Extract for keeping female health and application thereof

Abstract

The invention discloses an extract for promoting health and application thereof, and belongs to the technical field of medical and edible dual-purpose and traditional Chinese medicine health care, and the extract is a special extract obtained from beautiful millettia roots. The beautiful millettia root extract exerts new activity for promoting female health through a new mechanism, and the clinical effect is obviously improved. Experimental research proves that the extract promotes self-renewal of female germline stem cells and directional differentiation of the female germline stem cells into oocytes by promoting expression of Notch1, HES1 and HES5 in a Notch signal channel and regulates the female progestational hormone level, and shows the effects of promoting and recovering development of a second sexual character and preventing symptoms of polycystic ovarian syndrome on ovarian development disorder, and has a good application prospect. The radix millettiae speciosae extract is capable of promoting female physical development and maturation, delaying premature ovarian failure, relieving female depression and anxiety states and keeping female health, belongs to a new target and new mechanism, and makes up the mechanism blank of the radix millettiae speciosae extract in the aspect of keeping female health. The application has innovativeness.

Description

Technical Field

[0001] The present invention relates to the technical fields of food and traditional Chinese medicine health care, and particularly relates to an extract for maintaining female health and its uses. Background Art

[0002] Millettia speciosa Champ., mainly produced in Guangdong and Guangxi regions, is a plant of the genus Millettia in the family Fabaceae. Its roots are used as medicine, and it is also known as Zhujiaoli, Shanlianou, Jinzhonggen, Daodiao Jinzhong, Dalisu. It has a sweet taste and a neutral nature, and has the effects of tonifying deficiency and moistening the lungs, strengthening muscles and activating collaterals. Millettia speciosa Champ. has a long history of medicinal use. As early as in "Herbal Medicine Properties", it was recorded that: "Strengthen muscles and activate collaterals, tonify deficiency and moisten the lungs. Treat low back pain, rheumatic arthralgia, chronic hepatitis, pulmonary tuberculosis". "Luchuan Materia Medica" also recorded it: "Clear the lungs and relieve cough, clear heat and detoxify. Treat hemoptysis, dysentery, fever and thirst in febrile diseases, dizziness". Modern analytical chemistry research shows that all parts of Millettia speciosa Champ., such as roots, stems, leaves, and flowers, contain bioactive components, especially the active components in the roots are extremely rich. The main active components of Millettia speciosa Champ. are polysaccharides, terpenoids, flavonoids, steroids, alkaloids, etc., which have various physiological activities and pharmacological effects such as immunomodulation, anti-fatigue, anti-inflammatory, antioxidant, and anti-tumor. Clinically, it can be used to treat lumbar muscle strain, spermatorrhea, leucorrhea, and weakness after illness. As a medicinal and edible plant widely used by the people in southern China, Millettia speciosa Champ. is not only used as medicine in fresh form, but also made into various Chinese patent medicines and health foods, such as Zhuangyao Jianshen Pills, Guilong Ointment, Huoluo Zhitong Pills, Shujin Jianyao Pills, etc. It is rich in nutrition and has high medicinal value. At the same time, Millettia speciosa Champ. is often used to make medicated diets and medicated wines. In Guangdong and Guangxi regions, it is more commonly used as soup ingredients, with the effects of tonifying deficiency and moistening the lungs, strengthening bones and muscles, and invigorating the spleen, and has high edible value. The fermentation method can promote the transformation and release of active components in medicinal materials through the participation of microorganisms, generating more compounds with antioxidant and other effects. Aspergillus sydowii can degrade cellulose and has great development potential in industrial applications and pesticide degradation fields. At the same time, this fungus is a safe and effective medicinal source fungus, capable of producing a variety of bioactive metabolites, and has great development value in the applications in the fields of medicine and food. By consulting relevant literature, it is found that there is little research on Millettia speciosa Champ. at home and abroad, and there is less in-depth research on its physiological activities. It has been reported that a traditional Chinese medicine formula with Millettia speciosa Champ. as the monarch drug has the effect of preventing and treating premature ovarian failure. At the same time, there are also studies showing that the extract of Millettia speciosa Champ. can enhance sexual function, regulate follicular development, effectively slow down apoptosis of cells in the ovary and polarization of macrophages to pro-inflammatory M1 macrophages, play a protective role in the survival and maturation of oocytes, thereby improving ovarian reserve capacity, indicating that Millettia speciosa Champ. is beneficial to female health.

[0003] Due to genetic factors, endocrine disorders, environmental factors, and lifestyle factors, women's health is in the threatened area. Abnormal menstruation (primary amenorrhea, amenorrhea, or oligomenorrhea) caused by ovarian developmental disorders, accompanied by high levels of follicle-stimulating hormone (>25 U / L), luteinizing hormone, and low levels of estradiol, and decreased fertility. Hormonal disorders and the accumulation of negative emotions are manifested as polycystic ovary syndrome. Clinical symptoms such as insomnia and mood swings lead to a series of psychological problems such as anxiety and depression in patients, seriously affecting the quality of life. Maintaining women's health and its impact on the social environment and social progress make it a major challenge in contemporary medicine. Follicles are the basic structural and functional units of the ovary. Female germline stem cells differentiated from primordial germ cells can be induced to differentiate into oocytes by estrogen, continuously replenishing the continuously depleted primordial follicle pool and maintaining the healthy development of the ovary. Therefore, promoting the self-renewal and directed differentiation of female germline stem cells is particularly important for ovarian development.

[0004] Current studies have found that by regulating multiple signaling pathways such as PI3K / Akt, Wnt / β-catenin, and TGF-β / MAPKs, the functions of regulating ovarian blood supply, reducing the degree of follicular oxidative stress and inflammatory response, and stimulating follicular proliferation can be exerted to promote the recovery of ovarian function. The Notch signaling pathway is a highly conserved cell signaling pathway that participates in regulating processes such as cell proliferation, differentiation, apoptosis, and cell fate determination. The activation of Notch downstream effector factors, such as genes in the HES family, these genes promote cells to enter the proliferative state by inhibiting the expression of cell cycle inhibitors, and the activation of a higher level of Notch signal promotes the differentiation of ovarian germline stem cells into mature oocytes. Studies have found that Millettia speciosa Champ. can enhance the restoration of the ovarian cortical region structure and promote the proliferative activity of FGSCs in the cortex, but the specific mechanism and its impact on the differentiation ability of female germline stem cells are still unclear, and no studies have been found on the treatment of ovarian developmental disorders and abnormal development of secondary sexual characteristics with this mechanism of Millettia speciosa Champ., the prevention of polycystic ovary syndrome, and the improvement of female anxiety-like behaviors.

[0005] Consulting the literature and data, it is found that there are currently studies on the compound related to Millettia speciosa Champ. for ovarian function, but only the ovarian reserve capacity of mice is studied. The effects of the extract of Millettia speciosa Champ. on ovarian development and the emotional problems after female diseases are not comprehensively analyzed, and no novel mechanism is seen. There is a lack of in-depth research on new targets and molecular mechanisms. This experiment found that the extract of Millettia speciosa Champ. promotes the expression of Notch1, HES1, and HES5 in the Notch signaling pathway, so as to increase the expression of the reproductive stem cell and germ cell markers Oct4 and Mvh in the ovary, as well as the expression of the meiosis markers Stra8 and Sycp3, promotes the self-renewal of female reproductive stem cells and the directional differentiation into oocytes, regulates the levels of estrogen and progesterone, shows effects on ovarian developmental disorders, promotes or restores the development of secondary sexual characteristics, prevents the symptoms of polycystic ovary syndrome, promotes the physical development and maturity of women, and relieves the depressive and anxious states of women, maintaining women's health, which belongs to new targets and new mechanisms.

[0006] With the changes in the living environment, the acceleration of the rhythm, the influence of many factors such as surgery and drug use, the incidence of gynecological diseases has been increasing year by year, and women's health has received great attention. However, our research on women's health is still quite limited so far. Given its importance, developing products with novel mechanisms, outstanding activities, significant curative effects, and high safety is one of the hotspots in biological research. Summary of the Invention

[0007] The purpose of the present invention is to solve the above deficiencies and provide an extract with new targets, new mechanisms, and high physiological activity and its uses.

[0008] In order to achieve the above purpose, the technical solution of the present invention is as follows: The present invention provides an extract for maintaining women's health and its uses, and the extract is the extract of Millettia speciosa Champ.

[0009] Furthermore, the present invention also provides that the extract is obtained by subjecting Millettia speciosa Champ. to conventional crushing, fermentation processing method, microwave-assisted extraction and then drying.

[0010] Furthermore, the extract can be made into a traditional Chinese medicine preparation by adding excipients.

[0011] Furthermore, the excipients adopt liquid preparation excipients, semi-solid preparation excipients or solid preparation excipients.

[0012] Furthermore, the preparation is oral liquid, syrup, tablet, capsule, powder, dripping pill, granule, etc.

[0013] The extract in this invention promotes the expression of Notch1, HES1, and HES5 in the Notch signaling pathway, promotes the self-renewal and directed differentiation of female reproductive stem cells into oocytes, regulates estrogen and progesterone levels, and shows effects on ovarian development disorders, promotes or restores the development of secondary sexual characteristics, prevents symptoms of polycystic ovary syndrome, promotes female physical development and maturation, and alleviates female depression and anxiety, thus maintaining women's health.

[0014] Compared with existing technologies, the beneficial effects of this solution are: The extract of this invention can be used to maintain women's health.

[0015] This invention utilizes an innovative method that combines fermentation and microwave-assisted extraction to improve the extraction efficiency of bioactive components.

[0016] The extract of this invention possesses novel mechanisms, targets, and activities, demonstrating innovation. It can promote the expression of Notch1, HES1, and HES5 in the Notch signaling pathway, thereby promoting the self-renewal and directed differentiation of female reproductive stem cells into oocytes, regulating estrogen and progesterone levels, and exhibiting effects on ovarian development disorders, promoting or restoring the development of secondary sexual characteristics, preventing symptoms of polycystic ovary syndrome, promoting female physical development and maturation, delaying premature ovarian failure, and alleviating female depression and anxiety, thus maintaining women's health. This constitutes a novel mechanism of action of this extract. Attached Figure Description

[0017] Figure 1 This invention relates to the effect of *Achyranthes bidentata* extract on anxiety-like behavior in mice; (a) proportion of open arm time, (b) proportion of open arm number; Note: Compared with the blank group: *P<0.01; Compared with the model group: #P<0.05; ##P<0.01.

[0018] Figure 2 This invention relates to the effect of *Achyranthes bidentata* extract on the ovarian index of mice. Note: Compared with the blank group: *P<0.01; Compared with the model group: ##P<0.01.

[0019] Figure 3 The effects of *Achyranthes bidentata* extract on the proliferation of female reproductive stem cells in the embodiments of this invention are shown in the following figures: (a) 12h, (b) 24h, (c) 48h; Note: Compared with the negative control group: *P<0.01; Compared with the DAPH group: ##P<0.01.

[0020] Figure 4 This invention relates to the effects of *Achyranthes bidentata* extract on the levels of T, E2, and AMH hormones in the peripheral blood serum of mice; (a) E2, (b) T, (c) AMH; Note: Compared with the blank group: *P<0.01; Compared with the model group: ##P<0.01.

[0021] Figure 5 This invention relates to the effects of *Achyranthes bidentata* extract on female reproductive stem cell markers MvH and Oct4, as well as meiotic markers Stra8 and Sycp3 proteins; (a) Stra8, (b) Sycp3, (c) MvH, (d) Oct4; Note: Compared with the blank group: *P<0.01; Compared with the model group: ##P<0.01.

[0022] Figure 6 The effects of *Smilax glabra* extract on Notch1, HES1, and HES5 proteins in this embodiment of the invention are shown in the figures. (a) Notch1, (b) HES1, (c) HES5. Note: Compared with the blank group: *P<0.01; Compared with the model group: ##P<0.01. Detailed Implementation

[0023] The present invention and its embodiments are described below. This description is not restrictive, and actual embodiments are not limited thereto. Based on the embodiments of the present invention, if those skilled in the art are inspired by them and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0025] The experimental study on the regulation of ovarian function by the fermented extract of *Achyranthes bidentata* provided by this invention: Fresh *Achyranthes bidentata* medicinal material was taken, and after surface cleaning and sterilization, it was inoculated with a mixed bacterial solution of *Aspergillus* strains. Liquid fermentation was carried out under constant temperature shaking conditions for 14 days. After fermentation, the bacterial cells were removed by centrifugation, and the fermentation product was extracted by ethanol-water extraction. The obtained extract was concentrated under reduced pressure and freeze-dried to prepare an extract rich in active ingredients such as polyketides, polysaccharides, flavonoids, and alkaloids.

[0026] The extract was dissolved in dimethyl sulfoxide (DMSO) and treated with different concentrations (100 μg / mL, 200 μg / mL, 400 μg / mL) of mouse ovarian granulosa cell line (KK-1). The secretion levels of estradiol (E2) and progesterone (P4) in the cell supernatant were detected by enzyme-linked immunosorbent assay (ELISA), and the expression changes of Notch1, HES1, and HES5 genes in the Notch signaling pathway were analyzed by quantitative real-time PCR (qRT-PCR).

[0027] The results showed that the extract could significantly increase the secretion levels of E2 and P4 in granulosa cells (P<0.05) and promote the expression of Notch1, HES1 and HES5 in the Notch signaling pathway (P<0.05), demonstrating a significant regulatory effect on ovarian function.

[0028] Combined with Western blot results, Notch1 protein expression was significantly upregulated in the extract-treated group (P<0.05), consistent with gene expression results. This indicates that the fermented extract of *Smilax glabra* may promote oocyte generation and differentiation and regulate ovarian function by activating the expression of related genes and proteins in the Notch signaling pathway.

[0029] In summary, the fermented extract of *Smilax glabra* exhibits significant activity in regulating ovarian function through synergistic effects across multiple pathways and targets, and has the potential value of being developed as a treatment for ovarian dysfunction-related diseases.

[0030] A study on the therapeutic effect of fermented extract of *Smilax glabra* on polycystic ovary syndrome: The extract was freeze-dried and prepared into a 10 mg / mL solution, which was then further diluted for animal experiments. PCOS model rats (treated with diethylstilbestrol and nickel chloride as inducers) were selected and divided into a model group, a positive control group (treated with metformin), and low-dose groups (50 mg / kg, medium-dose group, 100 mg / kg, and high-dose group) of *Achyranthes bidentata* fermented extract. The extract was administered by gavage daily for 30 consecutive days.

[0031] During the experiment, the body weight and ovarian volume of the rats were measured regularly. The levels of LH, FSH, E2, P4, and testosterone (T) in the rat serum were detected using enzyme-linked immunosorbent assay (ELISA). Ovarian tissue from each group of rats was stained with hematoxylin-eosin (HE) and immunofluorescence (IF) to observe follicular development and the expression and distribution of banded synapses.

[0032] After the experiment, the expression levels of Notch1, HES1, and HES5 in the Notch signaling pathway were detected by real-time quantitative PCR (qRT-PCR) and Western blot analysis. The results showed that the medium and high doses of the fermented extract of *Smilax glabra* significantly improved the reduction in ovarian volume and weight gain in rats with polycystic ovary syndrome (P<0.05).

[0033] Serological analysis showed that the medium and high doses of the extract significantly reduced LH and T levels and increased the secretion of FSH, E2 and P4 (P<0.05), with effects comparable to or better than the positive control group (metformin).

[0034] Meanwhile, the expression levels of Notch1, HES1, and HES5 in the Notch signaling pathway were significantly upregulated in the medium and high dose groups of the extract (P<0.05), indicating that its therapeutic effect on PCOS rats may be achieved by regulating the activation of the Notch signaling pathway.

[0035] In summary, the fermented extract of *Smilax glabra* can effectively improve endocrine disorders and ovarian developmental abnormalities in PCOS model rats. Its mechanism of action is closely related to the regulation of the Notch signaling pathway, showing good therapeutic potential. Example

[0036] S1: Clean the fresh raw material of *Smilax glabra*, cut it into slices with a diameter of 0.4 cm using a slicer, steam for 45 minutes, dry at a medium-low temperature of 40℃, and then pulverize it into powder that can pass through an 8-mesh sieve. S2: Weigh 25g of *Achyranthes bidentata* powder, add 0.5% of the total weight of K2HPO4, mix well, place in a 250mL Erlenmeyer flask, add 1.5 times the amount of water, and sterilize. After sterilization, inoculate the *Aspergillus polyploid* seed culture into the sterilized *Achyranthes bidentata* (1:1) under a sterile operating table, and incubate at 28℃. After 14 days, remove the fermentation product, inactivate it, and dry it at a medium-low temperature.

[0037] S3: Mix the dried fermented product of *Smilax glabra* with 70% ethanol at a ratio of 1:15 and perform microwave extraction. The extraction conditions are: temperature 60℃, microwave frequency 2500MHz, microwave power 500W, extraction time 30min. After extraction, the solid and liquid are separated, and the extraction is repeated twice. The extracts are combined and concentrated to an extract paste with a relative density of 1.25. S4: Freeze in a -80℃ freezer for 24 hours, and then freeze-dry the extract to obtain the final extract. Example

[0038] S1: Clean the fresh raw material of *Smilax glabra*, cut it into slices with a diameter of 0.4cm using a slicer, steam for 45 minutes, dry at a medium-low temperature of 40℃, and then pulverize it into powder that can pass through an 8-mesh sieve. S2: After crushing the oxtail, mix it with 70% ethanol at a ratio of 1:15 and perform microwave extraction. The extraction conditions are: temperature 60℃, microwave frequency 2500MHz, microwave power 500W, extraction time 30min. After extraction, the solid and liquid are separated, and the extraction is repeated twice. The extracts are combined and concentrated to an extract paste with a relative density of 1.25. S3: Freeze in a -80℃ freezer for 24 hours, and then freeze-dry the extract to obtain the final extract. Example

[0039] S1: Clean the fresh raw material of *Smilax glabra*, cut it into slices with a diameter of 0.4 cm using a slicer, steam for 45 minutes, then pulverize at a medium-low temperature of 40℃ until it can pass through an 8-mesh sieve. S2: Weigh 25g of *Achyranthes bidentata* powder, add 0.5% of the total weight of K2HPO4, mix well, place in a 250mL Erlenmeyer flask, add 1.5 times the amount of water, and sterilize. After sterilization, inoculate the *Aspergillus polyploid* seed culture into the sterilized *Achyranthes bidentata* (1:1) under a sterile operating table, and incubate at 28℃. After 14 days, remove the fermentation product, inactivate it, and dry it at a medium-low temperature.

[0040] S3: Mix the dried fermented product of *Achyranthes bidentata* with water at a ratio of 1:15 and perform microwave extraction. The extraction conditions are: temperature 60℃, microwave frequency 2500MHz, microwave power 500W, extraction time 30min. After extraction, the solid and liquid are separated, and the extraction is repeated twice. The extracts are combined and concentrated to an extract paste with a relative density of 1.25. S4: Freeze in a -80℃ freezer for 24 hours, and then freeze-dry the extract to obtain the final extract.

[0041] Pharmacodynamic studies conducted in connection with this invention.

[0042] 1. Experimental Materials SPF-grade C57BL / 6 female mice, 4 weeks old, were acclimatized for 7 days in an animal facility environment before the experiment, with an ambient temperature of 20–25°C, relative humidity of 40–70%, and alternating light and dark for 24 hours per day. The samples were extracts obtained in Examples 1 and 2.

[0043] 2. Experimental Methods 2.1 Modeling of mice with unhealthy ovaries The specific method involves injecting cyclophosphamide into the peritoneal cavity of mice at a dose of 120 mg / kg·d on the first day, followed by continued injection at a dose of 15 mg / kg·d for the next 14 days.

[0044] 2.2 Experimental Grouping Eighty-four SPF-grade female mice were randomly divided into seven groups based on their administration methods: a blank control group, a model group, high-, medium-, and low-dose groups of *Achyranthes bidentata* fermented extract, a *Achyranthes bidentata* extract group, and a positive control group, with 12 mice in each group. Control group: 4-week-old mice were injected intraperitoneally with physiological saline for 15 consecutive days, followed by oral administration of physiological saline for 6 weeks; Model group: 4-week-old mice were intraperitoneally injected with cyclophosphamide at a dose of 120 mg / (kg·d) on the first day, followed by continuous injection at a dose of 15 mg / (kg·d) for 2 weeks; Low-dose group of fermented extract of *Smilax glabra*: 4-week-old mice were first injected intraperitoneally with cyclophosphamide for 15 days, and then administered fermented extract of *Smilax glabra* at 200 mg / (kg·d) by gavage for 6 weeks. Medium-dose group of fermented extract of *Smilax glabra*: 4-week-old mice were first injected intraperitoneally with cyclophosphamide for 15 days, and then continuously administered fermented extract of *Smilax glabra* at 400 mg / (kg·d) by gavage for 6 weeks; High-dose group of fermented extract of *Smilax glabra*: 4-week-old mice were first injected intraperitoneally with cyclophosphamide for 15 days, and then administered fermented extract of *Smilax glabra* at 800 mg / (kg·d) by gavage for 6 weeks. In the *Smilax glabra* extract group, 4-week-old mice were first injected intraperitoneally with cyclophosphamide for 15 days, followed by continuous gavage administration of *Smilax glabra* extract at 600 mg / (kg·d) for 6 weeks. Positive drug control group: 4-week-old mice were first injected intraperitoneally with cyclophosphamide for 15 days, and then administered Zuogui Pill 1.5 g / (kg·d) by gavage for 6 weeks.

[0045] 2.3 Elevated Cross Maze Experiment The elevated cross maze was used to assess anxiety-like behaviors in animals. Each group underwent the elevated cross maze test 30 minutes after the last administration of saline or other medications. The number of times rats entered open arms, the time spent entering open arms, the number of times they entered closed arms, and the time spent entering closed arms were recorded within 5 minutes. Formulas: Open arm count percentage = (Number of open arms / (Number of open arms + Number of closed arms)) × 100%. Open arm time percentage = Open arm dwell time / (Open arm dwell time + Closed arm dwell time) × 100%.

[0046] 2.4 Ovarian Index After euthanasia by neck dislocation, the ovaries were removed, cleaned in PBS solution, and weighed after removing surface liquid with absorbent paper. The formula for calculation is: Ovarian Index (‰) = Ovarian weight / Body weight × 1000.

[0047] 2.5 Detection of sex hormones in serum Serum from each group of mice was collected via the abdominal aorta according to the sample collection method, aliquoted into disposable EP tubes, and centrifuged to obtain serum. The levels of testosterone (T), estradiol (E2), and anti-Müllerian hormone (AMH) in the serum of each group of mice were detected using enzyme-linked immunosorbent assay (ELISA). The specific procedures are as follows: Add 50 μL of sample diluent to the blank wells, 50 μL of standard to the standard wells, and 50 μL of the sample to be tested to the remaining sample wells, with three replicates for each sample. Immediately add 50 μL of HRP enzyme-conjugated antibody working solution to each well, mix well, cover the plate with a membrane, and incubate at 37°C for 60 min. After removing from the oven, shake off the liquid in the wells and pat dry any excess liquid. Add 350 μL of washing buffer to each well, soak for 30 s, and then shake off the liquid in the plate. Repeat the above steps 5 times. Add 90 μL of substrate solution (TMB) to each well in the microplate, then seal it with a membrane to protect it from light and incubate at 37°C for 10 min. Add 50 μL of stop solution to each well in the microplate. Immediately afterwards, measure the OD value of the cells in each well, with the microplate reader set to 450 nm. The levels of testosterone, estradiol, and anti-Müllerian hormone in the serum of each group of mice were then calculated based on the OD values ​​of the standard samples.

[0048] 2.6 Tissue embedding and sectioning (1) Fixation: The intact ovaries were washed in PBS solution and then soaked in 4% paraformaldehyde and placed in a refrigerator at 4°C overnight; (2) Dehydration: Place the tissue in a dish containing PBS and soak and wash it 3 times for 5 min each time; wrap the ovary with lens paper and place it in the embedding box, and then soak the embedding frame in 70%, 80%, 95% I, 95% II, 100% I and 100% II alcohol for 30 min each in sequence. (3) Transparency: The embedding cassette was immersed in xylene I and II for 1 h each; (4) Impregnation: The embedding boxes were immersed in xylene paraffin for 30 min, and paraffin I and II for 2 h each; (5) Embedding: Take out the fully paraffin-impregnated tissue and place it in the embedding frame. Pour in liquid paraffin and let it solidify naturally at room temperature. (6) Sectioning: After removing the embedded wax block from the embedding frame, fix it on the microtome. Adjust the position of the wax block according to the slicing results to cut the maximum cross section of the wax block. Slice continuously with a thickness of 5 μm. Spread the cut wax slices fully on a water surface at 45 ℃-50 ℃. Use a glass slide (mark the wax block group on the glass slide with a pencil) to pick up the slices and bake them at 37 ℃ overnight for later use.

[0049] 2.7 Isolation, extraction, and culture of female germline stem cells Place the sterilized mouse back-up on a laminar flow hood. Make an incision in the lower third of the spine, near the kidneys. Using ophthalmic surgical forceps, remove the entire ovarian tissue and place it in a 35 mm dish containing D-Hanks solution. Separate and remove fat and the ovarian cyst, ensuring the integrity of the ovary. Finally, transfer the ovary to another dish containing D-Hanks solution to rinse away any remaining impurities. Add 1 mL of collagenase to a 2 mL centrifuge tube. Transfer the ovarian tissue to the tube using surgical forceps. Add 1 mL of collagenase to the tube and mix thoroughly with the ovary. Tightly cap the centrifuge tube and seal it completely with sealing film. Place the tube in a 37°C water bath and shake for 15 minutes for digestion. After digestion, centrifuge the tube at 2000 rpm for 5 minutes at 4°C and discard the supernatant. Add 1 mL of trypsin to the centrifuge tube and agitate the bottom to separate the tissue fragments, ensuring maximum contact area between the tissue and the trypsin. Place the tube in a 37°C water bath for 3 minutes. After digestion, quickly add 1 mL of medium containing 10% FBS to a centrifuge tube and centrifuge at 2000 rpm for 5 minutes at 4 ℃. Discard the supernatant. Add 200 µL of FGSCs culture medium to resuspend the cells. Carefully aspirate the cell suspension and remove the ovarian tissue fragments. Add the cell suspension to a 48-well culture plate incubated with 0.5% gelatin, and then incubate at 37 ℃ with 5% CO2. After 4 h of culture, take advantage of the difference in cell adhesion and carefully pipette to collect the supernatant and transfer it to a new 48-well culture plate incubated with 0.5% gelatin. At this time, the isolated cells are approximately 10-20 μm in diameter. Change the culture medium every 12 h and passage the cells according to their condition.

[0050] 2.8 CCK-8 cell proliferation assay 2.8.1 Preparation of drug-containing serum Four-week-old mice were administered Zuogui Wan (1.5 g / kg) by gavage, while the control group was administered an equal volume of distilled water by gavage. *Achyranthes bidentata* fermented extract was administered at doses of 200 mg / kg, 400 mg / kg, and 800 mg / kg by gavage, respectively, and *Achyranthes bidentata* extract at 600 mg / kg by gavage, once daily for 7 consecutive days. One hour after the last administration, the mice were anesthetized, and blood was collected from the abdominal aorta. The blood was left at room temperature for 2 hours, then centrifuged at 3000 r / min for 20 min at 4 ℃ to separate the serum. The serum was collected, filtered, sterilized, inactivated, and stored at -80 ℃ for later use.

[0051] 2.8.2 Detection of cell viability After digesting the cells with trypsin and centrifuging, the cells were repeatedly resuspended in an appropriate amount of culture medium to ensure even distribution, and the cell density was confirmed to be 2 × 10⁶ using a cell counting chamber.4 / mL.

[0052] Cell suspension (100 μL / well) was seeded into 96-well plates. Wells were divided according to the experimental design into: blank group (no cells, only culture medium), negative control group (same volume without *Achyranthes bidentata* extract), positive control group (20% *Zuogui Wan*), low-dose *Achyranthes bidentata* fermented extract group (20%), medium-dose *Achyranthes bidentata* fermented extract group (20%), high-dose *Achyranthes bidentata* fermented extract group (20%), *Achyranthes bidentata* extract group (20%), and DAPH (Notch signaling pathway inhibitor) (10 μM). Each group had 5 replicates. After 0 h, 12 h, 24 h, and 48 h of culture, 10% CCK-8 medium was added to each well of the plate by changing the medium. The plates were incubated in the dark for 1 hour. After incubation, the OD value of the samples at 450 nm was quickly measured using a microplate reader. The final OD value was obtained by subtracting the OD value of the blank group from the obtained OD value. Cell proliferation curves were plotted using statistical software.

[0053] 2.9 Western blotting was used to detect differentiation markers of female germline stem cells and protein expression in the Notch pathway. 2.9.1 Extraction of proteins from female germline stem cells (1) Use a pipette to remove and discard the old culture medium from the cells. Then add 2 mL of PBS to each culture dish to wash the cells thoroughly, slowly and gently. After washing 3 times, remove the PBS and place the dish on ice.

[0054] (2) Add 300 µL of protein lysis buffer to the culture dish. The protein lysis buffer consists of lysis buffer and PMSF mixed in a 100:1 ratio. After thoroughly mixing the culture dish, place it on ice for about 30 min to allow the cells to lyse.

[0055] (3) Collect cells using a new cell scraper according to the experimental group and place them on ice. Scrape the cells off and blow them off the bottom of the culture dish with a pipette. Transfer the resulting cell suspension to another new 1.5 mL EP tube.

[0056] (4) After centrifuging the cells at 12,000 rpm for 20 min at low temperature, carefully transfer the protein supernatant in the tube to another new 1.5 mL EP tube using a pipette. It can be stored at -20℃ for a long time or the concentration can be detected immediately.

[0057] (5) After the protein extraction of each group of cells was completed, the protein concentration was determined by BCA kit.

[0058] 2.9.2 Determination of protein concentration by BCA method (1) Prepare the protein standard according to the kit instructions to a final concentration of 0.5 mg / mL. Based on the number of protein samples to be tested, calculate and prepare the BCA working solution according to the instructions, wherein the dosage ratio of BCA working solution A to solution B is 1:50.

[0059] (2) Add the protein standards to the 96-well plate in sequence according to the concentration gradient shown in the instructions. Set up 3 sub-wells for each standard. Then add ddH2O to make up the total volume of each well to 20 µL.

[0060] (3) Then add protein samples. After diluting the protein samples to an appropriate ratio according to the protein concentration, add 1 µL and 19 µL ddH2O to each well of each group of cell protein samples to make the final total volume 20 µL. Ensure 3 sub-wells for each sample.

[0061] (4) Then add 200 µL of freshly prepared BCA working solution to each well, including standards and samples, and incubate the 96-well plate in a 37°C incubator for 30 min. Measure the OD value of the protein in the 96-well plate at a wavelength of 562 nm using a microplate reader, and then calculate the protein concentration of each group of samples.

[0062] 2.9.3 Western-Blot Detection Procedure (1) Preparation of SDS-PAGE gel: Prepare the required SDS-PAGE gel according to the instructions.

[0063] (2) Add 5×bμffer to the protein sample and heat at 100 degrees Celsius for 15 minutes.

[0064] (3) Sample loading: Add the cooled protein sample into the gel well as required.

[0065] (4) Electrophoresis: Connect the equipment as needed and set the voltage to 80V and the time to 80 minutes.

[0066] (5) Transfer: Immerse the PVDF membrane in methanol, remove the gel, arrange them neatly in a "sandwich" pattern and insert them into the transfer tank, fill with transfer buffer, place the transfer tank on ice, and transfer at 80V for 1.5h. The entire transfer process requires an ice bath.

[0067] (6) Sealing: Seal the PVDF membrane with 5% skim milk powder for 1 hour.

[0068] (7) Primary antibody incubation: After complete blocking, discard the blocking solution and wash four times with TBST buffer to remove loosely bound proteins. Prepare the primary antibody according to the antibody instructions, add the transfer membrane and 2 mL of antibody to the sealing membrane, and place the sealing membrane on a shaker and incubate overnight at 4°C.

[0069] (8) Secondary antibody incubation: After the primary antibody incubation is complete, the primary antibody is recovered and washed four times with TBST to remove the poorly bound primary antibody. Prepare the secondary antibody according to the antibody instructions, add the transfer membrane and 2 mL of antibody into the sealing membrane, place the sealing membrane on the bed, incubate at room temperature for 1 hour, and then wash three times with TBST for 15 minutes each time.

[0070] (9) Development: Incubate with freshly prepared colorimetric solution for 2 minutes under light-protected conditions, then expose and develop. The experimental results were analyzed for grayscale values ​​using ImageJ and plotted using GraphPad.

[0071] 2.10 Data Processing and Analysis Each experiment was repeated at least three times. Data were statistically analyzed using GraphPad Prism 5.0 software, and the analysis methods included analysis of variance and t-test. P <0.05 is statistically significant.

[0072] 3. Experimental Results 3.1 Effects of *Smilax glabra* extract on anxiety-like behavior in mice As shown in Table 1, compared with the control group, the proportion of open-arm time and the proportion of open-arm number in the elevated cruciate maze were significantly reduced in the model group mice. P <0.01). Compared with the model group, the proportion of open-arm time and the proportion of open-arm number were significantly increased in mice in the high-dose fermentation group of *Smilax glabra* extract. P <0.01). This indicates that the extract can effectively alleviate anxiety-like behavior in mice following ovarian developmental disorders.

[0073] Table 1. Effects of the extract of *Smilax glabra* from the present invention on anxiety-like behavior in mice. Note: Compared with the blank group: * P <0.01; Compared with the model group: # P <0.05; ## P <0.01.

[0074] 3.2 Effects of *Smilax glabra* extract on ovarian index As shown in Table 2, compared with the blank group, the model group had a significantly lower ovarian index ( P <0.01), compared with the model group, different doses of *Achyranthes bidentata* fermented extract increased the ovarian index in a dose-dependent manner ( P <0.01), compared with the model group, the ovarian index of the unfermented extract group of *Smilax glabra* was significantly increased (P <0.01). This indicates that both the fermented and unfermented extracts of *Achyranthes bidentata* in this invention can effectively improve ovarian quality.

[0075] Table 2. Effects of the extract of *Smilax glabra* from the present invention on the ovarian index of mice. Group Animal number Mouse body weight (g) Ovarian weight (g) Ovarian index (‰) Blank group 12 38.8±0.63 0.027±0.001 0.69±0.03 Model group 12 24.64±0.65 0.007±0.001 0.27±0.03* Low-dose group of fermented extract of *Smilax glabra* 12 30.02±0.59 0.013±0.001 <![CDATA[0.44±0.03 ## ]]> Medium dose group of fermented extract of *Smilax glabra* 12 31.57±0.61 0.016±0.002 <![CDATA[0.5±0.05 ## ]]> High-dose group of fermented extract of *Smilax glabra* 12 32.68±0.49 0.018±0.001 <![CDATA[0.55±0.03 ## ]]> Niu Dali extract group 12 31.58±0.78 0.016±0.002 <![CDATA[0.5±0.07 ## ]]> Positive drug control group 12 29.22±0.63 0.012±0.002 <![CDATA[0.40±0.05 ## ]]> Note: Compared with the blank group: * P <0.01; Compared with the model group: ## P <0.01.

[0076] 3.3 Effects of *Smilax glabra* extract on the proliferation of female reproductive stem cells like Figure 3 The results showed that, compared with the negative control group, the model group had significantly reduced cell viability ( P <0.01), compared with the model group, different doses of *Achyranthes bidentata* extract significantly increased cell proliferation activity at different time points. P <0.01). This indicates that the present invention can promote the proliferation of female reproductive stem cells.

[0077] 3.4 Effects of *Smilax glabra* extract on serum T, E2, and AMH levels in mouse peripheral blood like Figure 4 As shown, compared with the blank group, the peripheral blood serum T hormone of mice in the model group was significantly increased ( P <0.01), both E2 and AMH hormones decreased significantly ( P <0.01), compared with the model group, *Achyranthes bidentata* extract significantly reduced T hormone levels ( P <0.01), increases E2 and AMH hormone levels ( P <0.01). This indicates that the present invention can effectively regulate ovarian hormone levels.

[0078] 3.5 Effects of *Smilax glabra* extract on female reproductive stem cell markers MvH and Oct4, and meiotic markers Stra8 and Sycp3 proteins. like Figure 5 As shown, compared with the model group, the protein expression of MVH, Oct4, Stra8, and Sycp3 in the *Achyranthes bidentata* extract group was significantly increased. P <0.01). This indicates that after intervention with *Achyranthes bidentata* extract, the cell characteristics and differentiation capacity of female germline stem cells in the ovaries of model mice were maintained and enhanced, further demonstrating the effect of *Achyranthes bidentata* extract on maintaining and improving the proliferation and differentiation function of female germline stem cells.

[0079] 3.6 Effects of *Smilax glabra* extract on Notch1, HES1, and HES5 proteins like Figure 6As shown, compared with the blank group, the model group significantly reduced the Notch1 level in the Notch signaling pathway ( P <0.01), significantly reduced the levels of HES1 and HES5 related proteins ( P <0.01), compared with the model group, *Achyranthes bidentata* extract significantly reduced the levels of related proteins in the Notch signaling pathway and promoted protein expression ( P <0.01).

[0080] In summary, the experiments demonstrated that both fermented and unfermented extracts of *Achyranthes bidentata* significantly improved efficacy. Furthermore, the experiments revealed for the first time that *Achyranthes bidentata* extract can activate the Notch signaling pathway, promoting Notch1 protein expression, downstream factors HES1 and HES5 protein expression, promoting the self-renewal and directed differentiation of female reproductive stem cells into oocytes, and regulating estrogen and progesterone levels. This demonstrates its ability to address ovarian developmental disorders, promote or restore the development of secondary sexual characteristics, prevent symptoms of polycystic ovary syndrome, promote female physical development and maturation, delay premature ovarian failure, alleviate depression and anxiety in women, and maintain women's health. This represents a novel mechanism of the present invention.

[0081] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. An extract, characterized in that, The extract is prepared from the fermentation products of *Achyranthes bidentata*, and contains secondary metabolites produced during fermentation, including polyketides, polysaccharides, flavonoids, and alkaloids.

2. The extract as described in claim 1, characterized in that, The extract was prepared by fermentation with Aspergillus strains, extracted by ethanol-water extraction, and obtained by vacuum concentration and freeze-drying.

3. The extract as described in claim 1, characterized in that, The extract contains polyketide compounds that account for 0.2% to 20% of the total mass of the extract.

4. The use of a fermented extract of *Smilax glabra*, characterized in that, The extract can promote the self-renewal and directed differentiation of female reproductive stem cells into oocytes, and regulate estrogen and progesterone levels.

5. The use as described in claim 4, characterized in that, The extract can regulate ovarian development disorders and promote and restore the development of secondary sexual characteristics.

6. The use as described in claim 4, characterized in that, The extract can prevent symptoms of polycystic ovary syndrome and promote female physical development and maturation.

7. The use as described in claim 4, characterized in that, The extract can alleviate depression and anxiety in women; the extract can delay premature ovarian failure.

8. The use as described in claim 4, characterized in that, The dosage forms of the preparations include oral liquids, syrups, tablets, capsules, powders, pellets, or granules.

9. A method for extracting *Achyranthes bidentata*, characterized in that, The method includes the following steps: (1) Slice fresh *Smilax glabra*, steam it, dry it at low temperature, and then grind it into powder; (2) Mix with 70% ethanol at a ratio of 1:15 and extract using microwave-assisted extraction; (3) Freeze-dry the extract to obtain the final extract.

10. The extraction method according to claim 9, characterized in that, The method combines fermentation processing technology, using Aspergillus polypolysaccharide seed liquid to ferment the raw material of Achyranthes bidentata, promoting the production and transformation of polysaccharides, flavonoids and secondary metabolites.

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