Application of Millettia speciosa Champ. extract in improving ovarian reserve capacity of mice

Abstract

The present invention relates to the use of a Herba Bovis extract in improving the ovarian reserve capacity of mice. The present invention also discloses a preparation method of the Herba Bovis extract. The Herba Bovis extract of the present invention has the effect of improving sexual function, and specifically can solve the problem of decreased ovarian reserve capacity due to repeated superovulation; the Herba Bovis extract can effectively alleviate follicular hypoplasia, prevent abnormal changes in serum hormones caused by repeated superovulation, and reduce the activation of primordial follicles, thereby slowing down the process of ovarian aging. Not only that, the Herba Bovis extract also effectively slows down cell apoptosis in the ovary and the polarization of macrophages to pro-inflammatory M1 macrophages, plays a protective role in the survival and maturation of oocytes, thereby protecting the reserve capacity of the ovary; the present invention develops a new function of Herba Bovis, and provides a new idea for the development of drugs to improve sexual function.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine and relates to application of a Herba Lycopodii extract in improving the ovarian reserve capacity of mice. Background Art

[0002] In humans, assisted reproductive technology (ART) has become the most effective clinical treatment for infertility and subfertility. Superovulation or controlled ovarian stimulation using exogenous hormones is one of the most important treatments in human ART, with PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin) being widely used for superovulation in mammals. Studies have shown that repeated superovulation affects levels of hormones such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), inducing ovarian cell apoptosis and adversely affecting folliculogenesis, leading to a decrease in ovarian reserve function.

[0003] Niu Da Li (Schizonepeta tenuifolia) is the dried root of the Welchweed genus of the Leguminosae family. It is primarily found in Hainan, Guangxi Zhuang Autonomous Region, Guangdong, Hunan, and Guizhou provinces of my country. It is a medicinal and edible crop with benefits such as nourishing the lungs, strengthening tendons, and activating blood circulation. Studies have shown that Niu Da Li promotes the secretion of immune-related cytokines such as IL-2, IL-4, IL-10, TNF-α, and IgG. Its active ingredient, the novel polysaccharide MSCP2, can induce macrophage activation, while its total flavonoids have potent anti-inflammatory and immune-enhancing properties. Furthermore, studies have shown that a water extract of Niu Da Li can effectively alleviate drug-induced liver fibrosis in zebrafish by inhibiting hepatocyte apoptosis and reducing collagen deposition.

[0004] Therefore, there is a need to develop a drug that can enhance ovarian reserve. Summary of the Invention

[0005] The first purpose of the present invention is to provide a use of a Herba Lycopodii extract in improving the ovarian reserve capacity of mice, thereby overcoming the current problem of decreased ovarian reserve capacity due to repeated superovulation.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned Herba Lycopodii extract.

[0007] The present invention is achieved through the following technical solutions:

[0008] 1. Application of a Herba Bovis extract in improving the ovarian reserve capacity of mice.

[0009] 2. According to the above application, the preparation method of the Herba Lycopodii extract comprises the following steps:

[0010] The fresh Herba Lycopodii raw materials were cleaned and sliced into slices with a diameter of 0.5-2 cm using a slicer to obtain Herba Lycopodii finely crushed products. Purified water was added 7 times by mass for secondary crushing to obtain Herba Lycopodii finely crushed pulp and finely crushed residue. The finely crushed pulp was supplemented with 3 times by mass of water to make up the extraction amount, and emulsified and stirred in an emulsifying homogenization tank at a circulating pressure of 0.1-0.2 MPa. The Herba Lycopodii homogenate was collected and the obtained homogenate was centrifuged at a speed of 3800 rpm / min to obtain Herba Lycopodii separated liquid and separated residue. Then, 10 times by mass of 55% ethanol was added to the separated residue, and low-temperature high-pressure difference continuous extraction was carried out under the conditions of a pressure of 25 MPa and a temperature of 30°C. The separation was carried out at a speed of 3800 rpm / min. rpm / min centrifugation to obtain the Herba Dioscoreae alcohol solution; the Herba Dioscoreae separated liquid and the alcohol solution are respectively microfiltered using a 500nm microfiltration membrane; the separated liquid and the alcohol solution after microfiltration are sequentially subjected to membrane concentration, vacuum concentration, and plate and frame filtration operations to obtain alcohol extract and water extract, respectively; the combined extraction of the two extracts yields the Herba Dioscoreae extract.

[0011] The positive effects of the above technical solution: The Herba Lycopodii extract of the present invention has the effect of improving sexual function, specifically solving the problem of decreased ovarian reserve capacity caused by repeated superovulation; the Herba Lycopodii extract can effectively alleviate follicular hypoplasia, prevent abnormal changes in serum hormones caused by repeated superovulation, and reduce the activation of primordial follicles, thereby slowing the process of ovarian aging. Moreover, the Herba Lycopodii extract also effectively slows down cell apoptosis in the ovaries and the polarization of macrophages into pro-inflammatory M1 macrophages, playing a protective role in the survival and maturation of oocytes, thereby protecting the reserve capacity of the ovaries; the present invention has developed a new function of Herba Lycopodii, providing new ideas for the development of drugs to improve sexual function. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a schematic diagram of the results of HE staining of mouse ovarian tissue sections;

[0013] Figure 2 This is a schematic diagram of the results of ELISA testing of mouse serum, where A: LH (Luteinizing hormone; luteinizing hormone) level; B: FSH (Follicle stimulating hormone; follicle stimulating hormone) level; C: P4 (Progesterone; progesterone) level;

[0014] Figure 3Schematic diagram of the immunofluorescence staining results of mouse ovarian tissue sections, including: A: F480 localization map (red: F480; blue: DAPI: cell nucleus); B: CD206 localization map (red: CD206; blue: DAPI: cell nucleus); C: MHC II localization map (red: MHC II; blue: DAPI: cell nucleus); D: FOXO3a localization map (red: FOXO3a; blue: DAPI: cell nucleus); E: caspase3 localization map (red: caspase3; blue: DAPI: cell nucleus);

[0015] Figure 4 This is a schematic diagram of the results of real-time fluorescence quantitative analysis in KGN cells, where A: caspase3 mRNA level; B: Bax mRNA level;

[0016] Figure 5 The figure is a schematic diagram of the results of TUNEL assay in mouse ovarian tissue sections, wherein A: schematic diagram of apoptosis detection results; B: quantification of fluorescence intensity of TUNEL positive signals;

[0017] Figure 6 This is a schematic diagram of the results of caspase3 immunofluorescence signal expression in KGN cells. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below with reference to specific embodiments and test examples, but they should not be construed as limiting the present invention: Example

[0019] This example illustrates the preparation of the extract of Herba Lycopodii.

[0020] Fresh Herba Cynanchii raw materials were cleaned and sliced into 0.5 cm diameter slices using a slicer to obtain Herba Cynanchii finely chopped products. Purified water was added 7 times by mass for secondary crushing to obtain Herba Cynanchii finely chopped pulp and finely chopped residues. The finely chopped pulp was supplemented with 3 times by mass of water to make up the extraction amount, and emulsified and stirred in an emulsifying homogenization tank at a circulating pressure of 0.1 MPa. The Herba Cynanchii homogenate was collected and centrifuged at a speed of 3800 rpm / min to obtain Herba Cynanchii separated liquid and separated residues. Subsequently, 10 times by mass of 55% ethanol was added to the separated residue, and low-temperature high-pressure differential continuous extraction was performed under the conditions of a pressure of 25 MPa and a temperature of 30°C. Herba Cynanchii alcohol solution was obtained after centrifugation at a speed of 3800 rpm / min. 500 The Herba Dioscoreae extract and the alcohol solution are respectively microfiltered using a 1000 nm microfiltration membrane. The separated solution and the alcohol solution after microfiltration are sequentially subjected to membrane concentration, vacuum concentration, and plate and frame filtration operations to obtain alcohol extract and water extract, respectively. The combined extraction of the two extracts yields the Herba Dioscoreae extract. Example

[0021] This example illustrates the preparation of the extract of Herba Lycopodii.

[0022] Fresh Herba Lycopersicum var. amara raw material was cleaned and sliced into 2 cm diameter slices using a slicer to obtain Herba Lycopersicum var. amara finely ground product, which was then secondary crushed by adding 7 times the amount of purified water by mass to obtain Herba Lycopersicum slurry and fine residue. The fine pulp was supplemented with 3 times the amount of water to make up the extraction amount, and emulsified and stirred in an emulsification homogenization tank at a circulating pressure of 0.2 MPa. The Herba Lycopersicum slurry was collected and centrifuged at a speed of 3800 rpm / min to obtain Herba Lycopersicum separated liquid and separated residue. The separated residue was then added with 10 times the amount of 55% ethanol by mass, and low-temperature high-pressure differential continuous extraction was performed under the conditions of a pressure of 25 MPa and a temperature of 30°C. After centrifugation at a speed of 3800 rpm / min, Herba Lycopersicum ethanol solution was obtained. The Herba Lycopersicum separated liquid and alcohol solution were respectively microfiltered using a 500 nm microfiltration membrane. The separated liquid and alcohol solution after microfiltration were sequentially subjected to membrane concentration, vacuum concentration, and plate and frame filtration to obtain alcohol extract and water extract, respectively. The combined extraction of the two extracts yielded Herba Lycopersicum var. amara extract. Example

[0023] This example illustrates the preparation of the extract of Herba Lycopodii.

[0024] Fresh Herba Lycopersicum var. amara raw material was cleaned and sliced into 1 cm diameter slices using a slicer to obtain Herba Lycopersicum var. amara finely ground product, which was then secondary crushed by adding 7 times the amount of purified water by mass to obtain Herba Lycopersicum slurry and fine residue. The fine pulp was supplemented with 3 times the amount of water to make up the extraction amount, and emulsified and stirred in an emulsification homogenization tank at a circulating pressure of 0.15 MPa. The Herba Lycopersicum slurry was collected and centrifuged at a speed of 3800 rpm / min to obtain Herba Lycopersicum separated liquid and separated residue. The separated residue was then added with 10 times the amount of 55% ethanol by mass, and low-temperature high-pressure differential continuous extraction was performed under the conditions of a pressure of 25 MPa and a temperature of 30°C. After centrifugation at a speed of 3800 rpm / min, Herba Lycopersicum ethanol solution was obtained. The Herba Lycopersicum separated liquid and alcohol solution were respectively microfiltered using a 500 nm microfiltration membrane. The separated liquid and alcohol solution after microfiltration were sequentially subjected to membrane concentration, vacuum concentration, and plate and frame filtration to obtain alcohol extract and water extract, respectively. The combined extraction of the two extracts yielded Herba Lycopersicum var. amara extract.

[0025] The mice used in this study were 6-week-old ICR mice weighing 25 ± 5 g. They were purchased from Guangdong Sijiajingda Biotechnology Co., Ltd. and housed in the Experimental Animal Center of South China Agricultural University at a constant temperature of 25°C. The mice were given a photoperiod of 12 h of light and 12 h of darkness. During the breeding period, the mice had free access to food and water.

[0026] In this experiment, the ovulation promotion of mice was divided into 4 cycles, with an interval of 5 days between each cycle.

[0027] (1) R1 group (single ovulation induction): In the first three cycles, mice corresponding to the R4 group were given 0.2 ml of normal saline by intraperitoneal injection. At the beginning of the fourth cycle, each female mouse was injected with PMSG 10 IU (0.2 ml) at 19:00 in the afternoon. 48 hours later, HCG 10 IU / mouse (0.2 ml) was injected to induce ovulation. At the same time, pure water was given by gavage at 11:00 every day at the beginning of the first cycle.

[0028] (2) R4 group (4 ovulation inductions): PMSG 10 IU (0.2 ml) was intraperitoneally injected at 7:00 PM on the first day of each cycle. 48 hours later, HCG 10 IU / head (0.2 ml) was injected to induce ovulation. The above hormone injection process was repeated every 5 days for four cycles. At the same time, pure water was gavage administered at 11:00 AM every day at the beginning of the first cycle.

[0029] (3) Group L (low-dose Niu Da Li group): The hormone injection process was the same as that of Group R4, and low-dose Niu Da Li (400 mg / kg) was given by gavage at 11:00 every day at the beginning of the first cycle.

[0030] (4) Group H (high-dose Niu Da Li group): The hormone injection process was the same as that of Group R4, and high-dose Niu Da Li (800 mg / kg) was given by gavage at 11:00 every day at the beginning of the first cycle.

[0031] 15 hours after the last round of superovulation HCG injection, the mouse ovaries were collected and made into frozen sections and serum according to conventional processes for subsequent tests.

[0032] This example illustrates HE staining.

[0033] Heat-fix the frozen sections on a 37°C slide table for 3 minutes, then fix them in 4% paraformaldehyde solution for 10 minutes. Rinse with distilled water and stain with hematoxylin for 3-5 minutes. Rinse excess stain with tap water and rinse again with distilled water. Differentiate with hydrochloric acid for 3 seconds, rinse with distilled water, and bluing with ammonia for 1 minute, then rinse with distilled water. Place the slides in 50% alcohol, 70% alcohol, 80% alcohol, and 90% alcohol for 1 minute each. Stain with eosin for 20 seconds, dehydrate with anhydrous ethanol for 30 seconds twice, and then clear with xylene for 2-3 minutes before mounting with neutral gum. Allow to air dry and photograph under a microscope.

[0034] In order to observe whether repeated superovulation affects the histological structure of the ovary and the development of follicles, we used HE staining to stain and observe the ovarian sections of different groups of mice. The results are as follows: Figure 1As shown in the results, we can observe more developing large follicles in the ovaries of mice that have only been superovulated once. However, as the number of superovulation increases, the number of developing large follicles in the ovaries of mice that have undergone four rounds of superovulation decreases significantly. This suggests that after the ovaries have undergone multiple controlled stimulations, normal follicular development is affected, which may adversely affect the number and quality of subsequent ovulation. The number of large follicles in the ovaries of mice in the H group increased significantly compared to the R4 group, indicating that Niu Dali has a rescuing effect on the follicular development arrest caused by repeated superovulation in mouse ovaries.

[0035] This example illustrates an enzyme-linked immunosorbent assay (ELISA).

[0036] Mouse serum samples were tested using Shanghai ELISA kits for mouse FSH and LH. The original standard was serially diluted to 40 ng / L, 20 ng / L, 10 ng / L, 5 ng / L, and 2.5 ng / L. 40 μl of sample diluent was added to the test sample wells, followed by 10 μl of the test sample, and gently shaken to mix. The standard sample was added to the blank control wells at the corresponding concentration. No sample was added to the blank control wells. The plate was sealed with film and incubated in a 37°C oven for 30 minutes. After removing the film, the liquid was discarded and the wells were shaken dry. 250 μl of wash solution was added to each well, allowed to stand for 30 seconds, and then discarded. Repeat this wash five times. 50 μl of enzyme-labeled reagent was added to the standard sample wells and the test sample wells. No sample was added to the blank control wells. A new seal was applied to the plate and incubated in a 37°C oven for 30 minutes. After removing from the oven, the liquid was discarded and shaken dry. The wells were then washed five times with wash solution as described above. Add 50 μl of Color Developing Reagent A to each well, followed by 50 μl of Color Developing Reagent B. Gently shake to mix thoroughly, then incubate in a dark oven at 37°C for 10 minutes. Terminate the reaction by adding 50 μl of Stop Solution to each well. Within 15 minutes, measure the OD of each well at 450 nm using a microplate reader. Use a blank well as a zero value to calculate the sample concentration.

[0037] In the later stages of development, insufficient secretion of follicle-stimulating hormone (FSH) in females may also lead to atretic follicles. Considering that FSH and luteinizing hormone (LH) play an important role in folliculogenesis, we used ELIASA kits to detect the serum FSH and LH levels of mice in each group. The results showed that the serum FSH and LH levels in the R4 group were significantly higher than those in the R1 group, while the hormone levels in the Niu Dali treatment group rebounded, among which the L group (low-dose group) had a more significant effect ( Figure 2A, B). Progesterone secreted by the ovaries has a negative feedback effect on the hypothalamus and pituitary gland, inhibiting the secretion of gonadotropins. Excessive progesterone indicates an early onset of a latent LH surge, which may affect oocyte quality and lead to a decrease in embryonic development potential. Therefore, we tested serum progesterone levels by ELISA and found that the serum progesterone level of mice in the R4 group was significantly higher than that in the R1 group, while the progesterone level in the H group was significantly lower than that in the R4 group ( Figure 2 C). This indicates that Niu Dali is beneficial for restoring abnormal changes in hormone levels after repeated superovulation.

[0038] This example illustrates immunofluorescence staining.

[0039] Frozen sections were heat-fixed on a 37°C slide block for 3 minutes, followed by fixation in 4% paraformaldehyde for 10 minutes. Following fixation, sections were perforated three times in PBST for 5 minutes. Tissue circles were circled with a histochemical pen, and blocking solution was applied to the sections before incubation at 37°C for 30 minutes. Primary antibodies were then applied to sections containing caspase 3 (1:200, rabbit), MHC II (1:500, rat), F480 (1:500, rat), CD206 (1:500, rat), and FOXO3a (1:200, rabbit) and incubated overnight at 4°C. The following day, the primary antibodies were removed by washing with 0.1% Triton-X 100 in PBST three times for 5 minutes. Secondary antibodies (1:200) and 4',6-diamidino-2-phenylindole (DAPI, 1:1000) were applied in blocking solution and incubated in the dark for 40 minutes. The tissue surface was washed with PBS to remove excess secondary antibody and DAPI for 5 minutes three times. The slides were then mounted with antifade reagent and photographed using a fluorescence microscope.

[0040] Monocyte-derived macrophages (moMΦ) can polarize into two phenotypes of macrophages, M1 and M2. M1 macrophages can secrete proinflammatory cytokines and chemokines such as IL-1β and TNF-α, present antigens, and thus participate in positive immune responses and play a role in immune monitoring; M2 macrophages are mainly divided into anti-inflammatory cytokines such as IL-10 and TGF-β, which have the functions of reducing inflammation, promoting tumor growth and immunosuppression. The ovulation surge caused by gonadotropins will cause a corresponding inflammatory response in mature follicles. In order to understand whether repeated super-accelerated ovulation induced by PMSG and hCG will aggravate the inflammatory response in the ovaries and induce the recruitment of macrophages, we used immunofluorescence technology to detect macrophages in the ovaries. The test results showed that there was no significant difference in the total macrophage marker molecule F480 and the M2 macrophage marker molecule CD206 between different groups ( Figure 3A, B), while the number of positive cells labeled with MHCII, a marker molecule of M1 macrophages, increased significantly in the R4 group, indicating that repeated superovulation may induce an aggravation of the inflammatory response, thereby causing macrophages to polarize toward the M1 type and be recruited in the ovaries. The expression of MHCII in the ovaries of mice in the high-dose Niu Da Li group was significantly lower than that in the R4 group ( Figure 3 C). The results suggest that Rhizoma Niu Dali may prevent the occurrence of inflammatory response to a certain extent and reduce the polarization of macrophages to M1 type.

[0041] FOXO3a is an important transcription factor in follicle activation. It plays an important role in maintaining the number of primordial follicles, the physiological function of ovarian reserves, and female fertility. FOXO3a protein regulates follicle growth and atresia by promoting apoptosis of granulosa cells and oocytes in the mammalian ovary. In activated primordial follicles, FOXO3a is expressed in the cytoplasm of oocytes. Due to the limited number of follicles in the follicular pool in the ovary, premature and irreversible activation of the primordial follicle pool will lead to accelerated depletion of the resting follicle reserve, resulting in premature ovarian failure in mice. Through immunofluorescence detection, we found that the expression of FOXO3a in the R4 group was significantly higher than that in the R1 group, while the positive expression of FOXO3a was significantly downregulated in the high-dose Niu Da Li group ( Figure 3 D), which indicates that Rhizoma Dioscoreae can reduce the activation of FOXO3a induced by repeated superovulation, thereby slowing down the depletion of primordial follicles in the primordial follicle pool and thus slowing down the aging process of the ovary.

[0042] We also used immunofluorescence detection to detect caspase3, a marker of apoptosis in the ovaries. The results showed that the expression of caspase3 in the R4 group was significantly increased, while the positive signal of caspase3 in the ovaries of mice in the Niu Dali-administered group was significantly downregulated compared with the R4 group, indicating that Niu Dali has a positive effect on reducing the apoptosis of granulosa cells caused by repeated superovulation ( Figure 3 E).

[0043] This example illustrates real-time fluorescence quantitative PCR.

[0044] In this experiment, Trizol was used for RNA extraction. 500 μl of Trizol was added to every 30 mg of tissue, and the tissue was homogenized using a homogenizer on ice. The supernatant was taken for subsequent experiments. 500 μl of Trizol was added to each well of a 12-well plate for cell extraction. 100 μl of chloroform was added to the supernatant and shaken vigorously to mix the Trizol and chloroform thoroughly. The supernatant was allowed to stand at room temperature for 2-3 minutes to allow for stratification, followed by centrifugation at 13,000 rpm and 4°C for 15 minutes. 200 μl of the supernatant was transferred to a new 1.5 ml centrifuge tube, and an equal volume of isopropanol was added, mixed gently, and allowed to stand at room temperature for 10 minutes to allow the RNA to precipitate. Centrifuge at 13,000 rpm at 4°C for 15 minutes, discard the supernatant, then wash with 700 μl of ice-cold 75% ethanol, vortex, and centrifuge at 13,000 rpm at 4°C for 5 minutes. Repeat twice, discarding the alcohol. Centrifuge at 13,000 rpm at 4°C for 2 minutes, aspirate the remaining ethanol, and air-dry at room temperature to allow the ethanol to evaporate. Add an appropriate amount of DEPC water to dissolve the precipitate, vortex to mix, and measure RNA concentration using a photometer. Vazyme's HiScript II Q RT SuperMix for qPCR Reverse Transcription Kit was used, using 4 μl of 5× HiScript II Q RT SuperMix and 12 μl of RNA sample. Reverse transcription conditions were: 50°C for 15 minutes, 85°C for 5 seconds, and 4°C forever. After reverse transcription, the sample was stored at -20°C until use.

[0045] The experiment was performed using the Vazyme ChamQ SYBR qPCR Master Mix kit. The reaction system was as follows: 3 μL of cDNA template, 1 μL of upstream primer, 1 μL of downstream primer, and 5 μL of SYBR qPCR Master Mix. The reaction conditions were: initial denaturation at 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and 60°C for 35 s. Fluorescence signals were collected at 60°C. Results were analyzed based on melting curves.

[0046] To further validate the effect of Niu Dali on reducing granulosa cell apoptosis in ovarian cells, KGN cells, a human ovarian granulosa cell line, were used in vitro for validation. RT-PCR results showed that H2O2 treatment significantly upregulated the mRNA expression levels of apoptosis markers caspase3 and Bax, while Niu Dali-treated KGN cells showed a decrease in their mRNA expression levels. The effect was more pronounced with a high-dose Niu Dali pretreatment. Figure 4 A and B).

[0047] This example illustrates the TUNEL assay.

[0048] Heat fix the frozen sections with the tissue samples on a 37°C hotplate for 5 minutes. Then, immerse the slides in 4% paraformaldehyde solution and fix them at room temperature for 30 minutes. After fixation, wash the slides three times with PBS, each for 3-5 minutes. Use a histochemical crayon to draw a wax circle on the slide to enclose the tissue. Add 2% Triton X-100 permeabilization reagent inside the wax circle and perforate for 10 minutes. Block the slides with 5% BSA in PBS for 1 hour at room temperature. After blocking, wash the slides three times with PBS, each for 3-5 minutes. Add 1x Equilibration Buffer to the wax circle and equilibrate at room temperature for 20 minutes. Then, add TdT working solution and incubate the slides in a humidified chamber at 37°C for 60 minutes. After incubation, wash the slides three times with PBS, each for 3-5 minutes. Mount the slides with antifade reagent and photograph under a fluorescence microscope.

[0049] The growth factors and nutrients required for oocyte growth and maturation come from granulosa cells. Studies have found that repeated ovarian stimulation promotes apoptosis of granulosa cells and has a negative impact on ovarian cell function. Repeated superovulation increases the expression of apoptosis-related genes (Bax, caspase-3) in granulosa cells, disrupting the balance between granulosa cell growth and death. In order to explore whether Niu Dali can rescue the apoptosis of granulosa cells in ovarian antral follicles induced by repeated superovulation, we performed TUNEL staining on ovarian frozen sections. The results showed that there were more TUNEL-positive cells in the R4 group than in the R1 group (P<0.01), indicating that ovarian hyperstimulation led to increased cell apoptosis, while high-dose Niu Dali treatment significantly reduced the percentage of TUNEL-positive cells (P<0.05) ( Figure 5 A and B).

[0050] This example illustrates KGN cell culture and treatment.

[0051] KGN cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin-streptomycin) in an incubator at 37°C and 5% CO2.

[0052] Construction of H2O2-induced cell apoptosis model: The control group and H2O2 modeling group were cultured with DMEM / F12 medium containing 2% fetal bovine serum (FBS) and 1% double antibody (penicillin-streptomycin), respectively. The whole extract powder of Herba Lysimachiae was dissolved in DMEM / F12 and the bacteria were filtered using a 0.22μm filter membrane. The L group was pretreated with 2% DMEM / F12 containing 200μg / ml Herba Lysimachiae for 24h, and the H group was pretreated with 2% DMEM / F12 containing 400μg / ml Herba Lysimachiae. After 24h, the cells were treated with 200μM H2O2 for 4h to induce cell apoptosis to detect whether Herba Lysimachiae could slow down cell apoptosis.

[0053] Cells were plated onto 24-well plates. After cell attachment and treatment, the culture medium was discarded from the wells, the plates were washed three times with PBS, and fixed with 500 μl of 4% paraformaldehyde (PFA) solution for 10 min. The PFA was discarded, and the plates were washed three times with PBS (5 min each). The wells were then perforated with PBS containing 0.1% Triton X-100 (PBST) for 15 min at room temperature, followed by three washes with PBS (5 min each). Blocking buffer was then added for 1 hour at room temperature, followed by the addition of the primary antibody caspase 3 (1:200, rabbit) and incubation overnight at 4°C. The next day, the primary antibody was removed, the plates were washed three times with PBS for 5 min, and the plates were then incubated with a secondary antibody (1:200) and DAPI (1:1000) in blocking buffer. The plates were incubated for 30 min at room temperature in the dark, and then washed three times with PBS (5 min each) to remove any residual secondary antibody. Drop anti-fluorescence quenching agent on a clean glass slide, pick out the slide with tweezers, turn it upside down on the glass slide, seal it, and take pictures with a fluorescence microscope.

[0054] In addition, under the same treatment, cell immunofluorescence results showed that the caspase3 fluorescence expression intensity of KGN cells treated with Niu Da Li was significantly lower than that of cells induced apoptosis by H2O2 alone ( Figure 6 ), which further verified that Niu Dali can effectively alleviate granulosa cell apoptosis.

[0055] The Herba Lycopodii extract of the present invention has the effect of improving sexual function, specifically can solve the problem of decreased ovarian reserve capacity due to repeated superovulation; the Herba Lycopodii extract can effectively alleviate follicular hypoplasia, prevent abnormal changes in serum hormones caused by repeated superovulation, and reduce the activation of primordial follicles, thereby slowing the process of ovarian aging. Not only that, the Herba Lycopodii extract also effectively slows down cell apoptosis in the ovaries and the polarization of macrophages to pro-inflammatory M1 macrophages, plays a protective role in the survival and maturation of oocytes, thereby protecting the reserve capacity of the ovaries. The present invention develops a new function of Herba Lycopodii, providing a new approach for the development of drugs to improve sexual function.

Claims

1. A method for preparing a drug for treating reduced ovarian reserve in mice using a Herba Lycopodii extract, wherein the Herba Lycopodii is the dried root of the Herba Lycopodii plant of the genus Welch, Leguminosae. The method for preparing the Herba Lycopodii extract comprises the following steps: Fresh Herba Cynanchii raw materials were cleaned and sliced into slices with a diameter of 0.5-2 cm using a slicer to obtain Herba Cynanchii finely crushed products. Purified water was added 7 times by mass for secondary crushing to obtain Herba Cynanchii finely crushed pulp and finely crushed residues. The finely crushed pulp was supplemented with 3 times by mass of water to make up the extraction amount, and emulsified and stirred in an emulsifying homogenization tank at a circulating pressure of 0.1-0.2 MPa. The Herba Cynanchii homogenate was collected and the obtained homogenate was centrifuged at a speed of 3800 rpm / min to obtain Herba Cynanchii separated liquid and separated residues. Then, 10 times by mass of 55% ethanol was added to the separated residue, and low-temperature high-pressure difference continuous extraction was performed under the conditions of a pressure of 25 MPa and a temperature of 30°C. After centrifugation at a speed of 3800 rpm / min, Herba Cynanchii alcohol solution was obtained. 500 The Herba Dioscoreae extract and the alcohol solution are respectively microfiltered using a 1000 nm microfiltration membrane. The separated solution and the alcohol solution after microfiltration are sequentially subjected to membrane concentration, vacuum concentration, and plate and frame filtration operations to obtain alcohol extract and water extract, respectively. The combined extraction of the two extracts yields the Herba Dioscoreae extract.

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