Traditional Chinese medicine composition for improving spleen-kidney deficiency type ovarian reserve hypofunction as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition for improving spleen-kidney deficiency type ovarian reserve hypofunction as well as a preparation method and application of the traditional Chinese medicine composition. The traditional Chinese medicine composition provided by the invention comprises the following medicinal materials in parts by weight: semen cuscutae, herba taxilli, radix dipsaci, wine-processed fructus ligustri lucidi, rhizoma dioscoreae, bulbus lilii and herba lycopi. The traditional Chinese medicine composition for improving spleen-kidney deficiency type ovarian reserve hypofunction (DOR) is suitable for improving the ovarian reserve function, adjusting the sex hormone level and relieving symptoms such as soreness and weakness of waist and knees, insomnia and dreaminess and the like related to the DOR, and belongs to a traditional Chinese medicine innovative preparation in the field of gynecological reproductive health.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, specifically relating to a traditional Chinese medicine composition for ovarian reserve dysfunction caused by spleen and kidney deficiency, its preparation method and application. Background Technology

[0002] Diminished ovarian reserve (DOR) is a common gynecological disease that refers to a decrease in the number or quality of oocytes in the ovaries, accompanied by a decrease in anti-Müllerian hormone (AMH) and an increase in follicle-stimulating hormone (FSH), leading to a decline in fertility. Its incidence is increasing year by year with increasing social pressure, seriously affecting women's physical and mental health.

[0003] Currently, there are no specific drugs in Western medicine to improve ovarian reserve function in the treatment of DOR. For women diagnosed with DOR who wish to conceive, common treatment methods include ovulation induction, assisted reproductive technology (ART), and hormone replacement therapy. However, existing treatment methods have certain limitations, mainly: ovulation induction only intervenes in follicle development within a single cycle and cannot improve ovarian reserve function, while potentially leading to risks such as ovarian hyperstimulation syndrome (OHSS) and multiple pregnancies; hormone replacement therapy has limitations such as increased tumor risk and first-pass effect; and adjuvant medications (such as coenzyme Q10) only play an auxiliary role with limited efficacy.

[0004] Traditional Chinese medicine treatment adheres to the core concept of "tonifying the kidney and strengthening the spleen," but most existing Chinese patent medicines do not have a clear indication for "DOR of spleen and kidney deficiency," and cannot specifically improve the complex symptoms of DOR patients such as insomnia, dreaminess, and anxiety. Traditional decoctions have problems such as complicated decoction preparation, inaccurate dosage, and low patient compliance, which limit the clinical application effect. Summary of the Invention

[0005] The purpose of this invention is to provide a traditional Chinese medicine composition for improving ovarian reserve (DOR) of spleen and kidney deficiency type. This composition is suitable for improving ovarian reserve function, regulating sex hormone levels, and relieving symptoms such as lower back and knee pain, insomnia and dreaminess related to DOR. It belongs to the innovative traditional Chinese medicine in the field of gynecological reproductive health.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a traditional Chinese medicine composition comprising the following medicinal materials in parts by weight: 6-12 parts of Cuscuta chinensis, 9-15 parts of Taxillus chinensis, 9-15 parts of Dipsacus asper, 6-12 parts of Ligustrum lucidum, 15-30 parts of Dioscorea opposita, 6-12 parts of Lilium brownii, and 6-12 parts of Lycopus lucidus.

[0008] The traditional Chinese medicine composition comprises the following medicinal materials in parts by weight: 12 parts of Cuscuta chinensis, 12 parts of Taxillus chinensis, 12 parts of Dipsacus asper, 10 parts of Ligustrum lucidum, 10 parts of Dioscorea opposita, 12 parts of Lilium brownii, and 9 parts of Lycopus lucidus.

[0009] Secondly, the present invention provides a traditional Chinese medicine composition, which is extracted from the following medicinal materials: 6-12 parts of Cuscuta chinensis, 9-15 parts of Taxillus chinensis, 9-15 parts of Dipsacus asper, 6-12 parts of Ligustrum lucidum, 15-30 parts of Dioscorea opposita, 6-12 parts of Lilium brownii, and 6-12 parts of Lycopus lucidus.

[0010] Preferably, the traditional Chinese medicine composition is extracted from the following medicinal materials: 12 parts of Cuscuta chinensis, 12 parts of Taxillus chinensis, 12 parts of Dipsacus asper, 10 parts of Ligustrum lucidum, 10 parts of Dioscorea opposita, 12 parts of Lilium brownii, and 9 parts of Lycopus lucidus.

[0011] The extraction is carried out as follows: Mix the medicinal materials, add 6-10 times the amount of water, preferably 8 times the amount of water, soak for 0.5-1.5 hours, extract 1-3 times, concentrate, and dry to obtain the final product.

[0012] The concentration conditions are: reduced pressure concentration, concentration temperature of 60-80℃, and concentration relative density of 1.06-1.07 (50℃).

[0013] The drying conditions are as follows: vacuum drying is used, the drying temperature is 60-80℃, and the hyperoside retention rate is ≥90%.

[0014] Thirdly, the present invention provides a traditional Chinese medicine preparation comprising the above-mentioned traditional Chinese medicine composition.

[0015] The dosage form of the traditional Chinese medicine preparation is one of the following: granules, decoction, capsules, pills, powders, tablets, and oral liquids.

[0016] Fourthly, the present invention provides a granule comprising: an active ingredient and excipients; The active ingredient is the traditional Chinese medicine composition; the excipients are dextrin, soluble starch, and maltodextrin; dextrin is preferred.

[0017] The mass ratio of the active ingredient to the excipients is (1-5):1, and granules can be formed within this range. Based on experience, the higher the proportion of excipients, the easier the granulation process; therefore, formulations can be formed when the proportion of dextrin is at least 16.7%.

[0018] The granules also include a wetting agent; the wetting agent is 80%-90% ethanol.

[0019] The granules are produced using a wet granulation process.

[0020] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) Prescription innovation: The inventor's prescription follows the principle of "tonifying the kidney and strengthening the spleen as the main method and promoting blood circulation and calming the mind as the auxiliary method". The prescription not only strengthens the tonification of the kidney and spleen to improve ovarian reserve function, but also improves sleep and mood through the synergistic effect of lily. It solves the "single effect" defect of existing drugs with similar effects, and achieves the dual effects of "ovarian protection + sleep aid". It is suitable for the complex symptoms of DOR patients, with definite curative effect and high safety.

[0021] (2) Dosage optimization: The present invention adjusts the dosage of Cuscuta chinensis from 15g to 12g in the existing technology, and removes the redundant medicinal taste of Glehnia littoralis and Leonurus japonicus, which reduces the cost of medication and reduces the risk of "stagnation due to tonification". Combined with toxicological verification, it is more suitable for long-term use by DOR patients (no adverse reactions in clinical application).

[0022] (3) Stable process: The extraction and molding processes optimized by orthogonal experiments make the hyperoside transfer rate and granule yield better than the traditional process, and the heavy metal content is <7ppm and the arsenic salt content is <0.9ppm, with the quality control level higher than the industry standard; the granule dosage form solves the problems of cumbersome decoction and inaccurate dosage of traditional decoction, which can improve patient compliance. Attached Figure Description

[0023] Figure 1 Cell morphology images of vaginal smears from rats at different stages of the estrous cycle.

[0024] Figure 2 Photos of the ovaries and uterus of rats in each group after administration of the drug.

[0025] Figure 3 Effects of BA2023007 intermediate on ovarian follicles in DOR rats (HE, 200). Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0029] Example 1 This embodiment provides a traditional Chinese medicine composition, which, based on the daily dosage of raw herbs, has the following formula: Cuscuta chinensis 12g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum 10g, Dioscorea opposita 10g, Lilium brownii 12g, and Lycopus lucidus 9g.

[0030] All of the above-mentioned medicinal ingredients meet the standards of Part I of the 2020 edition of the Chinese Pharmacopoeia. There are no toxic medicinal materials, no incompatibilities of the Eighteen Incompatibilities or Nineteen Antagonisms, and the dosage of each medicinal ingredient does not exceed the upper limit stipulated in the Pharmacopoeia.

[0031] Example 2 This embodiment provides a method for preparing the traditional Chinese medicine composition described in Example 1, and the specific steps are as follows: (1) Raw material pretreatment: All medicinal ingredients were treated in accordance with the requirements of Part I of the 2020 edition of the Chinese Pharmacopoeia. Cuscuta chinensis, the principal ingredient, was crushed and then decocted.

[0032] (2) Extraction process: Add 8 times the amount of water, extract 3 times, 1 hour each time.

[0033] (3) Concentration: The extract is concentrated using the commonly used vacuum concentration method. The concentration temperature is 60~80℃ (specifically 70℃), and the concentration relative density is set to 1.06-1.07 (50℃).

[0034] (4) Drying process: The vacuum drying method is adopted, and the drying temperature is set at 60-80℃ to ensure that the hyperoside retention rate is ≥90% and dry extract powder is obtained.

[0035] Example 3 This embodiment provides a granule formulation, the specific steps of which are as follows: wet granulation is used, with dextrin as an excipient. The ratio of the dry extract powder obtained in Example 2 to dextrin is (1-5):1. 80%-90% ethanol is selected as the wetting agent to form granules. The packaging specification is 12g / bag, and the yield of the finished product is ≥90%.

[0036] Investigation Experiment 1. Evaluation of the application effect of the formula Comparative Example 1 The clinical efficacy evaluation of the kidney-tonifying and fertility-aiding formula for treating recurrent miscarriage (2017) records a traditional Chinese medicine composition with the following formula: Cuscuta chinensis 15g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum 10g, Dioscorea opposita 12g, Lilium brownii 10g, Lycopus lucidus 12g, Adenophora stricta 12g, and Leonurus japonicus 10g.

[0037] In clinical application, the prescription in Comparative Example 1 has the effects of tonifying the kidney and spleen, promoting blood circulation and calming the mind, and nourishing yin. For patients with recurrent spontaneous abortion (RSA) due to kidney deficiency and deficiency heat syndrome, it can effectively relieve their core symptoms such as soreness and weakness of the waist and knees, tinnitus, and shortness of breath. At the same time, it can improve symptoms of deficiency heat such as dry mouth and dry tongue. It plays an important role in regulating the luteal function and hormone levels of RSA patients and improves the success rate of pregnancy preservation in patients with recurrent spontaneous abortion.

[0038] However, the yin-nourishing effect of North American ginseng in the prescription is only suitable for a small number of patients with concurrent deficiency-heat syndrome, and the suitable population is relatively narrow. Those with weak spleen and stomach are prone to abdominal distension after taking it. Moreover, patients who want to have children often do not use contraception. If an unplanned pregnancy occurs during the medication period and is not detected in time, the motherwort in the prescription has a blood-activating effect, which poses a risk of harming the fetus.

[0039] Comparative Example 2 The clinical study on the effect of kidney-tonifying and fertility-enhancing formula on endometrial receptivity in patients with recurrent miscarriage due to kidney deficiency (2020) records a traditional Chinese medicine composition with the following formula: Cuscuta chinensis 15g, Dipsacus asper 12g, Ligustrum lucidum 10g, Dioscorea opposita 12g, Lilium brownii 10g, Citrus aurantium 12g, Lycopus lucidus 12g, and Leonurus japonicus 15g.

[0040] In clinical application, the prescription in Comparative Example 2 has the effects of tonifying the kidney and spleen, soothing the liver and regulating qi, promoting blood circulation and calming the mind. It increases the dosage of motherwort to promote blood circulation and adds bitter orange to soothe the liver and relieve depression. It is especially suitable for patients with kidney deficiency type RSA, especially those with blood stasis (menstrual blood clots, lower abdominal distension) and liver depression. It can improve the menstrual cycle, increase menstrual flow, redder menstrual color and reduce blood clots in patients with kidney deficiency type RSA. It can significantly improve the TCM symptoms of kidney deficiency such as soreness and weakness of the waist and knees, increase the thickness of the endometrium, significantly improve the endometrial type and the distribution of blood flow in and under the endometrium, and significantly reduce the RI and PI of uterine artery blood flow.

[0041] However, it also has significant drawbacks: First, the core medicinal ingredient is missing, as the mulberry mistletoe, which "nourishes the liver and kidneys and strengthens tendons and bones," is removed, resulting in a weakening of its core efficacy in tonifying the kidneys and consolidating essence. Second, the accessibility of the medicine is poor, as the herb bitter orange flower is often difficult for patients from other regions to obtain conveniently, affecting the actual promotion and application of the prescription. Third, many patients with fertility needs do not use contraception, and may become pregnant unexpectedly during the medication period without knowing their situation, as motherwort may pose a risk of promoting blood circulation and harming the fetus.

[0042] Comparative Example 3 The following two papers, "Analysis of the Molecular Mechanism of Kidney-Tonifying and Fertility-Assisting Formula in the Treatment of Recurrent Miscarriage" (2021) and "Clinical Study on the Influence of Kidney-Tonifying and Fertility-Assisting Formula on Endometrial Receptivity and Pregnancy Outcome in Patients with Recurrent Miscarriage" (2024), describe a traditional Chinese medicine composition with the following formula: Cuscuta chinensis 15g, Taxillus chinensis 12g, Dipsacus asper 15g, Ligustrum lucidum 10g, Lilium brownii 10g, Dioscorea opposita 12g, Lycopus lucidus 12g, and Leonurus japonicus 15g.

[0043] In actual treatment, the prescription in Comparative Example 3 has the effects of tonifying the kidney and spleen, promoting blood circulation and calming the mind. The dosage of Dipsacus asperoides is increased and Loranthus parasiticus is retained, which makes the kidney-tonifying effect more prominent. It can improve the menstrual condition and endometrial receptivity of patients with kidney deficiency type RSA. It can significantly improve symptoms of kidney deficiency such as soreness of the waist and knees, dizziness and tinnitus, and increase the clinical pregnancy rate of RSA patients.

[0044] However, many patients who wish to have children do not use contraception and may become pregnant unintentionally while taking the medication without knowing it. Motherwort may pose a risk of promoting blood circulation and harming the fetus.

[0045] Application and Effect Description: The prescriptions in Comparative Examples 1-3 mentioned above, as "kidney-tonifying and fertility-aiding formulas," have been used clinically (from 2017 to 2024) with certain effects. Their core advantages are concentrated in three aspects: first, they have a clear effect on tonifying the kidney and strengthening the spleen, effectively improving symptoms related to kidney deficiency; second, they optimize endometrial receptivity, increasing endometrial thickness and improving blood perfusion; and third, they can regulate pregnancy hormone levels, improving pregnancy-related indicators in RSA patients. However, all three prescriptions contain Leonurus japonicus, posing a risk of miscarriage if unintended pregnancy occurs during medication for patients trying to conceive; additionally, non-core ingredients such as Adenophora stricta and Citrus aurantium increase the complexity of the prescriptions and limit the suitable patient population; furthermore, all three prescriptions are designed for RSA patients and do not consider the treatment needs of similar kidney deficiency-related diseases such as diminished ovarian reserve (DOR).

[0046] Compared with Comparative Examples 1-3, Example 1 retains the core "kidney-tonifying" effect of Comparative Examples 1-3, and makes the following key optimizations targeting the core pathogenesis of DOR due to spleen and kidney deficiency, kidney deficiency and essence depletion, and spleen dysfunction leading to ovarian malnourishment: ① Refined dosage adjustment to improve long-term safety: The dosage of Cuscuta chinensis was adjusted to 12g, Dipsacus asperoides to 12g, and Lycopus lucidus to 9g, which improves the safety of long-term use and better meets the needs of patients for long-term conditioning.

[0047] ② The formula is streamlined and optimized to focus on the core pathogenesis and avoid safety risks: North American ginseng is removed to avoid its greasy and stomach-irritating properties and the limited range of suitable patients; Motherwort is removed to avoid the risk of miscarriage during the preconception period; and the formula is adapted to the preconception needs of DOR patients. The classic kidney-tonifying and spleen-strengthening combination of Loranthus parasiticus, Dipsacus asper, Cuscuta chinensis, and Dioscorea opposita is retained to strengthen the core efficacy of "tonifying the kidney, replenishing essence, and strengthening the spleen".

[0048] ③ Optimize the dosage of key medicinal ingredients and enhance the compatibility with concurrent symptoms: Increase the dosage of lily to 12g to enhance its calming and tranquilizing effects, and specifically improve the emotional problems such as anxiety and insomnia caused by decreased ovarian function in DOR patients.

[0049] 2. Investigation of extraction process An orthogonal experimental design was used, with the transfer rate of hyperoside as the evaluation index, to optimize the three factors affecting extraction efficiency: water addition ratio, extraction time, and extraction times. Three levels were selected for each factor, following the L9(3) model. 4 Experiments were conducted using orthogonal arrays.

[0050] The optimal extraction method was determined by orthogonal experiment: add 8 times the amount of water, extract 3 times, 1 hour each time; the extract was used as the indicator component with hyperoside (a representative component of Cuscuta chinensis), and the transfer rate RSD was <10%, indicating good process stability.

[0051] Table 1 Orthogonal Factor Level Table

[0052] Table 2 Orthogonal Experiment Table

[0053] Table 3 Analysis of Variance Table

[0054] Orthogonal analysis determined the optimal extraction process to be: adding 10 times the amount of water, extracting 3 times, 1 hour each time. The optimal transfer rate combination in the orthogonal experiment was: adding 8 times the amount of water, extracting 3 times, 1 hour each time. Therefore, levels 2 and 3 (adding water multiples) were compared. That is, process conditions A2B2C3 and A3B2C3 were compared.

[0055] Table 4 Comparison and Validation of Extraction Processes

[0056] The results showed that the transfer rates and yields of the two extraction processes were basically the same. Therefore, the extraction process with 8 times the amount of water was selected, and the extraction was performed 3 times, 1 hour each time. Moreover, the RSD of the yield and the RSD of the hyperoside transfer rate were both less than 10%, indicating that the process had good stability and reproducibility.

[0057] 3. Concentration process 3.1 Concentration Temperature Study The extract was concentrated using a common vacuum concentration method, and the effect of different concentration temperatures on the concentration process was investigated. The extract was concentrated under vacuum (vacuum degree not less than -0.08 MPa) at 60℃, 70℃ and 80℃ to a clear extract with a relative density of about 1.07 (50℃). The concentration temperature was optimized using the transfer rate of hyperoside as the indicator component.

[0058] Table 5 Concentration Temperature

[0059] The transfer rates of the index component hyperoside were not significantly different when concentrated under reduced pressure at 60℃, 70℃ and 80℃, respectively, proving that the concentration temperature of 60~80℃ has little effect on the transfer rate of the index component. Taking all factors into consideration, the concentration temperature was determined to be 60~80℃.

[0060] 3.2 Concentrated Relative Density Study The effect of different concentration relative densities on the concentration process was investigated, and the transfer loss rate of the index component hyperoside was used as an indicator to optimize the concentration relative density.

[0061] Table 6 Concentrated Relative Density

[0062] The results above show that when the relative density is 1.05 (50℃), the concentrate is thin and barely adheres to the walls, but the volume of the concentrate is large, which is not conducive to subsequent drying. When the relative density is 1.08 (50℃), the concentrate is relatively viscous, resulting in greater loss due to adhesion to the walls, and is also not conducive to subsequent operations. When the relative density is 1.06~1.07 (50℃), the concentrate is in good condition; therefore, the relative density of the concentrate is determined to be 1.06~1.07 (50℃).

[0063] 4. Drying process The extract was concentrated under reduced pressure at 70℃ to a relative density of 1.07 (50℃). The concentrate was divided into three equal portions and placed in a vacuum drying oven. The portions were then vacuum dried at 60℃, 70℃, and 80℃ (vacuum degree not less than -0.08 MPa). The content of hyperoside was determined, and its transfer rate was calculated. The drying temperature parameters were determined based on the results.

[0064] Table 7 Drying Process

[0065] According to the research results, the hyperoside transfer rate did not differ much at drying temperatures of 60℃, 70℃ and 80℃, so the drying temperature was determined to be 60~80℃.

[0066] 5. Formulation process (1) Screening of excipients: Three commonly used excipients, dextrin, maltodextrin and soluble starch, were investigated. Granulation, particle size and molding rate were used as evaluation indicators to select the best excipients for the formulation.

[0067] Table 8. Investigation of different excipients

[0068] Based on the research results, dextrin was selected as the excipient for the formulation.

[0069] (2) Wetting agent evaluation: Using ethanol of different concentrations as wetting agents, we evaluated three different concentrations of wetting agents: 80%, 85%, and 90%. Granulation conditions, particle size, and molding rate were used as evaluation indicators to select the best formulation wetting agent.

[0070] Table 9. Investigation of wetting agents at different concentrations

[0071] Based on the research results, 80%-90% ethanol was selected as the wetting agent for the formulation.

[0072] (3) Excipient ratio study: Using dextrin as a formulation excipient, the formulation effect of adding different proportions of excipients to dry extract powder was studied. Granulation, particle size, and molding rate were used as evaluation indicators to optimize the formulation excipient ratio.

[0073] Table 10. Investigation of different proportions of excipients

[0074] According to the research results, granules can be formed when the ratio of dry extract powder to excipients is between 5:1 and 1:1. Based on experience, the higher the proportion of excipients, the easier the granulation. Therefore, it is determined that formulations can be formed when the proportion of dextrin is not less than 16.7%.

[0075] Based on the above process research parameters, three batches of pilot-scale samples have been prepared. Each batch was fed with 60 kg of finished product granules, and the yield of all three batches was above 90%. The accelerated testing (6 months) and long-term testing (12 months) results of the three batches of pilot-scale samples all met the requirements of the "Internal Control Quality Standards," and compared with the 0-month test results, there was no significant trend of change. Based on the long-term test results, the shelf life of this product was determined to be 12 months. The research results indicate that the formulation preparation method determined in this invention is feasible, the process is stable, and it is suitable for large-scale production. Following the determined preparation process, the preparation of dry extract powder for pharmacodynamic and toxicological studies has been completed.

[0076] 6. Indications The functions and indications of the traditional Chinese medicine composition provided in Example 1 are: tonifying the kidney and strengthening the spleen, promoting blood circulation and calming the mind. It is used for ovarian reserve deficiency due to deficiency of both spleen and kidney, with symptoms such as soreness and weakness of the waist and knees, dizziness and tinnitus, fatigue, shortness of breath and reluctance to speak, sallow complexion, poor appetite, loose stools, restlessness, insomnia and dreaminess, pale and dark tongue, thin white coating, and deep and weak or slow pulse.

[0077] Effect verification Experiment 1: Effects on ovarian reserve function in DOR rats 1. Experimental materials: 1.1 The dry extract powder of traditional Chinese medicine prepared according to the formula of Example 1 of the present invention (Formula of Example 1) BA2023007 intermediate, batch number: 2408-00042, content: 5.5g crude drug / g dry extract powder, specification: 500g / bag, appearance: dark brown powder; slight odor, slightly bitter taste, provided by Chengdao Zhiji (Beijing) Technology Co., Ltd.

[0078] 1.2 Positive control drug Estradiol Valerate Tablets, Batch No.: 872A, DELPHARM lille SAS product.

[0079] 1.3 Laboratory Animals Eighty SPF-grade female SD rats, weighing 180–220 g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., with Experimental Animal Production License No.: SCXK (Xiang) 2021-0002 and Experimental Animal Quality Qualification No.: 430727251101794557. They were housed in Area D of the Barrier Environment Laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., with Experimental Animal Use License No.: SYXK (Xiang) 2025-0008.

[0080] 1.4 Main Reagents Tripterygium wilfordii polyglycosides, batch number: 20250202, product of Hunan Qianjin Xieli Pharmaceutical Co., Ltd.; isoflurane, batch number: 20240901, product of Jiangsu Hengfengqiang Biotechnology Co., Ltd.; Rat estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) ELISA kit, batch number: Jul2025, product of Huangshi Ains Company.

[0081] 2. Experimental Methods: 2.1 Grouping and Administration Eighty qualified SPF-grade female SD rats were selected. Vaginal exfoliated cells were analyzed to observe the estrous cycle. Rats with normal estrous cycles were randomly divided into a normal control group (n=10) and a model group (n=70). Except for the normal control group, all other groups were administered Tripterygium wilfordii polyglycosides 50 mg / kg by gavage for 14 consecutive days. Vaginal smears were examined daily to observe the estrous cycle. Disorders or prolongation of the estrous cycle were considered successful model establishment. Fifty successfully modeled rats were randomly divided into five groups based on their body weight: a model control group, an estradiol valerate tablet group (0.09 mg / kg), and low, medium, and high dose groups of the dry extract powder prepared in this invention (3.465, 6.93, and 13.86 g crude drug / kg, respectively), with 10 rats in each group. On the day of modeling, each group was administered the drug. The normal control group and the model control group were given an equal volume of pure water by gavage, while the other groups were given the corresponding drug solution by gavage. The administration volume was 10 mL / kg, once a day, for 14 consecutive days. After the last administration, the rats in each group were anesthetized by isoflurane inhalation, and blood was collected from the abdominal aorta to detect the levels of E2, LH, FSH, and AMH in the rat serum. The ovaries and uterus were dissected and weighed, and the organ coefficients of the ovaries and uterus were calculated. Histopathological examination of the ovaries was performed to observe the number of follicles and the histopathological changes in the ovarian tissue.

[0082] 2.2 Dosage setting The proposed clinical dosage of intermediate BA2023007 is 77 g crude drug per day. Based on the conversion of animal and human surface area, the equivalent clinical dose in rats is: 77 g × 0.018 / 0.2 kg. 6.93g crude drug / kg. Based on 0.5 and 2 times the clinically equivalent dose, the low and high doses of the BA2023007 intermediate are calculated as 3.465g crude drug / kg and 13.86g crude drug / kg, respectively. The clinical dose of estradiol valerate tablets is 1mg / tablet / day. Based on the animal and human surface area conversion, the equivalent clinical dose in rats is: 1mg × 0.018 / 0.2kg. 0.09 mg / kg. For details of the experimental dose design and grouping, please refer to Table 11.

[0083] Table 11 Animal grouping and dosage design

[0084] 2.3 Detection Indicators 2.3.1 Weight measurement: The rats were weighed weekly to observe changes in body weight.

[0085] 2.3.2 Monitoring of general condition and estrous cycle in rats: Rats were observed daily for their general condition, including mental state, diet, activity, and coat color, and their body weight was measured weekly to assess improvements in their overall health. During the modeling period, vaginal exfoliated cell smears were prepared and stained. The effect on the estrous cycle of rats was evaluated by observing the vaginal exfoliated cells in each group.

[0086] 2.3.3 Detection of serum E2, LH, FSH, and AMH levels: After the last administration, rats in each group were anesthetized with isoflurane, and blood was collected from the abdominal aorta. The blood was centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The levels of E2, LH, FSH, and AMH in rat serum were detected using an ELISA kit.

[0087] 2.3.4 Ovarian Index and Uterine Organ Coefficient: After the last administration, the rat ovaries and uterus were weighed, and the ovarian and uterine organ coefficients were calculated. Ovarian organ coefficient = ovarian weight / body weight 100%, Uterine Organ Coefficient = Uterine Weight / Body Weight 100%.

[0088] 2.3.5 Histopathological changes in ovarian tissue: After the last administration, ovarian tissue was taken, fixed in 10% formalin, stained with hematoxylin and eosin (HE), and the number of follicles and pathological changes in ovarian tissue were observed.

[0089] 3. Statistical methods The significant figures of the experimental data are rounded to the nearest whole number and in accordance with the following rules: Statistical Processing of Experimental Data (SOP-R-) d-003） Statistical analysis was required. SPSS 23.0 was used as the statistical software. Quantitative data were expressed as mean ± standard deviation (SD). This indicates that Leven's test is used to test for homogeneity of variance. If there is no statistical significance ( P If the variance is greater than 0.05, perform statistical analysis using one-way ANOVA. If the ANOVA is statistically significant (…), then… P If the variance is ≤0.05, use the LSD test (parametric method) for comparative analysis. If the variances are unequal ( P If the Kruskal-Wallis test result is ≤0.05, then the Kruskal-Wallis test is used. If the Kruskal-Wallis test is statistically significant ( P If the result is ≤0.05, then Dunnett's Test (a non-parametric method) is used for comparative analysis. Statistical results are expressed as α. = 0.05 is the test threshold, where P ≤0.05 indicates statistical significance. P ≤0.01 indicates that the difference being tested is highly significant.

[0090] 4 Results 4.1 Observation of the estrous cycle in rats like Figure 1 As shown, the cells in vaginal smears of rats under different estrous cycles exhibited changes. During D12-14, after continuous gavage, the rats in the model group remained in the interphase without any changes in the estrous cycle, suggesting that the modeling was successful.

[0091] 4.2 Effect of BA2023007 intermediate on body weight of DOR rats As shown in Table 12, there was no significant difference in body weight between the rats in the model control group and the normal control group, and there was no significant difference between the drug administration groups and the model control group.

[0092] Table 12 Effects of BA2023007 intermediate on body weight of DOR model rats ( (n=10)

[0093] 4.3 Effects of BA2023007 intermediate on serum E2, LH, FSH, and AMH levels in rats with reduced ovarian reserve As shown in Table 13, compared with the normal control group, the serum E2 and AMH levels of rats in the model control group were significantly reduced ( P ≤0.05), serum FSH levels were significantly elevated ( P ≤0.01). Compared with the model control group, the serum E2 level of rats in the low, medium and high doses of BA2023007 intermediate and estradiol valerate tablet groups was significantly increased ( P ≤0.05 or P ≤0.01), the serum FSH level in rats was significantly reduced (P ≤0.05 or P ≤0.01), serum AMH levels in rats in the high-dose estradiol valerate tablet group were significantly increased ( P ≤0.05).

[0094] Table 13 Effects of BA2023007 intermediate on serum sex hormone levels in DOR rats ( (n=10)

[0095] Note: Compared with the normal control group, + P ≤0.05; compared with the model control group, P ≤0.05, P ≤0.01.

[0096] 4.4 Effects of BA2023007 intermediate on ovarian and uterine organ coefficients in DOR rats As shown in Table 14, Figure 2 As shown, compared with the normal control group, there was no significant difference in the ovarian and uterine organ coefficients of the rats in the model control group, and there was no significant difference in the ovarian and uterine organ coefficients among the drug administration groups.

[0097] Table 14 Effects of BA2023007 intermediate on ovarian and uterine organ coefficients in DOR model rats ( (n=10)

[0098] 4.5 Effects of BA2023007 intermediate on ovarian histopathology in rats with reduced ovarian reserve As shown in Table 15, Figure 3 As shown, compared with the normal control group, the number of follicles in the model control group was significantly reduced ( P ≤0.05). Compared with the model control group, the number of follicles was significantly increased in the estradiol valerate tablet, BA2023007 intermediate medium and high dose groups (≤0.05). P ≤0.05 or P ≤0.01). Compared with the estradiol valerate tablet group, the number of follicles was significantly increased in the BA2023007 intermediate medium-dose group (≤0.01). P ≤0.05).

[0099] Table 15 Effects of BA2023007 intermediate on ovarian follicles in DOR rats ( (n=10)

[0100] Note: Compared with the normal control group, + P ≤0.05; compared with the model control group, P ≤0.01; compared with the estradiol valerate tablet group, # P ≤0.05.

[0101] 5. Summary Low, medium, and high doses of the BA2023007 intermediate significantly increased serum E2 levels and decreased serum FSH levels in rats; high doses of the BA2023007 intermediate significantly increased serum AMH levels in rats. Medium and high doses significantly increased the number of follicles, with the medium dose showing a significantly higher follicle count than estradiol valerate tablets.

[0102] Experiment 2: Effect of BA2023007 intermediate on the synergistic effect of suprathreshold doses of sodium pentobarbital 1. Experimental Materials 1.1 The dry extract powder of traditional Chinese medicine prepared by this invention BA2023007 intermediate, same as Experiment 1.

[0103] 1.2 Positive control Diazepam tablets, batch number: 230202, product of Shandong Xinyi Pharmaceutical Co., Ltd.

[0104] 1.3 Laboratory Animals Fifty SPF-grade ICR mice, weighing 18–22 g, half male and half female, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., Experimental Animal Production License No.: SCXK (Xiang) 2021-0002, Experimental Animal Quality Qualification No.: 430727251101802587. They were housed in Area D of the Barrier Environment Laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., Experimental Animal Use License No.: SYXK (Xiang) 2025-0008.

[0105] 1.4 Main Reagents 0.9% Sodium Chloride Injection, batch number: subject to actual use, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; purified water, self-made by our center; sodium pentobarbital, batch number: 030009008BA00220250108, product of Shanghai Yuansi Standard Technology Co., Ltd. / Shanghai Criminal Science and Technology Research Institute.

[0106] 2. Test Methods 2.1 Grouping and Dosing Fifty qualified ICR mice, half male and half female, were selected and randomly divided into a normal control group, a diazepam tablet group (1.3 mg / kg), and low, medium, and high dose groups of BA2023007 intermediate (5.005, 10.01, and 20.02 g crude drug / kg, respectively), with 10 mice in each group. The mice were administered the drugs by gavage for 7 consecutive days. Thirty minutes after the last administration, each group of mice was injected intraperitoneally with a suprathreshold dose of sodium pentobarbital of 35 mg / kg. The sleep latency and sleep time of the experimental animals in each group were then measured.

[0107] 2.2 Dosage setting The proposed clinical dosage of intermediate BA2023007 is 77 g crude drug per day. Based on the animal and human surface area conversion, the equivalent dose for mice is: 77 g crude drug × 0.0026 / 0.02 kg. 10.01 g crude drug / kg. Based on 0.5 and 2 times the clinically equivalent dose, the low and high doses of the BA2023007 intermediate were determined, i.e., 5.005 g crude drug / kg and 20.02 g crude drug / kg, respectively. The clinical dose of diazepam is 10 mg / day, and the mouse equivalent dose calculated based on body surface area is 10 mg. 0.0026 / 0.02kg 1.3 mg / kg. For details of the experimental dosage design and grouping, please refer to Table 16.

[0108] Table 16 Animal grouping and dosage design

[0109] 2.3 Detection Indicators 2.3.1 Sleep latency: The sleep latency of each group of mice was recorded. Evaluation criteria: Sleep latency refers to the time from the injection of suprathreshold dose of sodium pentobarbital to the disappearance of the righting reflex.

[0110] 2.3.2 Sleep time: The sleep time of each group of mice was observed and recorded. The evaluation criteria were: sleep duration refers to the time from the disappearance of the righting reflex to the recovery of the righting reflex.

[0111] 3. Statistical methods: Same as Experiment 1.

[0112] 4. Experimental Results 4.1 Effect of BA2023007 intermediate on the synergistic effect of suprathreshold doses of sodium pentobarbital Compared with the normal control group, the latency to sleep was significantly shortened in mice in the diazepam tablet, BA2023007 intermediate medium and high dose groups. P ≤0.05), significantly prolonging sleep onset time ( P ≤0.01).

[0113] Table 17. Effect of BA2023007 intermediate on the synergistic effect of suprathreshold doses of pentobarbital sodium. ( (n=10)

[0114] Note: Compared with the normal control group, + P ≤0.05, ++ P ≤0.01; compared with the diazepam group, # P ≤0.05, ## P ≤0.01.

[0115] 5. Summary Medium and high doses of the BA2023007 intermediate can significantly shorten the sleep latency and prolong sleep time in mice.

[0116] Experiment 3: Effect of BA2023007 intermediate on the hypnotic effect of subthreshold doses of sodium pentobarbital 1. Experimental Materials 1.1 The dry extract powder of traditional Chinese medicine prepared by this invention BA2023007 intermediate, same as Experiment 1.

[0117] 1.2 Positive control Diazepam tablets, batch number: 230202, product of Shandong Xinyi Pharmaceutical Co., Ltd.

[0118] 1.3 Laboratory Animals Fifty SPF-grade ICR mice, weighing 18–22 g, half male and half female, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., with Experimental Animal Production License No.: SCXK (Xiang) 2021-0002 and Experimental Animal Quality Qualification No.: 430727251101802653. They were housed in Area D of the Barrier Environment Laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., with Experimental Animal Use License No.: SYXK (Xiang) 2025-0008.

[0119] 1.4 Main Reagents 0.9% Sodium Chloride Injection, batch number: subject to actual use, product of Hunan Kangyuan Pharmaceutical Co., Ltd.; purified water, self-made by our center; sodium pentobarbital, batch number: 030009008BA00220250108, product of Shanghai Yuansi Standard Technology Co., Ltd. / Shanghai Criminal Science and Technology Research Institute.

[0120] 2. Test Methods 2.1 Grouping and Dosing Fifty SPF-grade ICR mice that passed quarantine were selected, with half males and half females. They were randomly divided into a normal control group, a diazepam group (1.3 mg / kg), and low, medium, and high dose groups of BA2023007 intermediate (5.005, 10.01, and 20.02 g crude drug / kg, respectively), with 10 mice in each group. The mice in each group were administered the drug by gavage. The normal control group was given pure water, while the mice in the other groups were given the corresponding drug solution. The administration was once a day for 7 consecutive days. 30 minutes after the last administration, the mice in each group were injected intraperitoneally with a subthreshold dose of sodium pentobarbital of 25 mg / kg. The number of mice that fell asleep and the sleep rate within 15 minutes after the intraperitoneal injection of sodium pentobarbital were then recorded.

[0121] 2.2 Dosage setting The proposed clinical dosage of intermediate BA2023007 is 77g crude drug / day. Based on the animal and human surface area conversion, the equivalent dose for mice is: 77g crude drug × 0.0026 / 0.02kg. 10.01 g crude drug / kg. Based on 0.5 and 2 times the clinically equivalent dose, the low and high doses of the BA2023007 intermediate were determined, i.e., 5.005 g crude drug / kg and 20.02 g crude drug / kg, respectively. The clinical dose of diazepam is 10 mg / day, which, converted to a mouse equivalent dose based on body surface area, is 10 mg. 0.0026 / 0.02kg 1.3 mg / kg. For details of the experimental dosage design and grouping, please refer to Table 18.

[0122] Table 18 Animal grouping and dosage design

[0123] 2.3 Detection Indicators Observe the mice's sleep within 15 minutes: If the righting reflex disappears and lasts for more than 1 minute, the mice are considered to have entered a sleep state. Calculate the sleep onset rate based on the number of sleeping mice: Sleep onset rate (%) = (Number of animals entering sleep / Total number of animals). 100%.

[0124] 3. Statistical methods: Same as Experiment 1.

[0125] 4. Experimental Results 4.1 Effect of BA2023007 intermediate on the hypnotic effect of subthreshold doses of sodium pentobarbital on sleep patterns in mice As shown in Table 19, the sleep rates of experimental animals in the normal control group, diazepam group, and low, medium, and high dose groups of BA2023007 intermediate were 10%, 90%, 50%, 60%, and 50%, respectively. Compared with the normal control group, the sleep rates of mice in the low, medium, and high dose groups of BA2023007 intermediate and the diazepam tablet group were significantly increased. P ≤0.05 orP ≤0.01).

[0126] Table 19. Effects of BA2023007 intermediate on the effect of subthreshold doses of sodium pentobarbital on sleep in mice. ( (n=10)

[0127] Note: Compared with the normal control group, + P ≤0.05, ++ P ≤0.01.

[0128] 5. Summary The sleep rates of experimental animals in the normal control group, diazepam group, and low, medium, and high dose groups of BA2023007 intermediate were 10%, 90%, 50%, 60%, and 50%, respectively. The results suggest that BA2023007 intermediate can significantly increase the sleep rate of mice.

[0129] Discussion and Summary Decreased ovarian reserve (DOR) is characterized by a reduction in the number of recruitable oocytes and a decrease in oocyte quality. With rapid societal development, women face increasing pressure from various aspects of life, coupled with unhealthy lifestyles, leading to a rising incidence of DOR and a high risk of developing premature ovarian failure. Sleep plays a crucial role in various physiological processes, including memory, learning, emotion, and behavior. Insomnia, besides reducing the quantity and quality of sleep and accompanied by daytime physical and mental symptoms, significantly increases the risk and mortality of neuropsychiatric diseases and cardiovascular diseases by affecting numerous physiological processes. This study established a model of decreased ovarian reserve in rats using Tripterygium wilfordii polyglycoside suspension administered via gavage. Results showed that low, medium, and high doses of BA2023007 intermediate significantly increased serum E2 levels and decreased serum FSH levels in rats; high dose significantly increased serum AMH levels. Medium and high doses significantly increased follicle count, with the medium dose of BA2023007 intermediate resulting in a significantly higher follicle count than estradiol valerate tablets.

[0130] The effects of BA2023007 intermediate on the synergistic effects of suprathreshold / subthreshold doses of sodium pentobarbital were investigated. The results showed that medium and high doses of BA2023007 intermediate significantly shortened the sleep latency and prolonged sleep time in mice. The sleep onset rates in the normal control group, diazepam group, and low, medium, and high dose groups of BA2023007 intermediate were 10%, 90%, 50%, 60%, and 50%, respectively. Low, medium, and high doses of BA2023007 intermediate significantly increased the sleep onset rate in mice.

[0131] In conclusion, the BA2023007 intermediate has a significant therapeutic effect on decreased ovarian reserve and has a synergistic sleep-aiding effect with sodium pentobarbital.

[0132] Experiment 4: Toxicological Studies 1. Single-dose toxicity test (acute toxicity) The experiment used SPF-grade SD rats, female, 465×300×200mm 3 Five animals were housed in cages. They were raised in accordance with the environmental conditions required for SPF-grade laboratory animals according to the national standard (GB14925-2023). After passing quarantine and acclimatizing to the rearing environment, the animals were weighed, grouped, and given medication.

[0133] Forty female SD rats, weighing 180.3–201.7 g and certified by quarantine, were randomly divided into two groups according to body weight: a blank control group and a BA2023007 intermediate group, with 20 rats in each group. Animals in the BA2023007 intermediate group were administered 20 mL / kg of the intermediate solution orally via gavage at the maximum reproducible concentration (0.7 g dry extract powder / mL). They were fasted for approximately 13 hours before administration, but water was allowed. The drug was administered twice daily, with a cumulative dose of 154.0 g crude drug / kg, approximately 140 times (based on body weight) and 22 times (based on body surface area) of the clinically intended dose. The blank control group was administered pure water using the same volume and method. Animals were closely observed and their toxic symptoms, characteristics, onset and recovery time of toxic reactions, and mortality were recorded within 0–4 hours after administration. Observation continued the following day, twice daily (morning and afternoon), for a total of 14 days. Animals were weighed on the day of administration, before administration, and on days 4, 7, 10, and 14 after administration, and their weight changes were recorded. After the observation period, all surviving animals were grossly dissected to observe for any abnormalities in their organs.

[0134] Experimental results: Effects on general activity, animal toxicity, and mortality: During the experiment, no significant abnormalities were observed in the blank control group after administration. In the BA2023007 intermediate group, fecal abnormalities were observed after the second administration, mainly manifested as mild to moderate soft and pasty stools. These abnormalities returned to normal the following day, and no animal deaths were observed throughout the experiment. These fecal abnormalities may be related to a physiological stress response in the gastrointestinal tract caused by high-dose gavage administration of a high-concentration drug solution. This was a transient reaction and did not cause further abnormalities; therefore, it is not considered to have toxicological significance.

[0135] Effect on body weight: Oral administration of BA2023007 intermediate at a cumulative dose of 154.0 g crude drug / kg to SD rats on the same day had no significant effect on body weight.

[0136] At the end of the experiment, gross anatomical observation revealed no obvious abnormalities on the surface or cut surface of any organ.

[0137] in conclusion: Under the conditions of this experiment, SD rats were administered BA2023007 intermediate solution (0.7g dry extract powder / mL) orally twice a day, with a cumulative dose of 154.0g crude drug / kg. No related acute toxic reactions or deaths were observed, suggesting that its maximum tolerated dose (MTD) is ≥154.0g crude drug / kg, which is approximately equivalent to 140 times (based on body weight) and 22 times (based on body surface area) the intended clinical dose for adults.

[0138] 2. Repeated-dose toxicity test over 6 consecutive months (long-term toxicity) 165 SD rats, SPF grade, half male and half female, 465×300×200mm 3 Five animals were housed in cages. They were raised in accordance with the environmental conditions required for SPF-grade laboratory animals according to the national standard (GB14925-2023). After the animals passed quarantine and acclimatized to the housing environment, they were weighed, grouped, and given medication.

[0139] One hundred and sixty qualified SD rats, weighing 179.2–220.4 g, were randomly divided into four groups of 40 animals each (half male and half female) according to sex and weight. These groups were the blank control group, and low, medium, and high dose groups of BA2023007 intermediate (14.85, 28.88, and 57.75 g crude drug / kg), approximately equivalent to 14, 26, and 53 times (based on body weight) and 2, 4, and 8 times (based on body surface area) of the clinically intended dose, respectively. Each test group was administered the drug by gavage at a volume of 15 mL / kg, while the blank control group received the same volume of pure water. The administration was once daily, seven days a week, for six months (26 weeks). At the mid-term (end of week 13), the end of the administration period (end of week 26), and the end of the recovery period (end of week 4), 40, 80, and 40 SD rats (half male and half female) were dissected as planned. The examination items include: general clinical observation, weight and food intake measurement, routine urine test, hematology, blood biochemistry, coagulation and electrolyte test, hormone index test, ophthalmological examination, bone marrow smear; organ coefficient, histopathological examination, etc.

[0140] Experimental results: SD rats were administered low, medium, and high doses (14.85, 28.88, and 57.75 g crude drug / kg) of BA2023007 intermediate via oral gavage for 6 consecutive months. During the experiment, no obvious abnormalities were observed in the clinical observation of SD rats, no animal deaths were observed, and there were no significant effects on the weight, food intake, ophthalmological examination, hematology, coagulation, blood biochemistry, and serum electrolytes of SD rats.

[0141] Oral administration of medium and high doses (28.88 and 57.75 g crude drug / kg) of BA2023007 intermediate to SD rats for 6 consecutive months increased the number of PRO positive results in urinary routine tests and increased the liver coefficient in SD rats. High dose (57.75 g crude drug / kg) of BA2023007 intermediate also increased the kidney coefficient in SD rats. Whether this has toxicological significance needs to be determined in conjunction with subsequent histopathological results.

[0142] Experiment 5: Typical Cases Case 1: Ms. Cong, female, 32 years old, initial consultation: September 3, 2024 Chief complaint: Decreased ovarian reserve diagnosed 9 months ago. Present Illness: In December 2023, the patient experienced intermenstrual bleeding. Multiple basal hormone level tests at another hospital indicated decreased ovarian reserve: FSH: 15.6 mlU / ml, LH: 7.9 mlU / ml, E2: 52 pg / ml, AMH: 0.45 ng / ml. The patient was treated with dydrogesterone at the other hospital but discontinued the medication on her own. Currently, the menstrual cycle is 25-28 days, with moderate menstrual flow, blood clots, and mild dysmenorrhea. LMP: August 19, 2024. On August 29, 2024, the patient presented with slight vaginal bleeding, without abdominal pain. Current symptoms include: fatigue, lower back pain, cold lower abdomen, cold hands and feet, vivid dreams, good appetite, and normal bowel movements. The tongue is pale and dark with a thin white coating, and the pulse is deep and weak.

[0143] Auxiliary examinations: Basic hormone levels were measured on June 13, 2024: FSH: 23.25 mlU / ml, LH: 8.83 mlU / ml, E2: 46 pg / ml, AMH < 0.06 ng / ml, and vaginal ultrasound showed no obvious abnormalities.

[0144] Reproductive history: G1P0, miscarriage at 5+ weeks of gestation in July 2024.

[0145] Past medical history: In August 2023, Peking University Third Hospital performed a combined laparoscopic endometrial polyp resection, pelvic adhesion lysis, broad ligament myoma removal, and bilateral fallopian tube mesentery cyst resection.

[0146] Family history: Mother went through menopause at age 46.

[0147] Western medical diagnosis: Decreased ovarian function, history of adverse pregnancy outcomes.

[0148] Traditional Chinese Medicine diagnosis: Intermenstrual bleeding, spleen and kidney deficiency syndrome.

[0149] Treatment: Strengthen the spleen and kidneys, nourish blood and regulate menstruation.

[0150] Prescription: Cuscuta chinensis 12g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum (processed with wine) 10g, Dioscorea opposita 10g, Lilium brownii 12g, Lycopus lucidus 9g, Cinnamomum cassia twig 12g, Liquidambar formosana 12g, Atractylodes macrocephala (fried) 15g. Decoct in water and take one dose daily for 5 weeks. The patient desires to conceive; she was advised to discontinue medication after becoming pregnant to ensure a healthy pregnancy.

[0151] Second visit (October 9, 2024): LMP: August 19, 2024. Menstruation delayed, no menstruation; self-test urine HCG negative. Current condition: Lower abdomen and hands and feet warm, appetite and sleep good, bowel movements regular. Tongue pale and dark, with teeth marks on the edges, thin white coating, deep and weak pulse. Prescription: Increase the dosage of cinnamon twig to 20g, add calcined oyster shell (decocted first) 25g, take for 4 weeks.

[0152] Third visit (November 13, 2024): LMP: November 7, 2024, moderate flow, no discomfort during menstruation. Current condition: Feeling bloated in the stomach, sticky stools, no other discomfort, tongue and pulse as before. Prescription: Formula 9.3 plus 12g of stir-fried radish seeds, and increase the amount of stir-fried Atractylodes macrocephala to 20g. Take for 4 weeks.

[0153] Four diagnostic methods (December 25, 2024): LMP: Basic hormone levels checked on December 1 and 3, 2024: FSH: 7.84 mIU / ml, LH: 1.98 mIU / ml, E2: 68.85 pg / ml, AMH: 0.11 ng / ml. Self-tested urine HCG on December 23, 2024 was positive. Current symptoms: Lower abdominal pain (sharp, stabbing), mild nausea, good appetite, insomnia, light sleep with easy awakening, normal bowel movements. Pale and dark tongue with a thin white coating, weak and deep pulse. Auxiliary examinations: β-HCG on December 25, 2024: 192.8 mIU / ml, E2: 254.73 pg / ml, P: 18.27 ng / ml. No significant abnormalities were found in biochemistry, blood, or urine routine tests. Prescription: Cuscuta chinensis 12g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum (processed with wine) 10g, Dioscorea opposita 10g, Lilium brownii 12g, Margarita (decocted first) 30g, Polygala tenuifolia 12g. Take for one week. Doctor's advice: Follow up if there is significant abdominal pain or heavy vaginal bleeding.

[0154] Fifth visit (December 31, 2024): β-HCG: 2591 mIU / mL, E2: 459.31 pg / ml, P: 27.17 ng / ml. Mild bloating, slightly dry stools (one bowel movement per day), sleep good. Pale and dark tongue with little coating, wiry and slippery pulse. Prescription: Add 10g of cypress seed to the prescription from December 25th. Continue for one week. Follow-up visits weekly thereafter, with prescriptions adjusted according to symptoms. β-HCG doubling is good, no significant adverse reactions during pregnancy, fetal development is normal.

[0155] Case 2: Ms. Liu, female, 37 years old, initial consultation: September 1, 2024 Chief complaint: Decreased ovarian reserve for 10 months, with one miscarriage. Present Illness: In December 2023, due to lighter menstrual flow, tests at another hospital showed FSH: 17.5 mU / ml, LH: 6.8 mU / ml, E2: 42 pg / ml, AMH: 0.2 ng / ml, leading to a diagnosis of diminished ovarian reserve. No systematic treatment was given. Currently, the menstrual cycle is 28-30 days, with light menstrual flow (one sanitary napkin per day), blood clots, breast tenderness, and significant dysmenorrhea. Lmp: May 25, 2024. At 8 weeks of gestation, a miscarriage was diagnosed at another hospital, resulting in a spontaneous abortion on August 4, 2024. Embryo chromosome analysis showed trisomy 20. Currently, there is no menstruation. Current symptoms include: fatigue, excessive sweating, low mood, lower abdominal coldness, occasional abdominal distension and lower back pain, good appetite, light sleep with frequent awakenings, regular bowel movements, and frequent urination. The tongue is pale and dark with teeth marks on the edges, a thin white coating, and a deep, weak pulse.

[0156] Auxiliary examinations: On January 13, 2024, sex hormone levels were checked: FSH: 12.41 mlU / ml, LH: 6.55 mlU / ml, E2: 23 pg / ml, AMH: 0.18 ng / ml. Pelvic ultrasound showed an en 1.1 cm diameter with heterogeneous echogenicity. In November 2023, AMH was 0.27 ng / ml.

[0157] Reproductive history: G1P0, miscarriage at 8+ weeks of gestation in August 2024.

[0158] Past medical history: Congenital single kidney.

[0159] Family history: Denies any family medical history.

[0160] Western medical diagnosis: Hypoovarian insufficiency, history of adverse pregnancy outcomes Traditional Chinese Medicine Diagnosis: Scanty menstruation, spleen and kidney deficiency syndrome Treatment: Tonify the kidneys and strengthen the spleen Prescription: Cuscuta chinensis 12g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum (processed with wine) 10g, Dioscorea opposita 10g, Lilium brownii 12g, Lycopus lucidus 9g, Triticum aestivum 30g, Glycyrrhiza uralensis 6g, Ziziphus jujuba 15g, Nelumbo nucifera stamens 12g, Cinnamomum cassia 4g, Margarita (decocted first) 30g, Fennel seeds (for external use) 20g. Decoct in water and take one dose daily for 5 weeks. Apply the fennel seeds and dregs externally to the abdomen (30 minutes / time / day). The patient desires pregnancy and is advised to discontinue medication after conception to ensure a healthy pregnancy.

[0161] Second visit (October 9, 2024): LMP: September 28, 2024, flow increased compared to before, dysmenorrhea and breast distension improved. Lower abdomen still feels cold as before, occasional dizziness, appetite is good, sleep is still light as before, stool is slightly sticky, urination frequency improved. Tongue is pale and dark, coating is thin and white, pulse is deep and weak. Prescription: Remove lotus stamen from the previous prescription, add 8g of cinnamon, take for 4 weeks.

[0162] Third visit (November 13, 2024): LMP: October 24, 2024, moderate flow, dysmenorrhea improved. Lower abdominal coldness lessened compared to before, occasional lower limb coldness, improved light sleep, pale and dark tongue, thin white coating, deep and weak pulse. Prescription: Add 10g of Eucommia ulmoides (salted) to the previous prescription. Take for 4 weeks.

[0163] Four diagnostic methods (December 11, 2024): LMP: November 19, 2024, normal flow and color, occasional dysmenorrhea. Lower abdominal coldness lessened compared to before, lower limb coldness relieved, mood improved, no other discomfort, pale and dark tongue, thin white coating, deep and weak pulse. Continue with the original prescription. November 21, 2024, sex hormone tests: FSH: 7.85 mlU / ml, LH: 5.94 mlU / ml, E2: 65 pg / ml, AMH: 1.52 ng / ml. Biochemistry and routine blood and urine tests showed no significant abnormalities. Continue medication for 4 weeks.

[0164] Fifth visit (January 14, 2025): LMP: December 12, 2024, 33 days of missed period, positive on self-pregnancy test. β-HCG: 2038 mIU / ml, E2: 239.97 pg / ml, P: 17.01 ng / ml on the same day. Symptoms: small amount of brown vaginal discharge, occasional lower back pain and abdominal pain, mild bloating and nausea, light sleep, loose stools, one bowel movement per day. Pale and dark tongue, slightly greasy white coating, wiry and slippery pulse. Prescription: Add 12g of bamboo shavings and 12g of dried tangerine peel to the previous prescription, and increase the dosage of yam to 20g. Continue for one week. Follow-up visits weekly thereafter, with prescriptions adjusted according to symptoms. The brown discharge gradually disappeared, the lower back pain and abdominal pain subsided, and all pregnancy indicators returned to normal.

[0165] Case 3: Ms. Chang, female, 34 years old, first visit: October 23, 2024 Chief complaint: Scanty menstruation for over 2 years, with ovarian reserve deficiency diagnosed 3 months ago. Present Illness: Previously, menstruation was regular, with a cycle of 28-31 days and moderate menstrual flow (3 sanitary napkins per day). Dysmenorrhea with blood clots and breast tenderness was present. Two years ago, due to high emotional stress, menstrual flow became scanty, requiring only 1 sanitary napkin per day, with a cycle of 25 days. This was not taken seriously. From July to September 2024, menstruation ceased. The patient consulted a local hospital, where hormone levels were tested: FSH: 32.7 mlU / ml, LH: 17.8 mlU / ml, E2: 54 pg / ml, AMH: 0.02 ng / ml. A diagnosis of DOR was made. Menstruation resumed after taking dydrogesterone tablets and astragalus capsules. LMP: October 19, 2024. Menstruation is still ongoing, with scanty flow (1 sanitary napkin per day), dark in color, accompanied by dysmenorrhea and lower back pain. Symptoms include: lower back and knee weakness, fatigue, occasional palpitations, cold intolerance, good appetite, restless sleep with vivid dreams, anxiety, sticky stools, and normal urination. The tongue is pale and dark with a thin white coating, and the pulse is deep and weak.

[0166] Auxiliary examinations: September 30, 2024: Sex hormones: FSH: 107.71 mlU / ml, LH: 71.1 mlU / ml, E2: 70 pg / ml, AMH: 0.02 ng / ml. TSH: 0.49 mlU / ml. October 10, 2024: Pelvic ultrasound showed En 5.5 cm.

[0167] Reproductive history: G2P0, self-induced abortion in 2018, natural miscarriage in March 2024.

[0168] Past medical history: Denies any history of previous illnesses.

[0169] Family history: Denies any family medical history.

[0170] Western medical diagnosis: Menstrual disorders, decreased ovarian reserve.

[0171] Traditional Chinese Medicine diagnosis: Scanty menstruation, spleen and kidney deficiency syndrome.

[0172] Treatment: Tonify the spleen and kidneys, nourish blood and regulate menstruation.

[0173] Prescription: Cuscuta chinensis 12g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum (processed with wine) 10g, Dioscorea opposita 10g, Lilium brownii 12g, Lycopus lucidus 9g, Achyranthes bidentata 12g, Nardostachys chinensis 9g, stir-fried white hyacinth bean 20g, pearl powder (decocted first) 30g. Decoct in water and take one dose daily for four weeks. Doctor's instructions: Apply the dregs externally to the abdomen.

[0174] Second visit (November 20, 2024): LMP: October 19, 2024. Currently on day 33 of the menstrual cycle, no menstruation. Denies pregnancy. Currently taking Femoston orally. Symptoms: Palpitations resolved, appetite good, light sleep as before, anxiety, occasional acid reflux, slightly sticky stools, normal urination. Pale and dark tongue with a thin white coating, deep and weak pulse. Prescription: Add 30g of cuttlebone and 18g of yam to the previous prescription, to be taken for 4 weeks.

[0175] Third visit (December 22, 2024): LMP: November 20-25, 2024, scanty and dark in color, denies pregnancy, dysmenorrhea improved. Occasional heartburn, good appetite, sleep with many dreams, still has bad breath, loose stools. Pale and dark tongue, thin white coating, deep and weak pulse. Sex hormone tests on November 21, 2024: FSH: 13.96 mlU / ml, LH: 17.71 mlU / ml, E2: 90 pg / ml, AMH: 0.9 ng / ml. No significant abnormalities were found in the biochemical tests. Prescription: Remove Nardostachys jatamansi from the previous prescription, increase the dosage of Dioscorea opposita to 30g. Take for 4 weeks.

[0176] Fourth consultation (April 6, 2025): The patient reported discontinuing Femoston from January 2025, with regular menstrual cycles of 26-31 days. LMP: March 31, 2025, still not finished, with heavier flow than before, dark red in color, and occasional dysmenorrhea. Occasionally experiencing stomach pain and bloating, acid reflux resolved, appetite and sleep good, no other discomfort, pale and dark tongue, thin white coating, deep and weak pulse. Prescription: The previous prescription, minus cuttlebone, plus 15g of Perilla stem. Take for 4 weeks. Instructions on sexual intercourse; discontinue medication for pregnancy maintenance if pregnancy occurs.

[0177] Follow-up (July 2025): The patient reported conceiving naturally and underwent subsequent prenatal checkups at a local hospital, where the fetus was developing well.

[0178] Case 4: Ms. Wang, female, 37 years old, initial consultation: July 19, 2023 Chief complaint: Three adverse pregnancies, three failed IVF-ET attempts, and a history of diminished ovarian reserve for four months. Present Illness: Miscarriage at 8 weeks gestation in 2017, biochemical pregnancy in 2019, fetal arrest at 6 weeks gestation in 2020, and three IVF-embryo transfers between August 2022 and March 2023, all without implantation. Hormone levels checked in March 2023: FSH: 15.6 mIU / ml, LH: 8.9 mIU / ml, E2: 89 pg / ml, AMH: 0.35 ng / ml, diagnosed as DOR. Menstrual cycle 28 days, menstrual flow reduced (2 sanitary napkins per day), dysmenorrhea accompanied by breast tenderness and blood clots. LMP: July 14, 2023, lasted 4 days, no discomfort during menstruation. Symptoms include lower back pain, fatigue, lower abdominal coldness, occasional headache, vivid dreams, poor appetite, loose stools, normal urination. Pale and dark tongue with thin white coating, deep and weak pulse.

[0179] Auxiliary examinations: July 16, 2023: Sex hormones: FSH: 11.9 mlU / ml, LH: 4.43 mlU / ml, E2: 60 pg / ml, AMH: 0.24 ng / ml. Pelvic ultrasound showed no obvious abnormalities. TSH: 2.09 mlU / ml, all biochemical tests showed no obvious abnormalities.

[0180] Reproductive history: G3P0, miscarriage at 8 weeks of gestation in 2017, biochemical pregnancy in 2019, and fetal arrest at 6 weeks of gestation in 2020.

[0181] Past medical history: Hypothyroidism for 4 years, currently being treated with levothyroxine.

[0182] Family history: Denies family history.

[0183] Western medical diagnosis: Decreased ovarian reserve and adverse pregnancy history.

[0184] Traditional Chinese Medicine diagnosis: Recurrent miscarriage, spleen and kidney deficiency syndrome.

[0185] Treatment: Tonify the kidneys and strengthen the spleen.

[0186] Prescription: Cuscuta chinensis 12g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum (processed with wine) 10g, Dioscorea opposita 10g, Lilium brownii 12g, Lycopus lucidus 9g, Cinnamomum cassia 4g, Angelica sinensis 12g, Curcuma longa 12g, Vitex trifolia (fried) 9g. Decoct in water and take one dose daily for 5 weeks. The patient desires to conceive; she was advised to discontinue medication after becoming pregnant to ensure a healthy pregnancy.

[0187] Second visit (August 12, 2023): LMP: July 14, 2023, no menstruation, denies pregnancy, self-test for urine HCG is negative. Current condition: headache relieved, lower abdominal coldness improved, still has lower back pain, appetite is good, sleep is improved, bowel movements are normal. Tongue is pale and dark, coating is thin and white, pulse is deep and weak. Prescription: The previous prescription is modified by removing Vitex trifolia and Curcuma longa, and adding Notopterygium incisum 9g, to be taken for 4 weeks.

[0188] Third visit (October 14, 2023): LMP: September 1, 2023, 4 days of bleeding, normal flow, no menstruation, denies pregnancy. Eggs were retrieved at another hospital in September, resulting in 3 frozen embryos. Lower back pain improved, felt cold in the stomach, abdominal distension, frequent flatulence, appetite and sleep were good, regulated twice. Tongue and pulse were the same as before. September 2023: FSH: 7.8 mlU / ml, LH: 5.13 mlU / ml, E2: 48 pg / ml, AMH: 1.46 ng / ml. Biochemical tests, blood and urine routine tests showed no significant abnormalities. Prescription: The previous prescription, omitting Angelica sinensis, adding 12g of stir-fried radish seeds and 6g of prepared ginger. Take for 4 weeks. On the day of embryo transfer, remove Lycopus lucidus and Notopterygium incisum.

[0189] Fourth consultation (December 16, 2023): One frozen embryo was transferred on November 29th. On December 16th, blood β-HCG was 3578 mIU / ml. Currently, the patient experiences lower back pain, no abdominal pain, no vaginal bleeding, occasional nausea, good sleep, slightly loose stools, and normal urination. The tongue is pale and dark with little coating, and the pulse is wiry and slippery. Prescription: Cuscuta chinensis 12g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum (processed with wine) 10g, Dioscorea opposita 10g, Lilium brownii 12g, Amomum villosum (added later) 6g, Aucklandia lappa 6g, Atractylodes macrocephala (fried) 30g. Continue for one week. Follow-up visits are scheduled weekly, with prescriptions adjusted according to the patient's condition. Continue the Chinese medicine treatment until the 12th week of pregnancy if β-HCG doubles. No complications during pregnancy, and fetal development is normal.

[0190] Case 5: Ms. Wang, female, 35 years old. Initial consultation date: February 23, 2023. Chief complaint: Unable to conceive for 3 years despite unprotected sex, and diagnosed with diminished ovarian reserve 1 year ago. Present Illness: The patient attempted pregnancy for three years from 2020 to 2023 without success. In the past two years, she experienced significant stress from work and life. One year ago, she began experiencing early menstrual cycles. Tests at another hospital showed FSH: 12.6 mlU / ml, LH: 4.5 mlU / ml, E2: 32 pg / ml, AMH: 0.96 ng / ml, leading to a diagnosis of hypoovarian reserve, but she did not receive systematic treatment. Her past menstrual cycles were 22-26 days, lasting 2-3 days, with moderate, bright red flow containing clots, and significant dysmenorrhea and lower back pain. LMP: January 14, 2023. Current Symptoms: Lower back pain and fatigue, coldness in the lower back, good appetite, difficulty falling asleep, anxiety, dry stools, pale and dark tongue with a thin white coating, and a deep, weak pulse.

[0191] Auxiliary examinations: Sex hormone tests on January 15, 2023: FSH: 13.5 mlU / ml, LH: 3.82 mlU / ml, E2: 58 pg / ml, AMH: 0.8 ng / ml. No obvious abnormalities were found in biochemistry and routine blood and urine tests.

[0192] Reproductive history: G0P0. Past history: Past health.

[0193] Family history: Denies family history.

[0194] Western medical diagnosis: Decreased ovarian reserve, infertility.

[0195] Traditional Chinese Medicine diagnosis: Infertility, spleen and kidney deficiency syndrome.

[0196] Treatment principle: Tonify the kidneys and replenish essence, strengthen the spleen and nourish the blood. Prescription: Cuscuta chinensis 12g, Taxillus chinensis 12g, Dipsacus asper 12g, Ligustrum lucidum (processed with wine) 10g, Dioscorea opposita 10g, Lilium brownii 12g, Lycopus lucidus 9g, Codonopsis pilosula 9g, Astragalus membranaceus (processed with honey) 12g, Eucommia ulmoides (processed with salt) 12g. Decoct in water and take one dose daily for four weeks. Instruct the patient to relax, adjust her lifestyle, and have her female hormone levels checked again next menstrual cycle.

[0197] Second visit (April 1, 2023): After taking the above prescription, the dysmenorrhea improved, the lower back pain subsided, and the fatigue and weakness were relieved. The lower limbs were slightly cold, and the patient experienced poor sleep, frequent awakenings, and vivid dreams. A follow-up female hormone test showed FSH: 11.7 mlU / ml, LH: 5.4 mlU / ml, E2: 60 pg / ml, and AMH: 0.7 ng / ml. The roasted astragalus was removed, and 18g of salt-processed eucommia was added, along with 20g of raw fossilized teeth (decocted first).

[0198] Third visit (May 27, 2023): After taking the above prescription, sleep improved, and there were no other discomforts. A follow-up female hormone test showed FSH: 7.3 mlU / ml, LH: 5.6 mlU / ml, E2: 47 pg / ml, AMH: 1.9 ng / ml. Biochemistry and routine blood and urine tests showed no significant abnormalities. The above prescription was continued, and sexual intercourse was advised. In August 2023, pregnancy was discovered. The patient was given a modified version of Shou Tai Wan (a traditional Chinese medicine) to maintain the pregnancy. She subsequently gave birth to a healthy daughter without any drug-related adverse reactions.

[0199] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, The medicinal materials include the following parts by weight: Cuscuta chinensis 6-12 parts, Taxillus chinensis 9-15 parts, Dipsacus asper 9-15 parts, Ligustrum lucidum 6-12 parts, Dioscorea opposita 15-30 parts, Lilium brownii 6-12 parts, and Lycopus lucidus 6-12 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The ingredients include the following parts by weight: 12 parts of Cuscuta chinensis, 12 parts of Taxillus chinensis, 12 parts of Dipsacus asper, 10 parts of Ligustrum lucidum, 10 parts of Dioscorea opposita, 12 parts of Lilium brownii, and 9 parts of Lycopus lucidus.

3. A traditional Chinese medicine composition, characterized in that, It was extracted from the following medicinal materials: Cuscuta chinensis 6-12 parts, Taxillus chinensis 9-15 parts, Dipsacus asper 9-15 parts, Ligustrum lucidum 6-12 parts, Dioscorea opposita 15-30 parts, Lilium brownii 6-12 parts, and Lycopus lucidus 6-12 parts.

4. The traditional Chinese medicine composition according to claim 3, characterized in that, It was extracted from the following medicinal materials: 12 parts of Cuscuta chinensis, 12 parts of Taxillus chinensis, 12 parts of Dipsacus asper, 10 parts of Ligustrum lucidum, 10 parts of Dioscorea opposita, 12 parts of Lilium brownii, and 9 parts of Lycopus lucidus.

5. The traditional Chinese medicine composition according to claim 3 or 4, characterized in that, The extraction is carried out as follows: Mix the medicinal materials, add 6-10 times the amount of water, soak for 0.5-1.5 hours, extract 1-3 times, concentrate, and dry to obtain the final product.

6. The traditional Chinese medicine composition according to claim 5, characterized in that, The concentration conditions are: reduced pressure concentration, concentration temperature of 60-80℃, concentration relative density of 1.06-1.07, and 50℃.

7. The traditional Chinese medicine composition according to claim 5, characterized in that, The drying conditions are as follows: vacuum drying is used, the drying temperature is 60-80℃, and the hyperoside retention rate is ≥90%.

8. A traditional Chinese medicine preparation, characterized in that, It includes the traditional Chinese medicine composition according to claim 1 or 2, or the traditional Chinese medicine composition according to any one of claims 3-7.

9. The traditional Chinese medicine preparation according to claim 8, characterized in that, The dosage form of the traditional Chinese medicine preparation is one of the following: granules, decoction, capsules, pills, powders, tablets, and oral liquids.

10. A granule, characterized in that, It includes the following components: active ingredients and excipients; The active ingredient is the traditional Chinese medicine composition according to claim 1 or 2, or the traditional Chinese medicine composition according to any one of claims 3-7; The excipients are one or more of dextrin, soluble starch, and maltodextrin. The mass ratio of the active ingredient to the excipient is (1-5):1; The granules also include a wetting agent; the wetting agent is 80%-90% ethanol.

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