A traditional Chinese medicine composition for nourishing kidney and relieving depression, and a preparation method and application thereof

Abstract

The application relates to a traditional Chinese medicine composition for nourishing kidney and relieving depression as well as a preparation method and application thereof. The application relates to a traditional Chinese medicine composition and a preparation method and application thereof. In order to solve the problems that long-term hormone replacement therapy not only has more adverse reactions, but also has potential risks of causing endometrial lesions, thrombosis and tumors, the traditional Chinese medicine composition comprises radix codonopsis, radix ophiopogonis, taxilli herba, radix dioscoreae, ligustri fructus, anemarrhena asphodeloides, herba ecliptae, radix paeoniae alba, radix bupleuri, pericarpium citri reticulatae, fritillaria, poria cocos, semen ziziphi spinosae and schisandra chinensis. The traditional Chinese medicine composition is applied to preparation of a medicine for preventing and / or treating perimenopausal syndrome and premature ovarian failure. The application belongs to the technical field of medicines.

Description

Technical Field

[0001] This invention relates to a traditional Chinese medicine composition, its preparation method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] With the development of the economy and society and the aging population, the incidence of perimenopausal syndrome is increasing year by year. Its clinical symptoms are complex, involving dysfunction of multiple systems in the body. It not only causes physical and psychological discomfort and reduces the quality of life for patients, but also increases the long-term risk of metabolic syndrome, cardiovascular disease, osteoporosis, and other diseases. Statistics show that by 2030, China is expected to have approximately 280 million perimenopausal women, with an incidence rate of 60%–80%. In recent years, the age of onset has become earlier, and declining ovarian function and abnormal fluctuations in hormone levels are the main causes of this disease. Improving the quality of life of patients with perimenopausal syndrome and reducing its incidence has become an important public health issue that cannot be ignored, and it is receiving increasing attention and importance from the medical community. Around the age of 49 (79 years old), a woman's kidney qi gradually declines, her menstrual flow gradually decreases, and her Chong and Ren meridians gradually weaken, leading to the cessation of menstruation and menopause. During this physiological transition period, the influence of the internal and external environment, such as an inherent imbalance of yin and yang, a history of depression, pre-existing illnesses, or changes in family and social environments, can easily lead to an imbalance of kidney yin and yang, resulting in the onset of the disease. "The kidneys are the foundation of innate essence," and "the five viscera are interconnected, and when things reach an extreme, they will inevitably affect the kidneys." Therefore, imbalances in the yin and yang of the kidneys can easily affect other organs. The liver and kidneys share the same origin; the liver stores blood, and the kidneys store essence. Essence and blood both originate from the essence of food and water, and they can transform and nourish each other. Insufficient liver blood and deficiency of kidney essence often affect each other, leading to symptoms such as dizziness, tinnitus, and weakness in the lower back and knees, indicating a deficiency of both liver and kidney essence and blood.

[0003] Currently, modern medicine primarily treats perimenopausal syndrome and early-onset ovarian insufficiency with hormone replacement therapy. However, studies have shown that long-term hormone replacement therapy not only has numerous adverse reactions, but also carries significant risks of endometrial lesions, thrombosis, and tumors. Furthermore, hormone replacement therapy has many contraindications and requires caution, greatly limiting its clinical applicability. Traditional Chinese medicine (TCM) treatment for perimenopausal syndrome shows good efficacy, fewer adverse reactions, lower dependence, and higher patient acceptance. Summary of the Invention

[0004] This invention addresses the problems of long-term hormone replacement therapy, including numerous adverse reactions and the potential risks of endometrial lesions, thrombosis, and tumors. It proposes a traditional Chinese medicine composition for nourishing the kidneys and relieving depression, along with its preparation method and application.

[0005] The technical solution adopted by the present invention to solve the above problems is as follows: the traditional Chinese medicine composition for nourishing the kidney and relieving depression includes Codonopsis pilosula, Ophiopogon japonicus, Taxillus chinensis, Dioscorea opposita, Ligustrum lucidum, Anemarrhena asphodeloides, Eclipta prostrata, Paeonia lactiflora, Bupleurum chinense, Citrus reticulata, Lilium brownii, Poria cocos, Ziziphus jujuba var. spinosa, and Schisandra chinensis.

[0006] Furthermore, the mass fractions of each component in the herbal combination are as follows: Codonopsis pilosula 10 parts, Ophiopogon japonicus 15 parts, Taxillus chinensis 10 parts, Dioscorea opposita 20 parts, Ligustrum lucidum 20 parts, Anemarrhena asphodeloides 20 parts, Eclipta prostrata 15 parts, Paeonia lactiflora 30 parts, Bupleurum chinense 10 parts, Citrus reticulata 10 parts, Lilium brownii 10 parts, Poria cocos 15 parts, Ziziphus jujuba var. spinosa 10 parts, and Schisandra chinensis 10 parts.

[0007] The step of preparing the traditional Chinese medicine composition into an aqueous decoction in the method for preparing the traditional Chinese medicine composition for nourishing the kidney and relieving depression described in this invention is as follows: Step 1: Wash and dry the following herbs: Codonopsis pilosula, Ophiopogon japonicus, Taxillus chinensis, Dioscorea opposita, Ligustrum lucidum, Anemarrhena asphodeloides, Eclipta prostrata, Paeonia lactiflora, Bupleurum chinense, Citrus reticulata, Lilium brownii, Poria cocos, Ziziphus jujuba var. spinosa, and Schisandra chinensis. Grind them into coarse powder. Step 2: Place the coarse powder of medicinal materials obtained in Step 1 into a clay pot, add water to cover the medicinal materials and soak. Step 3: After soaking the herbs, bring them to a boil over high heat, then reduce to a simmer and cook slowly. Filter the liquid and collect the filtrate. Step 4: Add water to the residue again, boil, filter, and combine the two filtrates; Step 5: Concentrate the filtrate under reduced pressure to 200 mL to obtain the decoction of traditional Chinese medicine.

[0008] Furthermore, in step 1, the mass fractions of Codonopsis pilosula are 10 parts, Ophiopogon japonicus are 15 parts, Taxillus chinensis are 10 parts, Dioscorea opposita is 20 parts, Ligustrum lucidum is 20 parts, Anemarrhena asphodeloides is 20 parts, Eclipta prostrata is 15 parts, Paeonia lactiflora is 30 parts, Bupleurum chinense is 10 parts, Citrus reticulata is 10 parts, Lilium brownii is 10 parts, Poria cocos is 15 parts, Ziziphus jujuba var. spinosa is 10 parts, and Schisandra chinensis is 10 parts.

[0009] Furthermore, in step 2, the soaking time for the medicinal materials is 30 minutes.

[0010] Furthermore, in step 3, after soaking the medicinal herbs, bring them to a boil over high heat and then simmer over low heat for 30 minutes.

[0011] Furthermore, in step 4, the filter residue is boiled again with water for 30 minutes.

[0012] The process of preparing the traditional Chinese medicine composition into pills according to the method for preparing the traditional Chinese medicine composition for nourishing the kidney and relieving depression described in this invention is as follows: Codonopsis pilosula, Ophiopogon japonicus, Taxillus chinensis, Dioscorea opposita, Ligustrum lucidum, Anemarrhena asphodeloides, Eclipta prostrata, Paeonia lactiflora, Bupleurum chinense, Citrus reticulata, Lilium brownii, Poria cocos, Ziziphus jujuba var. spinosa, and Schisandra chinensis are ground into a fine powder, mixed with water, and then fed into a pill-making machine to form pills.

[0013] Furthermore, the mass fractions of Codonopsis pilosula are 10 parts, Ophiopogon japonicus are 15 parts, Taxillus chinensis are 10 parts, Dioscorea opposita is 20 parts, Ligustrum lucidum is 20 parts, Anemarrhena asphodeloides is 20 parts, Eclipta prostrata is 15 parts, Paeonia lactiflora is 30 parts, Bupleurum chinense is 10 parts, Citrus reticulata is 10 parts, Lilium brownii is 10 parts, Poria cocos is 15 parts, Ziziphus jujuba var. spinosa is 10 parts, and Schisandra chinensis is 10 parts.

[0014] The traditional Chinese medicine composition for nourishing the kidneys and relieving depression described in this invention is used in the preparation of a formula for the prevention and / or treatment of perimenopausal syndrome and early-onset ovarian insufficiency.

[0015] The beneficial effects of this invention are: 1. This invention mainly has the effects of soothing the liver and relieving depression, nourishing yin and nourishing the heart; it is used for dizziness and tinnitus, soreness and weakness of the waist and knees, hot flashes in the palms, soles and chest, hot flashes and sweating, insomnia and dreaminess, irritability and anger, dry mouth and throat; menopausal syndrome and early-onset ovarian insufficiency with the above symptoms. 2. This invention has significant effects on patients with perimenopausal syndrome and early-onset ovarian insufficiency. The formulation of this traditional Chinese medicine composition focuses on the pharmacological effects of "principal, assistant, adjuvant and guide" to regulate the physiological functions of the internal organs and the balance of Yin and Yang as a whole. It aims to provide patients with perimenopausal syndrome and early-onset ovarian insufficiency with a portable and effective product for nourishing the kidneys and relieving depression. 3. The advantages of this invention are: the Chinese medicine pills are easy to carry and take. The pills made by this invention are the size of mung beans, and the excipient is water. They can be taken with warm water after meals, which provides convenience for patients. It realizes the full utilization of Chinese medicine, gives full play to the effective role of the drug, and achieves the beneficial effects of nourishing the kidney and relieving depression, benefiting a large number of patients with perimenopausal syndrome and early-onset ovarian insufficiency. Attached Figure Description

[0016] Figure 1 This is a diagram comparing the total scores of TCM symptoms before and after treatment; Figure 2 This is a diagram comparing neurotransmitter levels before and after treatment; Figure 3 This is a diagram showing the comparison of sex hormone levels before and after treatment; Figure 4 This is a diagram showing the comparison of SDS and SAS self-rating scale scores before and after treatment; Figure 5 This is a diagram showing the comparison of MoCA and KMI scores before and after treatment; Figure 6 This is a schematic diagram comparing the SPI of rats in different time points and after treatment in Example 2; Figure 7 This is a schematic diagram comparing the TDT (transient thermal decompression) of rats in different time points and within 5 minutes in an open field after treatment in Example 2; Figure 8 These are the movement trajectories of rats in different groups at different time points in Example 2.

[0017] Example Example 1: The effect of the traditional Chinese medicine composition of the present invention on patients with menopausal syndrome and mood disorders; Materials and Methods Sixty patients with menopausal syndrome and mood disorders who visited the Menopause Clinic of the Department of Gynecology at the First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine between January 2020 and March 2023 were treated with the pills of this invention for 3 months.

[0018] test drug This invention relates to a traditional Chinese medicine pill formulation: 10 parts Codonopsis pilosula, 15 parts Ophiopogon japonicus, 10 parts Taxillus chinensis, 20 parts Dioscorea opposita (fried), 20 parts Ligustrum lucidum (wine-processed), 20 parts Anemarrhena asphodeloides, 15 parts Eclipta prostrata, 30 parts Paeonia lactiflora (wine-processed), 10 parts Bupleurum chinense, 10 parts Citrus reticulata peel, 10 parts Lilium brownii (honey-processed), 15 parts Poria cocos, 10 parts Ziziphus jujuba var. spinosa (fried), and 10 parts Schisandra chinensis. Each ingredient is weighed according to the above proportions, washed, and ground into a fine powder. Water is added, and the powder is fed into a pill-making machine to form pills the size of mung beans, which are then dried. The pills are taken 2-3 times daily (once in the morning and once in the evening), with a dosage of 5-10g each time, taken with warm water after meals.

[0019] result This product can improve the TCM syndrome scores and hormone levels in perimenopausal patients with mood disorders. After treatment, it increases serum levels of monoamine neurotransmitters 5-HT and DA, and decreases NE level; it also increases sex hormone E2 level and decreases FSH and LH levels. By collecting symptom scales (including SDS score, SAS score, MoCA score, and modified Kupperman score) from patients before and after treatment, it is shown that this product can significantly improve depression, anxiety, and sleep, and has a significant therapeutic effect.

[0020] Example 2: Effects of the herbal composition of the present invention on perimenopausal mood disorders in rats; Materials and Methods SPF-grade female SD rats aged 6-8 weeks, with a body weight of 200±20g, were provided by the Drug Safety Evaluation Center of Heilongjiang University of Traditional Chinese Medicine. Building Model A perimenopausal model was established using VCDs. Rats were intraperitoneally injected with VCD corn oil solution (160 mg / kg) at fixed times daily for 15 consecutive days. During injection, the operator wore sterile gloves, used their left hand to stabilize the rat's head, neck, and tail, positioning it in a head-down, abdomen-up position. The lower abdominal skin was disinfected with a 75% alcohol swab using a spiral motion. The right hand held a 1 mL syringe and slowly inserted the needle at a 45° angle 1-2 mm lateral to the abdominal midline. After penetrating the abdominal wall, aspiration confirmed the absence of blood, urine, or intestinal contents reflux. The drug solution was then injected at a uniform rate, and the rats were immediately returned to their original cages after injection. Rats with successfully established perimenopausal models were treated using the CUMS protocol for 28 consecutive days.

[0021] Stimulation methods included water deprivation, food deprivation, tail clamping, swimming in ice water, 45-degree tilted cages, damp bedding, day-night reversal, restraint, and hot air. To ensure the unpredictability of the stimulation methods, a randomization method was used to ensure that: ① the interval between the same stimulus was ≥3 days; ② circadian rhythm-related stimuli (such as light interference) were not implemented consecutively.

[0022] estrous cycle monitoring After VCD administration, vaginal smears were examined in rats for 10 consecutive days to monitor changes in their estrous cycle. Characteristic cytological changes were observed in each stage of the estrous cycle. In proestrus, numerous oval-shaped nucleated epithelial cells predominated, with deep blue nuclei and a significant nucleocytoplasmic ratio, and light pink cytoplasm with clear borders. In estrus, sheet-like anucleated keratinized epithelial cells were dominant, arranged in a fish-scale pattern, with homogeneous pink cytoplasm and strong refractive properties. In mesestrus, a mixture of nucleated epithelial cells, keratinized cells, and neutrophils was observed, with some cells retaining deep blue nuclei, and the cytoplasm showing pleomorphic staining. In estrus, neutrophils predominated, with occasional small numbers of epithelial cells distributed in a punctate pattern, and the cytoplasm showing no significant staining. Estrous cycle disorder was characterized by an estrous cycle >5 days, <4 days, or a single estrous phase lasting ≥3 days. Vaginal smears showed estrous cycle disorder in some model rats; some model rats showed interestrous phase characteristics for 5 consecutive days, namely, predominantly white blood cells with occasional small number of nucleated epithelial cells and no typical estrous keratinized cell layer formation, proving that the perimenopausal rat model was successfully established.

[0023] Animal grouping A perimenopausal rat model was established using intraperitoneal injection of VCD (volumetric cyclophosphamide). Forty-five healthy female SD rats aged 6-8 weeks were selected and administered VCD corn oil solution (160 mg / kg) intraperitoneally daily for 15 days. Two rats died during the injection period. The estrous cycle of the 43 surviving rats was assessed using vaginal exfoliative cytology, all showing predominantly diastolic cell characteristics or disordered estrous cycles, indicating successful entry into the perimenopausal state. Subsequently, the perimenopausal model rats underwent CUMS intervention, during which one rat died due to severe stress (mortality rate 2.3%), resulting in 42 rats completing the entire modeling process.

[0024] The next step, based on behavioral criteria and combined with changes in general conditions such as rat weight and coat, selected 8 stress-resistant rats that did not exhibit significant anxiety / depression-like behavioral phenotypes. These were designated as the perimenopausal non-emotional disorder group (n=8) and served as the control group for subsequent experiments. The remaining 34 stress-sensitive rats were designated as the perimenopausal emotional disorder group (n=34). Next, a stratified block randomization method was used to group the 34 perimenopausal emotional disorder rats. The specific procedure was as follows: using SPI as the stratification variable, the rats were sorted in ascending order of SPI value and divided into 9 blocks (the first 8 blocks each contained 4 rats, and the 9th block contained the remaining 2 rats). A computer random number generator was used to assign the 4 rats in each complete block to the model group and the low / medium / high dose group (1 rat each). The remaining 2 rats were randomly assigned to the model group and the high dose group, resulting in a grouping structure of 9 rats each in the model group and the high dose group, and 8 rats each in the low / medium dose group.

[0025] To address the heterogeneity of behavioral screening thresholds in previous studies and to systematically validate the scientific validity of the perimenopausal mood disorder grouping, this study employed a longitudinal tracking strategy. After the modeling procedure was completed, blood samples were collected again from all surviving rats (n=42) via orbital venous plexus puncture. The dynamic changes of behavioral parameters (SPI, CCT, TDT, IT, RC) and serum indicators (FSH, E2, 5-HT) before and after modeling in each group were statistically analyzed.

[0026] Experimental drugs and intervention protocols This invention relates to a decoction of traditional Chinese medicine: 10 parts Codonopsis pilosula, 15 parts Ophiopogon japonicus, 10 parts Taxillus chinensis, 20 parts Dioscorea opposita (fried), 20 parts Ligustrum lucidum (wine-processed), 20 parts Anemarrhena asphodeloides, 15 parts Eclipta prostrata, 30 parts Paeonia lactiflora (wine-processed), 10 parts Bupleurum chinense, 10 parts Citrus reticulata peel, 10 parts Lilium brownii (honey-processed), 15 parts Poria cocos, 10 parts Ziziphus jujuba var. spinosa (fried), and 10 parts Schisandra chinensis. Each ingredient is weighed according to the above proportions, washed, and boiled in 8 times the volume of water for 30 minutes. The mixture is filtered to obtain the filtrate. This process is repeated twice, and the two filtrates are combined and concentrated to a crude drug content of 1 g / mL. The filtrate is stored at 4℃ for later use. Based on the conversion ratio of the effective dose per unit area between experimental rats and humans, the daily dose for clinical adults (215 g crude drug / 70 kg) was converted to the equivalent dose for experimental rats. The results were: low dose group 4.61 g / kg, medium dose group 9.22 g / kg, and high dose group 18.45 g / kg. The intervention plans for each group are as follows: Based on routine feeding, The control group (n=8) received an equal volume of physiological saline (0.9% NaCl, 5 mL / kg / d). The model group (n=9) was given an equal volume of physiological saline (0.9% NaCl, 5 mL / kg / d). The low-dose group of traditional Chinese medicine (ZSJYD-L) (n=8) was given a decoction of this traditional Chinese medicine composition at a dose of 4.61 g / kg / d. The medium-dose group of traditional Chinese medicine (ZSJYD-M) (n=8) was given a decoction of this traditional Chinese medicine composition at a dose of 9.22 g / kg / d. The high-dose group of traditional Chinese medicine (ZSJYD-H) (n=9) was given an aqueous decoction of this traditional Chinese medicine composition at a dose of 18.45 g / kg / d. Rats were administered the medication via gavage at a fixed time each day (9:00-10:00). The intervention period for each group was 4 weeks. The body weight of each rat was measured before modeling, after VCD modeling, after CUMS modeling, and on the last day of each week starting with gavage. The medication solution was prepared fresh for each use, and the gavage dose was adjusted according to body weight (1mL / 100g).

[0027] Enzyme-linked immunosorbent assay (ELISA) detection steps: The expression levels of serum 5-HT, NE, DA, SIRT1, PGC-1α, SOD, MDA, and Aβ in subjects before and after treatment were detected by ELISA. Once all samples were collected, they were measured using ELISA. The operation was strictly performed in accordance with the instructions of the kit. Step 1, Sample Pretreatment and Reagent Preparation: Thaw the frozen serum sample at room temperature. If precipitate is observed after thawing, a second centrifugation is required to remove impurities. Meanwhile, take out the refrigerated test kit in advance and equilibrate it at room temperature for 20 minutes to ensure that the reagent activity is restored to the optimal state. Step 2, Preparation of Standard Gradients: Take the original concentration standard, mix it thoroughly with a vortex mixer, and then perform serial dilution at a volume ratio of 1:2. The specific operation is as follows: add 150 μL of stock solution and an equal volume of dilution buffer to the EP tube in sequence, and dilute stepwise to the lowest concentration standard to form 5 gradient concentration systems. Step 3, Sample loading on the ELISA plate: Set up blank control wells (add diluent only), standard wells and sample detection wells; add 50 μL of the corresponding concentration standard solution directly to the standard wells; add 40 μL of special diluent to the sample wells first, and then accurately add 10 μL of the treated serum sample to make the final dilution factor of 5 times. Step 4, Immunoassay and Signal Processing: Cover with a special sealing film and transfer the ELISA plate to a 37°C incubator for 30 minutes to promote full binding of antigen and antibody. After incubation, discard the reaction solution and wash 5 times with pre-diluted washing buffer (30-fold concentrate diluted proportionally). Fill each well with the wash buffer, let stand for 30 seconds, and then shake off the excess liquid. Finally, tap the plate to remove any remaining liquid. Then, add 50 μL of ELISA reagent to each well and repeat the incubation and elution steps to remove unbound material. Step 5, Colorimetric Analysis and Data Acquisition: Add 50 μL each of colorimetric reagent A and B to each well in sequence, and react at 37°C for 10 min under dark conditions. After complete color development, add 50 μL of stop solution to terminate the reaction. Use an ELISA reader to measure the absorbance value at a wavelength of 450 nm. All readings should be completed within 15 min after the reaction is terminated. Zero the blank well to correct for systematic errors.

[0028] Pathological examination Making paraffin slices (1) Fixation and trimming: Tissue samples were fixed with 4% paraformaldehyde at 4°C for 24 hours. The fixed tissue blocks were then removed and smoothed with a scalpel at the target site in a fume hood. The cut tissue and corresponding labels were placed in an embedding frame. (2) Dehydration and clearing: Place the dehydration box in the dehydrator and dehydrate with a gradient of alcohols in sequence: 75% ethanol overnight, 80% ethanol for 25 min, 85% ethanol for 25 min, 90% ethanol for 25 min, 95% ethanol for 25 min, 100% ethanol for 25 min, and 100% ethanol for 25 min; Xylene clearing: soak in xylene I for 25 min, and soak in xylene II for 15 min; (3) Placing and embedding: Place the transparent tissue into paraffin at 56~58℃ for ≥2.5h. After the paraffin is completely immersed, use a pre-cooled metal mold (-20℃) for directional embedding. Avoid air bubbles during embedding. (4) Sectioning, spreading, and baking: Chill the embedded paraffin block for 2 hours, fix it on a microtome, and cut 4μm thin sections. Use coronal sections for hippocampal tissue and sagittal sections for ovarian tissue. Place the sections in a tablet press and float them in 40℃ warm water to flatten them, then transfer them to poly-L-lysine coated slides. Preheat the slides at 50℃ for 30 minutes using a spreader, bake them at 60℃ for 2 hours and then at 95℃ for 15 minutes using a baking machine to ensure that the paraffin film is completely melted. Remove them at room temperature and store them for later use.

[0029] Hematoxylin-eosin (HE) staining (1) Dewaxing and hydration of paraffin sections: Xylene I for 5 min, Xylene II for 5 min for dewaxing; then hydrated in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 2 min, 85% ethanol for 2 min, 75% ethanol for 2 min, and distilled water for 2 min in sequence. (2) Staining: Stain with hematoxylin for 5 min, wash the slide twice with distilled water for 5 min each time, spin dry, differentiate with 1% hydrochloric acid ethanol for 5 s, wash the slide twice with distilled water for 5 min each time, and stain with eosin for 1 min; (3) Dehydration and transparency: rapid dehydration with gradient ethanol: 75% ethanol for 30s, 85% ethanol for 30s, 95% ethanol for 30s, anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, xylene II for 5min, xylene I for 5min to achieve transparency; (4) Mounting: Add 50 μL of neutral resin to each slide, cover it slowly with a coverslip at a 45° angle to avoid air bubbles, observe under a microscope, and acquire images.

[0030] Immunohistochemical staining (1) Dewaxing and hydration of paraffin sections: Xylene I for 5 min, Xylene II for 5 min for dewaxing; then hydrated in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 2 min, 85% ethanol for 2 min, 75% ethanol for 2 min, and distilled water for 2 min in sequence. (2) Antigen retrieval: Cover the tissue sections with citrate antigen retrieval buffer (pH 6.0), heat in a microwave oven on medium heat until boiling, keep warm for 8 minutes, then turn to medium-low heat and reheat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented to avoid drying the slides. After naturally cooling to room temperature, place the slides in PBS (pH 7.4) and wash them 3 times (5 minutes each time) on a decolorizing shaker to remove any residual retrieval solution. (3) Blocking endogenous peroxidase: The slices were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min. They were then washed three times in PBS (pH 7.4) on a decolorizing shaker for 5 min each time. (4) Serum blocking: Cover the tissue loop evenly with 3% BSA and block at room temperature for 30 min (for goat-derived primary antibodies, use rabbit serum for blocking and for other sources, use BSA for blocking). (5) Primary antibody incubation: Gently shake off the blocking solution, dilute the primary antibody in PBS at a certain ratio, and then drop it evenly onto the surface of the slide. Place the slide flat in a humidified chamber and incubate overnight at 4°C (add an appropriate amount of distilled water to the humidified chamber to prevent antibody evaporation). (6) Secondary antibody incubation: Place the slide in PBS at pH 7.4 and wash it three times on a decolorizing shaker for 5 min each time. After the slide is slightly dry, add the secondary antibody, which is of the species corresponding to the primary antibody and labeled with horseradish peroxidase (HRP), to fully cover the tissue area within the slide loop, and incubate at room temperature for 50 min; (7) DAB staining: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After the slide has dried slightly, add freshly prepared DAB staining solution to the tissue area. Observe the staining process closely under a microscope. Positive signals appear as brownish-yellow. When a light brown background appears, immediately rinse the slide with tap water to stop the staining reaction; (8) Nuclear counterstaining and blueing: stain with hematoxylin for 3 min, rinse with tap water, differentiate with 1% hydrochloric acid ethanol for 5 s, rinse with tap water, blue with dilute ammonia for 5 min, rinse with tap water. (9) Dehydration and sealing: Immerse the sections sequentially in 75% ethanol, 85% ethanol, anhydrous ethanol I, anhydrous ethanol II, and xylene I, treating each for 5 minutes to complete the dehydration and clearing process. After removing the sections from the xylene, allow them to air dry slightly, then mount them with neutral resin. (10) Observation, image acquisition and analysis using an optical microscope; (11) Interpretation of immunohistochemical results of paraffin sections: hematoxylin staining of cell nuclei is blue, and DAB positive expression is brownish-yellow; (12) Quantitative analysis: This study selected two positive area-related parameters, positive area ratio and H-score, for quantitative analysis: ① Positive area ratio = positive area / tissue area. It reflects the amount of positive area and is suitable for cases where positive areas are abundant and widely expressed. ② H-score: an abbreviation for Histochemistry score, is a histological scoring method for processing immunohistochemistry. It converts the positive area ratio and staining intensity in each section into corresponding numerical values, achieving a comprehensive semi-quantitative analysis of both the depth and quantity of positive staining in tissue immunostaining.

[0031] result Before modeling, the experimental animals were in a healthy baseline state during the acclimatization feeding phase, exhibiting smooth and glossy fur, symmetrical body shape, active spontaneous activity, and regular feeding and drinking. No abnormal social behavior or stress response was observed. After VCD injection, the rats developed dry and sparse fur, some with hair loss, increased weight, with significant fat accumulation in the abdomen and buttocks, decreased activity, and weakened resistance when grasped. After CUMS, the Model group rats showed reduced fur volume, significant weight loss, lethargy, fatigue, and a tendency to huddle together with little movement. They also exhibited reduced food and water intake and defensive postures such as piloerection and arched backs. Treatment with traditional Chinese medicine showed varying degrees of improvement in all groups. The rats' fur became thicker and shinier, hair loss decreased, body shape became more symmetrical, activity levels returned to normal, and food and water intake returned to normal.

[0032] like Figure 6As shown, baseline test results before modeling showed no statistically significant difference in SPI among the rat groups (P>0.05), indicating that the experimental animals had good biological homogeneity in their initial state and small individual differences, providing a basis for comparability in subsequent experiments. After VCD combined with CUMS intervention for modeling, compared with baseline, the SPI of the Model group and each dose treatment group decreased significantly (P<0.001), while the SPI of the Control group showed no significant change (P>0.05); at the same time, the SPI of the Model group and each dose treatment group was significantly lower than that of the Control group (P<0.001), which is consistent with the typical behavioral characteristics of anhedonia induced by chronic stress. After 4 weeks of intervention with the decoction solution of the traditional Chinese medicine composition of this invention, the SPI of the Model group was still significantly lower than that of the Control group (P<0.001), suggesting that the anhedonia behavior persisted. The various treatment groups of the traditional Chinese medicine showed a dose-dependent improvement trend. The SPI of the ZSJYD-M and ZSJYD-H groups was significantly higher than that of the Model group (P<0.001), while the SPI of the ZSJYD-L group was also elevated, but not statistically significant (P>0.05). Comparison between different dosage groups showed that the SPI of the ZSJYD-H group was significantly higher than that of the ZSJYD-M group (P<0.05) and the ZSJYD-L group (P<0.01), and the SPI of the ZSJYD-M group was also significantly higher than that of the ZSJYD-L group (P<0.05), further suggesting that the aqueous decoction solution of the traditional Chinese medicine composition of the present invention has a certain dose-dependent effect on improving anhedonia.

[0033] Open field experiment changes Total distance traveled (TDT) The changes in TDT within 5 minutes in the open field experiment of rats in each group are shown in Table 1-2. Figure 7As shown. During the baseline testing phase, there was no statistically significant difference in TDT among the rat groups (P>0.05), indicating that the animals' spontaneous activity ability was highly consistent before modeling and comparable. After modeling with VCD combined with CUMS intervention, the TDT in the Model group and each treatment group decreased significantly from baseline (P<0.001), while the TDT in the Control group showed no significant change (P>0.05). Simultaneously, compared with the Control group, the TDT in the Model group and each treatment group was significantly reduced (P<0.001), a result consistent with the behavioral characteristics of decreased spontaneous movement ability in animals due to chronic stress in previous studies. After 4 weeks of intervention with the decoction solution of the traditional Chinese medicine composition of this invention, the TDT in the Model group was still significantly lower than that in the Control group (P<0.001), the TDT in the ZSJYD-M and ZSJYD-H groups was significantly higher than that in the Model group (P<0.001), while the TDT in the ZSJYD-L group showed no statistically significant difference compared with the Model group (P>0.05). The comparison results between different dosage groups showed that the TDT of the ZSJYD-H group was significantly higher than that of the ZSJYD-L group (P<0.01), suggesting that the decoction solution of the traditional Chinese medicine composition of the present invention has a certain dose-dependent effect on improving animal movement behavior.

[0034] movement trajectory like Figure 8As shown, in the open field experiment conducted before modeling, the rats' movement tracks densely covered most of the area, with obvious activity traces in all corners and the central area, reflecting their high curiosity and adaptability to the new environment. After modeling, the control group rats' movement tracks in the open field remained relatively evenly distributed. Compared to other groups, the control group rats had a longer total movement distance and more time spent visiting the central area, indicating that the control group rats maintained good activity levels after VCD injection and CUMS stimulation, and did not exhibit significant anxiety / depression-like behavior. In contrast, the model group and each dose treatment group rats showed significant anxiety / depression-like behavioral changes, with a significant decrease in overall activity level and a significant increase in rest time, and a tendency to walk along the edges of the open field, avoiding the central area. In the open field experiment after treatment, the control group rats still exhibited normal behavioral patterns, with movement tracks covering the entire open field, including the central and peripheral areas, showing high activity levels and good spatial exploration ability. In contrast, the model group rats mainly walked along the edges, with almost no activity traces in the central area, and still exhibited obvious anxiety / depression-like behavior. Rats in the ZSJYD-L group showed improvement in anxiety / depression-like behavior, but their movement patterns remained predominantly peripheral, with less activity in the central area. Rats in the ZSJYD-M group showed significant signs of recovery, with movement patterns similar to the Control group, exhibiting frequent activity in both the central and peripheral areas and a marked increase in activity level. Rats in the ZSJYD-H group performed best, with movement patterns similar to the Control group, showing activity in both the central and peripheral areas and a significantly shortened resting time, demonstrating the best therapeutic effect.

[0035] Working principle The pharmacological properties and combined effects of the selected components from traditional Chinese medicine are as follows: Ophiopogon japonicus nourishes yin and generates fluids, relieves irritability and calms the mind; Codonopsis pilosula replenishes qi, generates fluids and nourishes blood; Taxillus chinensis tonifies the liver and kidneys, benefits essence and blood, strengthens tendons and bones, promotes hair growth and strengthens teeth, generates marrow, nourishes the brain and relieves dizziness; Dioscorea opposita tonifies qi and nourishes yin, tonifies the spleen, lungs and kidneys; Ligustrum lucidum nourishes liver and kidney yin essence; Anemarrhena asphodeloides clears heat and drains fire, nourishes yin and moistens dryness; Eclipta prostrata tonifies liver and kidneys, cools blood and stops bleeding; Paeonia lactiflora nourishes blood and regulates menstruation, astringes yin and stops sweating; Bupleurum chinense soothes the liver and relieves depression; Lilium brownii nourishes yin, clears the heart and calms the mind; Poria cocos calms the mind and soothes the mind; Ziziphus jujuba var. spinosa nourishes the heart and liver and calms the mind; Schisandra chinensis nourishes the kidneys, astringes essence and stops diarrhea, generates fluids internally, calms the mind and soothes the mind externally, strengthens the exterior and astringes to stop sweating; Citrus reticulata regulates qi and strengthens the spleen, making the whole formula nourishing without being greasy. The whole formula works together to soothe the liver and relieve depression, nourish yin and nourish the heart.

[0036] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for nourishing the kidneys and relieving depression, characterized in that, It includes Codonopsis pilosula, Ophiopogon japonicus, Taxillus chinensis, Dioscorea opposita, Ligustrum lucidum, Anemarrhena asphodeloides, Eclipta prostrata, Paeonia lactiflora, Bupleurum chinense, Citrus reticulata peel, Lilium brownii, Poria cocos, Ziziphus jujuba var. spinosa, and Schisandra chinensis.

2. The traditional Chinese medicine composition for nourishing the kidneys and relieving depression according to claim 1, characterized in that, The mass fractions of each component in the herbal combination are as follows: Codonopsis pilosula 10 parts, Ophiopogon japonicus 15 parts, Taxillus chinensis 10 parts, Dioscorea opposita 20 parts, Ligustrum lucidum 20 parts, Anemarrhena asphodeloides 20 parts, Eclipta prostrata 15 parts, Paeonia lactiflora 30 parts, Bupleurum chinense 10 parts, Citrus reticulata 10 parts, Lilium brownii 10 parts, Poria cocos 15 parts, Ziziphus jujuba var. spinosa 10 parts, and Schisandra chinensis 10 parts.

3. A method for preparing the traditional Chinese medicine composition according to claim 1, characterized in that, The steps for preparing the traditional Chinese medicine composition into an aqueous decoction are as follows: Step 1: Wash and dry the following herbs: Codonopsis pilosula, Ophiopogon japonicus, Taxillus chinensis, Dioscorea opposita, Ligustrum lucidum, Anemarrhena asphodeloides, Eclipta prostrata, Paeonia lactiflora, Bupleurum chinense, Citrus reticulata, Lilium brownii, Poria cocos, Ziziphus jujuba var. spinosa, and Schisandra chinensis. Grind them into coarse powder. Step 2: Place the coarse powder of medicinal materials obtained in Step 1 into a clay pot, add water to cover the medicinal materials and soak. Step 3: After soaking the herbs, bring them to a boil over high heat, then reduce to a simmer and cook slowly. Filter the liquid and collect the filtrate. Step 4: Add water to the residue again, boil, filter, and combine the two filtrates; Step 5: Concentrate the filtrate under reduced pressure to 200 mL to obtain the decoction of traditional Chinese medicine.

4. A method for preparing a traditional Chinese medicine composition for nourishing the kidneys and relieving depression according to claim 3, characterized in that, In step 1, the mass fractions of Codonopsis pilosula are 10 parts, Ophiopogon japonicus are 15 parts, Taxillus chinensis are 10 parts, Dioscorea opposita is 20 parts, Ligustrum lucidum is 20 parts, Anemarrhena asphodeloides is 20 parts, Eclipta prostrata is 15 parts, Paeonia lactiflora is 30 parts, Bupleurum chinense is 10 parts, Citrus reticulata is 10 parts, Lilium brownii is 10 parts, Poria cocos is 15 parts, Ziziphus jujuba var. spinosa is 10 parts, and Schisandra chinensis is 10 parts.

5. A method for preparing a traditional Chinese medicine composition for nourishing the kidneys and relieving depression according to claim 3, characterized in that, The soaking time for the medicinal herbs in step 2 is 30 minutes.

6. A method for preparing a traditional Chinese medicine composition for nourishing the kidneys and relieving depression according to claim 3, characterized in that, In step 3, after soaking the herbs, bring them to a boil over high heat and then simmer over low heat for 30 minutes.

7. A method for preparing a traditional Chinese medicine composition for nourishing the kidneys and relieving depression according to claim 3, characterized in that, In step 4, the residue is boiled again with water for 30 minutes.

8. A method for preparing the traditional Chinese medicine composition according to claim 1, characterized in that, The process of preparing the traditional Chinese medicine composition into pills is as follows: Codonopsis pilosula, Ophiopogon japonicus, Taxillus chinensis, Dioscorea opposita, Ligustrum lucidum, Anemarrhena asphodeloides, Eclipta prostrata, Paeonia lactiflora, Bupleurum chinense, Citrus reticulata, Lilium brownii, Poria cocos, Ziziphus jujuba var. spinosa, and Schisandra chinensis are ground into a fine powder, mixed with water, and then fed into a pill-making machine to form pills.

9. A method for preparing a traditional Chinese medicine composition for nourishing the kidneys and relieving depression according to claim 8, characterized in that, The weight percentages of Codonopsis pilosula are 10 parts, Ophiopogon japonicus are 15 parts, Taxillus chinensis are 10 parts, Dioscorea opposita is 20 parts, Ligustrum lucidum is 20 parts, Anemarrhena asphodeloides is 20 parts, Eclipta prostrata is 15 parts, Paeonia lactiflora is 30 parts, Bupleurum chinense is 10 parts, Citrus reticulata is 10 parts, Lilium brownii is 10 parts, Poria cocos is 15 parts, Ziziphus jujuba var. spinosa is 10 parts, and Schisandra chinensis is 10 parts.

10. The application of the traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is used in the preparation of a medicine for the prevention and / or treatment of perimenopausal syndrome and early-onset ovarian insufficiency.

Images