 0.01 ml / g). The test results are as follows: Take the unilateral ovaries of mice in each group, perform HE staining, observe under the microscope, compare and count the pathological changes of the ovarian tissues of mice in each group, and take pictures and record as follows Figures 26 - 27 shown in the figure: In the young control group, many developing follicles at different levels can be seen. Compared with the young control group, the number of developing follicles in the ovaries of mice in the old control group is extremely small; compared with the traditional Chinese medicine group of the present invention, the ovarian pathology of Comparative Example 1 shows fewer developing follicles. Further counting and statistics of follicles in each group show that: compared with the old control group, the number of follicles at all levels in Comparative Example 1 and the traditional Chinese medicine group of the present invention have increased. The number of primordial follicles, primary follicles and secondary follicles in Comparative Example 1 is slightly higher than that in the traditional Chinese medicine group of the present invention (increased by 26.8%, 28.7% and 26.6% respectively), while the number of antral follicles, mature follicles and corpora lutea in the traditional Chinese medicine group of the present invention has increased by 63.3%, 121.7% and 80.3% respectively compared with Comparative Example 1. In short, from the ovarian pathology and follicle counting results, it shows that the curative effect of the traditional Chinese medicine composition of the present invention is significantly higher than that of Comparative Example 1.

[0127] Comparative Example 2 The difference between Comparative Example 2 and Example 1 lies only in the different components of the traditional Chinese medicine composition, including 30 g of Astragalus membranaceus, 10 g of Angelica sinensis, 10 g of Polygonatum odoratum, 10 g of Morinda officinalis, 10 g of Rubus idaeus, 10 g of Dioscorea opposita, and 10 g of Morus alba. The model construction process and the administration dose are the same as those in Example 1. The administration dose in Comparative Example 2 is 90 g / 60 kg * 9.1 ≈ 13.7 g / kg, and the intragastric administration volume V of mice is calculated according to 10 ml / kg (i.e., 0.01 ml / g).

[0128] To explore the maintenance effect of the prescriptions of Comparative Example 2 and Example 1 on the follicular development of aging mice, pregnant mare serum gonadotropin and human chorionic gonadotropin were injected into mice for ovulation induction, and the number of oocytes obtained in each group was counted. The results are statistically shown as follows Figures 28 - 29 shown in the figure: Compared with the young control group, the number of oocytes obtained in the GV stage and MII stage in the old control group was significantly reduced (p < 0.001). Compared with the old control group, the traditional Chinese medicine group of the present invention significantly increased the number of oocytes in the GV stage and MII stage (p < 0.05, p < 0.01). Compared with the traditional Chinese medicine group of the present invention, the number of oocytes obtained in the GV stage and MII stage in the Comparative Example 2 group was significantly reduced (p < 0.05). The number of oocytes obtained by ovulation induction in mice is extremely important for evaluating its ovarian function and reproductive ability. From the statistical results of the number of oocytes in the GV stage and MII stage, it shows that the curative effect of the traditional Chinese medicine composition of the present invention is significantly higher than that of Comparative Example 2.

[0129] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Chinese medicine composition for repairing and / or activating the residual ovarian function of aged ovaries, characterized in that: The traditional Chinese medicine composition consists of the following components: astragalus, angelica, ophiopogon, polygonatum, dodder seed, morinda officinalis, raspberry, yam and mulberry leaf.

2. The Chinese medicine composition according to claim 1, characterized in that: The traditional Chinese medicine composition consists of the following components by weight: 20-50 parts of astragalus, 5-20 parts of angelica, 5-20 parts of ophiopogon, 5-20 parts of polygonatum, 10-30 parts of dodder seeds, 5-20 parts of morinda officinalis, 5-20 parts of raspberry, 5-20 parts of yam and 5-20 parts of mulberry leaves.

3. The Chinese medicine composition according to claim 2, characterized in that: The traditional Chinese medicine composition consists of the following components by weight: 20-40 parts of astragalus, 5-15 parts of angelica, 5-15 parts of ophiopogon, 5-15 parts of polygonatum, 15-30 parts of dodder seeds, 5-15 parts of morinda officinalis, 5-15 parts of raspberry, 5-15 parts of yam and 5-15 parts of mulberry leaves.

4. The Chinese medicine composition according to claim 3, characterized in that: The Chinese medicine composition consists of the following components by weight: 30 parts of astragalus, 10 parts of angelica, 10 parts of ophiopogon, 10 parts of polygonatum, 20 parts of dodder seeds, 10 parts of morinda officinalis, 10 parts of raspberry, 10 parts of yam and 10 parts of mulberry leaves.

5. The method for preparing the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Take astragalus, angelica, ophiopogon, polygonatum, dodder seed, Morinda officinalis, raspberry, yam and mulberry leaf in proportions by weight, add water and reflux for extraction; (2) Concentrating the extracted medicinal liquid to obtain a Chinese medicine composition.

6. The preparation method according to claim 5, 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 time of the reflux extraction is 60-150 minutes, and the number of reflux extractions is 1-3 times.

7. Use of the Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a drug for delaying ovarian aging.

8. The use according to claim 7, characterized in that: The medicine also includes a pharmaceutically acceptable carrier.

9. The use according to claim 7, characterized in that: The dosage form of the drug is drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, powders, emulsions, pellets, pills, buccal tablets, freeze-dried powder injections, gels, suppositories or aerosols.

10. Use of the Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a drug for repairing and / or activating the residual ovarian function of aged ovaries.

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