A new Chinese medicine component, chaishaosong capsule, for treating PMDD with liver-Qi stagnation and depression

Abstract

The application discloses a Chinese medicinal component new drug, safflower and paeony saponin soft capsules, for treating PMDD liver-Qi stagnation syndrome depression subtype. The safflower and paeony saponin soft capsules with stable quality are prepared by optimizing the process, and the mass ratio of safflower saponin and total paeony saponin is 0.05-0.075:0.04-0.06. The pharmacological and pharmacodynamic experiment results prove that the safflower and paeony saponin solution can significantly improve the depression-like emotion of Kunming mice in FST / TST; can significantly improve the depression emotion behavior appearance and pathological index changes of model rats in two PMDD liver-Qi stagnation syndrome depression subtypes, namely natural screening and progesterone withdrawal; and the pharmacological mechanism is to significantly increase the ALLO, P and E2 contents in peripheral serum of the model rats and to increase the ALLO content in the hippocampus brain area. The application further discloses a drug clinical test scheme for treating PMDD liver-Qi stagnation syndrome depression subtype. The application provides a Chinese medicinal component new safflower and paeony saponin soft capsule for treating PMDD liver-Qi stagnation syndrome depression subtype.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical technology, basic research and clinical application in psychiatry and gynecology, and specifically relates to a new Chinese medicine component, Chai Shao saponin soft capsule, for treating premenstrual dysmenorrhea (PMDD) with liver qi stagnation and depression subtype. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] PMDD is a severe form of premenstrual syndrome (PMS), referring to a mental illness syndrome in women of reproductive age characterized by mood disturbances, physical symptoms, behavioral changes, and decreased work capacity occurring 14-7 days before menstruation, with symptoms lessening and disappearing after menstruation. The American Diagnostic and Statistical Manual of Mental Disorders (DSM-5) classifies PMDD under the category of "Depressive Disorders." Due to its monthly premenstrual episodes, it causes significant psychological and physical distress to patients, impacting their work and interpersonal relationships, thus attracting considerable attention from the psychiatric community.

[0004] Internationally, the first-line drugs for treating PMDD are serotonin reuptake inhibitors (SSRIs) such as fluoxetine hydrochloride and sertraline, with a clinical efficacy rate of ≤60%. Domestically, the Western medicine treatments are the same as above; Traditional Chinese medicine treatments include "Jingqianshu Granules," a compound new drug for treating premenstrual syndrome (PMS) previously developed and marketed by our team, but there are currently no new drugs with clearly defined Chinese medicine components, efficacy, and mechanisms of action.

[0005] Large-scale, multicenter clinical studies on the efficacy of sertraline in treating PMDD have shown that it is significantly more effective than depressive symptoms in PMDD patients with the "Irritability Symptoms" subtype, as differentiated by the DRSP subscale. Given the significant limitations of sertraline as a first-line treatment for the depressive subtype of PMDD, the development of new drugs targeting this subtype has become a key international scientific and technological issue concerning PMDD.

[0006] Preliminary epidemiological surveys conducted by our team in three cities in Shandong Province and seven centers across four provinces and municipalities nationwide revealed that Liver Qi Reversal Syndrome and Liver Qi Stagnation Syndrome are the two main syndromes of PMS / PMDD. The main clinical manifestations of Liver Qi Reversal Syndrome are premenstrual irritability and anger, while Liver Qi Stagnation Syndrome is premenstrual depression and a tendency to withdraw from challenges. ICD-11 defines PMDD's main emotional types as irritability and depressive mood; this corroborates that Liver Qi Reversal Syndrome and Liver Qi Stagnation Syndrome correspond to the subtypes of irritability and depression, respectively.

[0007] The above indicates that developing new drugs for the liver qi stagnation syndrome and depression subtype, the main syndrome of PMDD, has become an urgent scientific and technological problem to be solved in the treatment of this syndrome subtype by both traditional Chinese and Western medicine, both domestically and internationally. Summary of the Invention

[0008] The purpose of this invention is to address the problems currently faced by medical technology by providing a new traditional Chinese medicine component, Chai Shao Saponin Soft Capsules, for treating PMDD liver qi stagnation syndrome and depression subtype.

[0009] The term "PMDD Liver Qi Stagnation Syndrome" refers to a syndrome that meets the diagnostic criteria for PMDD in Traditional Chinese Medicine (TCM) and is characterized by the pathogenesis and clinical manifestations of liver qi stagnation.

[0010] The term "PMDD depression subtype" refers to a clinical subtype characterized by depressed mood and withdrawal from challenges, as differentiated by the Daily Record of Symptom Severity Scale (DRSP) subscale scores in Western medicine based on the PMDD diagnostic criteria.

[0011] The term "traditional Chinese medicine components" refers to a group of pharmacologically active components with high homogeneity extracted from traditional Chinese medicine, i.e., a class of components.

[0012] The term "bupleurum saponins" is an abbreviation for bupleurum saponins and total peony glycosides. Total bupleurum glycosides refer to the pharmacologically active natural glycosides extracted from Bupleurum chinense; total peony glycosides refer to a class of pharmacologically active components, mainly paeoniflorin, extracted from Paeonia lactiflora.

[0013] The term "new drug" refers to a drug whose components and pharmacological effects differ from existing drugs and are not marketed in China. Chai Shao Saponin Soft Capsules fall under this definition as a new drug.

[0014] The term "bufossaponin soft capsules" refers to soft capsule preparations containing traditional Chinese medicine components such as bufossaponins and total glycosides of paeony.

[0015] To achieve the above objectives, the technical solution provided by this invention is as follows: The first aspect of this invention provides a method and process for preparing a bupleurum saponin soft capsule for treating PMDD liver qi stagnation syndrome depressive subtype. The bupleurum saponin soft capsule comprises contents and a capsule shell; by weight, the contents consist of bupleurum extract, soybean oil, beeswax, and soybean lecithin, with a soybean oil:bupleurum extract:beeswax:soybean lecithin ratio of 5~7:1:0.1~0.15:0.03~0.07. The dry extract of Paeonia lactiflora is composed of Paeonia lactiflora saponins and total glycosides of Paeonia lactiflora, with a mass ratio of Paeonia lactiflora saponins to total glycosides of Paeonia lactiflora of 0.05~0.075:0.04~0.06.

[0016] The present invention relates to a soft capsule containing bupleurum saponins, the main components of which are bupleurum saponins and total glucosides of paeony. The capsule shell is composed of commonly used glycerin, gelatin, water, and ethylparaben (an antibacterial preservative). The present invention solves the problem of content migration to the capsule shell by adding a surfactant to the contents, which solubilizes the drug and allows it to exist in capsule form, thus forming a larger molecular system and slowing down the diffusion of the contents.

[0017] In some specific embodiments of the present invention, the capsule shell is composed of gelatin, glycerin, water, and preservatives. Preferably, the ratio of gelatin, glycerin, and water is 1:0.4:1. The present invention solves the problem of uneven contents in soft capsules by adding soybean oil as a diluent to maintain the uniformity and fluidity of the contents for a longer period.

[0018] In some specific embodiments of the present invention, the preservative is ethylparaben. Ethylparaben is a broad-spectrum antibacterial preservative. Adding 0.2 wt% ethylparaben as a preservative for the soft capsule shell can effectively prevent the bupleuropaeoniflorin soft capsules from becoming moldy during storage.

[0019] Based on the above research, the present invention also provides a method for preparing the aforementioned peony saponin soft capsules, comprising the following steps: The dry extract of Paeonia lactiflora, beeswax, and soybean lecithin were dissolved in soybean oil, sieved, and the resulting liquid was obtained. Glycerin, water, and preservatives are heated to a certain temperature, gelatin is added to gelatinize, the mixture is dispersed evenly, degassed, and filtered to obtain a gel solution. The medicinal liquid and the gel were used to prepare soft capsules by compression, and then dried to obtain the peony saponin soft capsules.

[0020] In some specific embodiments of the present invention, the dried extract of Paeonia lactiflora is composed of Paeonia lactiflora saponins and total Paeonia lactiflora glycosides, with a mass ratio of Paeonia lactiflora saponins to total Paeonia lactiflora glycosides of 0.05~0.075:0.04~0.06.

[0021] The preparation method of saikosaponin is as follows: Bupleurum slices are refluxed twice with 6 times the amount of 70% ethanol for 2 hours each time. The extracts are combined and concentrated to 1 g crude drug / mL. The column is packed with macroporous resin D101 using the wet method. The sample volume of Bupleurum concentrate is 1.0 BV for dynamic adsorption. Then, it is eluted with 8.0 BV of 70% ethanol. The eluent is concentrated under reduced pressure and freeze-dried to obtain the saikosaponin extract. The preparation method of total glycosides of Paeonia lactiflora is as follows: Paeonia lactiflora slices are extracted three times with 8 times the amount of 30% ethanol, each time for 2 hours. The extracts are combined and concentrated to 0.75 g crude drug / mL. HPD-100 macroporous resin is packed into a column by wet loading at a diameter-to-height ratio of 1:10. A loading solution with a concentration of 0.75 g crude drug / mL is added at a loading flow rate of 7 BV / h for dynamic adsorption. Elution is performed with 50% ethanol at a flow rate of 7 BV / h and an elution volume of 2 BV. The eluent is concentrated under reduced pressure and freeze-dried to obtain the total glycosides extract of Paeonia lactiflora.

[0022] In some specific embodiments of the present invention, the temperature of the adhesive is 50~100℃, preferably 80~90℃.

[0023] In some specific embodiments of the present invention, degassing is performed by vacuuming, with a vacuum degree of -0.07 to -0.08 MPa. During the vacuuming process, the state of the adhesive should be carefully observed until no obvious bubbles are released from the surface and the adhesive surface shows a honeycomb-like flow, indicating that degassing is complete and the operation can be stopped.

[0024] In some specific embodiments of the present invention, the pressing is as follows: under the conditions of an ambient temperature of 16~18℃ and a relative humidity of ≤50%, the temperature of the glue box is preheated to 48~52℃, the temperature of the spray body is preheated to 38~42℃, the air compressor is turned on, and the glue liquid is pressed into the glue box through the glue delivery pipe; the blower is turned on, the thickness of the rubber sheet is adjusted, and a continuous tape is made; the medicine liquid is filled into the tape, and the soft capsule is obtained by rotating and pressing through a roller mold.

[0025] In some specific embodiments of the present invention, the drying is performed at 33~37°C and 28%~32% relative humidity for 3.5~4.5 h.

[0026] In a second aspect, the present invention provides pharmacodynamic and pharmacological studies of bupleuropaeoniflorin soft capsules conducted in two experimental animals and one experimental animal model of two disease syndromes, in accordance with the requirements of the "Classification and Application Materials for Registration of Traditional Chinese Medicine".

[0027] Specifically, three batches of experiments were conducted on Kunming mice to investigate the intervention of depressive-like mood with peony saponin soft capsules.

[0028] After 7 days of acclimatization, mice were administered the drug via gavage for 7 days. The bupleurum saponin solution was divided into three dose groups: high, medium, and low. The positive control group was the fluoxetine group. Forced swimming (FST) and tail suspension test (TST) were performed. The results of the three batches of experiments showed that, compared with the blank control group, the high and low dose groups of bupleurum saponin solution and the fluoxetine group significantly prolonged the suspension immobility latency and reduced the suspension immobility time in the third, second, and first batches of experiments, respectively. Among them, the medium dose group of bupleurum saponin solution and the fluoxetine group had the most significant effect on treating depressive-like mood in mice.

[0029] Furthermore, two disease-related animal models were established in female Wistar rats, and experiments were conducted on the efficacy and pharmacological mechanism of bupleuropaeoniflorin soft capsules in treating PMDD / PMDD-PWD liver qi stagnation syndrome depression subtype.

[0030] Specifically, Wistar female rats with naturally selected regular estrous cycles were used to establish a PMDD liver qi stagnation depression subtype rat model. Using FST, the difference between the non-receiving (NR) and receiving (R) periods of suspension immobility was calculated and ranked in descending order. The top 30% of the ranked rats met the evaluation criteria for the PMDD liver qi stagnation depression subtype rat model. The characteristic of increased NR suspension immobility time and significantly decreased R suspension immobility time in the model rats is consistent with the clinical manifestation of PMDD liver qi stagnation depression subtype symptoms appearing before menstruation and disappearing after menstruation, thus meeting the evaluation criteria for this disease model.

[0031] Furthermore, the results of gavage administration intervention showed that the combination of bupleuropaeoniflorin and fluoxetine significantly reduced the immobility time of NR suspension in model rats, thereby improving depressive mood. Among them, the high-dose bupleuropaeoniflorin group had the most similar therapeutic effect to the normal control group. Moreover, the pharmacological mechanism of this efficacy is that bupleuropaeoniflorin improves the symptoms of PMDD liver qi stagnation syndrome depressive subtype by increasing serum ALLO, P, and E2 levels, as well as ALLO levels in the hippocampus and P levels in the hypothalamus.

[0032] Given the small proportion of rats with regular estrous cycles and the instability of estrous cycles during the experiment, the preferred method was to replicate rats with regular estrous cycles using the ovariectomy and progesterone withdrawal method. A forced swimming test was then used to replicate the PMDD-PWD liver qi stagnation depression subtype rat model. Drug intervention results showed that, compared with the positive control drug fluoxetine, bupleuropaeoniflorin soft capsules significantly improved the emotional and behavioral manifestations of the liver qi stagnation depression subtype in the model rats. Furthermore, the pharmacological mechanism of this drug is that bupleuropaeoniflorin significantly increases the levels of ALLO, P, and E2 in the peripheral serum of the model rats, increases ALLO in the hippocampus, and decreases ALLO in the hypothalamus, thereby treating the symptoms of the PMDD liver qi stagnation depression subtype.

[0033] In a third aspect, the present invention provides a clinical trial research protocol for the use of bupleuropaeoniflorin soft capsules to treat PMDD liver qi stagnation syndrome depression subtype.

[0034] Specifically, the research protocol provides clear and complete regulations from case selection to the diagnosis of PMDD and its subtypes such as liver qi stagnation and irritability, including inclusion and exclusion criteria, treatment course, efficacy evaluation, and statistical methods. This provides the necessary application materials for subsequent registration of new traditional Chinese medicine drugs.

[0035] Furthermore, this study provides a research protocol for conducting clinical trials of bupleuropaeoniflorin saponins soft capsules to treat PMDD liver qi stagnation syndrome depression subtype.

[0036] Compared with the prior art, the above-described one or more technical solutions of the present invention have achieved at least the following beneficial effects: 1. This invention provides an animal model and clinical trial protocol for treating PMDD syndrome subtypes, specifically for the development of drugs for PMDD liver qi stagnation and depression subtypes. The development of drugs for treating PMDD syndrome subtypes requires suitable animal models and clinical trials. PMDD with liver qi stagnation and depression is a major syndrome subtype of PMDD, but existing technologies lack animal models and clinical trial protocols for this subtype. This invention establishes a naturally selected rat model of PMDD with liver qi stagnation and depression with stable estrous cycles. A model evaluation method was developed by calculating the difference between the non-receiving (NR) and receiving (R) periods of the FST in experimental rats and ranking them in descending order. The modeling experiment showed that the top 30% of the rats had increased NR suspension time and significantly reduced R suspension time. This is consistent with the clinical manifestation of depression in patients with PMDD with liver qi stagnation and depression, which appears before menstruation and disappears after menstruation. Therefore, this model is confirmed as a rat model of PMDD with liver qi stagnation and depression. To address the technical challenges of a low selection ratio of rats with regular estrous cycles and unstable estrous cycles during experiments, this invention further employs a forced swimming experiment using ovariectomy and progesterone withdrawal in experimental rats to establish a PMDD-PWD liver qi stagnation depression subtype rat model with stable estrous cycles and clinical characteristics and pathological changes of the PMDD liver qi stagnation depression subtype.

[0037] Furthermore, this invention discloses a clinical trial protocol for a drug to treat PMDD liver qi stagnation syndrome depression subtype.

[0038] This provides valuable animal models and clinical trial protocols for the development of new drugs for this disease, which can be referenced and applied in basic experimental and clinical research.

[0039] 2. The effective component ratio of the bupleuropaeoniflorin soft capsules of the present invention is clear and the preparation quality is stable. Pharmacological and efficacy experiments have shown that the drug has significant efficacy in treating PMDD liver qi stagnation and depression subtype and the pharmacological mechanism is clear.

[0040] The present invention provides a soft capsule formulation with advanced technology and stable quality by optimizing the process to determine the ratio of saikosaponin to total peony glycosides as 0.05~0.075:0.04~0.06.

[0041] Pharmacological and efficacy experiments of *Chaiyao* saponin soft capsules demonstrated: 1) Significant antidepressant effect: Experiments on three batches of Kunming mice using the FST / TST method showed that the medium-dose group of *Chaiyao* saponin solution had the most significant antidepressant-like effect. 2) Significant improvement in the apparent behavior and pathological indicators of two PMDD liver qi stagnation depression subtype rat models (natural selection and progesterone withdrawal), with a clear mechanism of action. Compared with the positive control drug fluoxetine, *Chaiyao* saponin soft capsules significantly improved the emotional and behavioral manifestations of the liver qi stagnation depression subtype in model rats. Furthermore, the pharmacological mechanism of this efficacy is that *Chaiyao* saponin significantly increases the levels of ALLO, P, and E2 in the peripheral serum of model rats, increases ALLO in the hippocampus, and decreases ALLO in the hypothalamus, thereby treating the symptoms of the PMDD liver qi stagnation depression subtype.

[0042] This invention provides a solution to the problem of new Chinese medicine components for which no specific components have been identified and whose efficacy and mechanism of action have been clearly defined in medical technology.

[0043] Furthermore, pharmaceutical, pharmacological, and efficacy application materials were prepared for the registration application of new traditional Chinese medicine drugs.

[0044] 3. The registration application for the bupleurum saponin soft capsule of this invention is for an innovative traditional Chinese medicine, and it is expected to obtain a drug clinical trial approval.

[0045] The total saponins of Paeonia lactiflora soft capsules have been included in the "Product Development Plan" of the pharmaceutical company cooperating with the applicant of this invention. For details, please refer to "Other Supporting Documents" "Research and Development Plan of Paeonia lactiflora Saponins Soft Capsules for External Use to Eliminate Breast Stasis".

[0046] Other beneficial effects and features of the present invention will be partly apparent from the following description of the accompanying drawings. Attached Figure Description

[0047] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0048] Figure 1 Technical roadmap for the preparation of paeoniflorin saponins soft capsules; Figure 2Flowchart of the molding process for peony saponin soft capsules; Figure 3 Comparison of suspension immobility time in mouse FST groups: first batch (A), second batch (B), and third batch (C); Control represents blank control group, Fluoxetine represents fluoxetine treatment group, ChaiShao-H represents high-dose ChaiShao-Paeonia lactiflora extract treatment group, ChaiShao-M represents medium-dose ChaiShao-Paeonia lactiflora extract treatment group, and ChaiShao-L represents low-dose ChaiShao-Paeonia lactiflora extract treatment group. Figure 4 Comparison of the first batch (A), the second batch (B), and the third batch (C) of the suspension immobility latency of mice in different FST groups; Control represents the blank control group, Fluoxetine represents the fluoxetine treatment group, ChaiShao-H represents the high-dose ChaiShao-Pei extract treatment group, ChaiShao-M represents the medium-dose ChaiShao-Pei extract treatment group, and ChaiShao-L represents the low-dose ChaiShao-Pei extract treatment group. Figure 5 Comparison of resting time in mouse TST groups in the first batch of experiments (A), the second batch of experiments (B), and the third batch of experiments (C); Control represents the blank control group, Fluoxetine represents the fluoxetine treatment group, ChaiShao-H represents the high-dose ChaiShao-Paeonia lactiflora extract treatment group, ChaiShao-M represents the medium-dose ChaiShao-Paeonia lactiflora extract treatment group, and ChaiShao-L represents the low-dose ChaiShao-Paeonia lactiflora extract treatment group. Figure 6 Comparison of the first three FST suspension immobility times (A) in the model group and the first three suspension immobility times (B) in the normal control group; Model-NR1 represents the non-acceptance period experiment in the first time period, Model-R represents the acceptance period experiment in the third time period, and Model-NR2 represents the non-acceptance period experiment in the fifth time period; * p <0.05; Figure 7 Comparison of the number of FST suspension immobilities in the model group during the first three administrations (A) and the number of suspension immobilities in the normal control group during the first three administrations (B); Model-NR1 represents the non-acceptance period experiment in the first time period, Model-R represents the acceptance period experiment in the third time period, and Model-NR2 represents the non-acceptance period experiment in the fifth time period; * p <0.05; Figure 8Comparison of FST suspension immobility time before and after administration in the normal control group (Control), model group (Model), fluoxetine group (Fluoxetine), high-dose ChaiShao saponin group (ChaiShao-H), medium-dose ChaiShao saponin group (ChaiShao-M), and low-dose ChaiShao saponin group (ChaiShao-L); NR1 represents the D1 phase experiment within the first time period, R1 represents the R phase experiment within the third time period, NR2 represents the D1 phase experiment within the fifth time period, NR3 represents the D1 phase experiment after administration, and R2 represents the R phase experiment after administration; compared with the suspension immobility time in R1 phase, * p <0.05; compared with the suspension time in R2, # p <0.05; Figure 9 Figure A: Comparison of OT% between the model group and the normal control group at each stage before drug administration (A); Comparison of OE% between the model group and the normal control group at each stage before drug administration (B); NR1 represents the D1 stage experiment within the first time period, R1 represents the R stage experiment within the third time period, and NR2 represents the D1 stage experiment within the fifth time period; Figure A: Comparison of OT% in the R stage between the model group and the normal control group, * p <0.05; Figure 10 Comparison of OT% changes before and after administration in the normal control group (Control), model group (Model), fluoxetine group (Fluoxetine), high-dose ChaiShao saponin group (ChaiShao-H), medium-dose ChaiShao saponin group (ChaiShao-M), and low-dose ChaiShao saponin group (ChaiShao-L); NR1 represents the D1 phase experiment within the first time period, R1 represents the R phase experiment within the third time period, NR2 represents the D1 phase experiment within the fifth time period, NR3 represents the D1 phase experiment after administration, and R2 represents the R phase experiment after administration; compared with the OT% of R1 phase, * p <0.05; Figure 11 Comparison of the N-phase modification time before drug administration between the normal control group and the model group (A), comparison of the number of N-phase modifications before drug administration between the normal control group and the model group (B), comparison of the N-phase modification time after drug administration between the normal control group and the model group (C), and comparison of the number of N-phase modifications after drug administration between the normal control group and the model group (D). Figure 12 Comparison of the N-phase modification time before administration (A) and after administration (B) among the normal control group (Control), fluoxetine group, high-dose ChaiShao saponin group (ChaiShao-H), medium-dose ChaiShao saponin group (ChaiShao-M), and low-dose ChaiShao saponin group (ChaiShao-L). Figure 13 Comparison of ALLO levels in peripheral blood serum, hypothalamus, and hippocampus of rats in different groups; Serum, Hippocampus, and Hypothalamus represent the comparison of ALLO levels in peripheral blood serum, hippocampus, and hypothalamus, respectively; compared with the model group, **** p <0.0001, *** p <0.001,** p <0.01, * p <0.05; Figure 14 Comparison of P content in peripheral blood serum, hypothalamus, and hippocampus of rats in different groups; Serum, Hippocampus, and Hypothalamus represent the P content in peripheral blood serum, hippocampus, and hypothalamus, respectively; compared with the model group, * p <0.05,** p <0.01,,*** p <0.001; Figure 15 Comparison of E2 levels in peripheral blood serum of rats in different groups; compared with the model group, ** p <0.01,* p <0.05,*** p <0.001; Figure 16 The experimental protocol for establishing a PMDD-PWD rat model of liver qi stagnation and depression subtype includes two experimental parts: A. Schedule of Experiment Part 1; B. Schedule of Experiment Part 2; D1, estrus stage 1; D2, estrus stage 2; P / E, preestrus / endometrial stage; M, metestrus; FST, forced swimming test; intragastric injection; Figure 17 Results of the forced swimming test in rats treated with fluoxetine and induced by hormones; A represents the resting time, B represents the number of resting rats, C represents the resting latency; N represents the test in the non-acceptance phase; R represents the test in the acceptance phase;** p <0.01 compared to the control group, *** p <0.001 compared to the control group, **** p <0.0001 compared to the control group, ### p <0.001 and model group, #### p <0.0001 and model group (n=8 per group; two-way ANOVA followed by post-hoc Sidak multiple comparison test); Figure 18The study included: Model group, Control group, Fluoxetine group, ChaiShao-H high-dose ChaiShao saponin group, ChaiShao-M medium-dose ChaiShao saponin group, ChaiShao-L low-dose ChaiShao saponin group, ShameOperation group, and Normal group. The suspension immobility time of each group was compared before administration, before administration, after administration, and after administration. Figure 19 Comparison of suspension immobility time in model group rats; Figure 20 Comparison of suspension immobility time in normal control group rats; Figure 21 Comparison of suspension immobility time in rats of different drug-treated groups; Figure 22 Results of detection of NE, GABA, 5-HT, and Glu in the left hippocampus; Figure 23 : Detection results of NE, GABA, 5-HT, and Glu in the right hippocampus; Figure 24 Results of GABA and Glu content detection in the hypothalamus; Figure 25 Results of detection of NE, GABA, 5-HT and Glu content in peripheral serum. Detailed Implementation

[0049] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0050] Example 1 Preparation Study of Paeonia lactiflora saponins soft capsules 1. Extraction and purification process of paeony saponins The extraction and purification method of saikosaponins is as follows: Bupleurum slices were refluxed twice with 6 times the amount of 70% ethanol for 2 hours each time. The extracts were combined and concentrated to 1 g crude drug / mL. A macroporous resin D101 was used for wet column packing. The concentrated Bupleurum extract was loaded with a volume of 1.0 BV for dynamic adsorption, followed by elution with 8.0 BV of 70% ethanol. The eluent was concentrated under reduced pressure and freeze-dried to obtain the saikosaponin extract.

[0051] The extraction and purification method of total glycosides of Paeonia lactiflora is as follows: Paeonia lactiflora slices are extracted three times with 8 times the amount of 30% ethanol, each time for 2 hours. The extracts are combined and concentrated to 0.75 g crude drug / mL. HPD-100 macroporous resin is packed into a column by wet loading at a diameter-to-height ratio of 1:10. A loading solution with a concentration of 0.75 g crude drug / mL is added at a loading flow rate of 7 BV / h for dynamic adsorption. Elution is performed with 50% ethanol at a flow rate of 7 BV / h and an elution volume of 2 BV. The eluent is concentrated under reduced pressure and freeze-dried to obtain the total glycosides extract of Paeonia lactiflora.

[0052] 2. Preparation process of Paeonia lactiflora saponins soft capsules Preparation and quality control indicators of paeony saponins soft capsules This invention discloses an oral pharmaceutical composition for treating PMDD (pericardium-induced liver qi stagnation syndrome). The main components include bufonin and total glucosides of paeony, as well as glycerin, gelatin, water, and ethylparaben (an antibacterial preservative), which are commonly used in soft capsule shells. This invention addresses the problem of contents migration to the capsule shell by adding a surfactant to solubilize the drug and allow it to exist in capsule form, thus creating a larger molecular system and slowing down the diffusion of the contents. Furthermore, the addition of soybean oil as a diluent allows the contents to remain for a longer period, thereby solving the problem of uneven contents distribution in soft capsules.

[0053] Its specific preparation steps are as follows: 2.1 Preparation of soft capsule contents (1) Selection of diluent Diluents increase the flowability of drugs to ensure accurate dosage and smooth filling. The components in the Chai Shao soft capsules have poor water solubility; using PEG400 can lead to uneven dispersion, and the prepared soft capsules may experience shell hardening, discoloration, deformation, and prolonged disintegration time during storage. After screening, soybean oil was finally selected as the diluent. Simultaneously, using a comprehensive score of uniformity (30%), flowability (30%), and sedimentation volume ratio (40%) as evaluation indicators, the optimal feed ratio of dry extract to soybean oil was investigated at ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7 to determine the optimal feed ratio.

[0054] Table 1. Investigation of Soybean Oil Ratio

[0055] Conclusion: The highest comprehensive score was achieved when the ratio of medicinal powder (i.e., dry extract of Paeonia lactiflora, saikosaponin and total peony glycosides in the form of 0.05~0.075:0.04~0.06): soybean oil was 1:5. Therefore, this ratio was selected.

[0056] (2) Selection of suspending agents In preparing suspensions of soft capsule contents, it is usually necessary to add an appropriate amount of suspending agent to increase the viscosity of the dispersion medium, thereby improving the uniformity of the suspension and enhancing the stability of the soft capsule contents. This experiment used uniformity (30%), flowability (30%), and sedimentation volume ratio (40%) as indicators to investigate the suspending effect of dry extract:beeswax at ratios of 5:1, 10:1, 15:1, 20:1, and 25:1.

[0057] Table 2. Investigation of beeswax ratio

[0058] Conclusion: The highest overall score was achieved when the ratio of medicinal powder (i.e., dry extract of peony root extract) to beeswax was 10:1, therefore this ratio was chosen.

[0059] (3) Selection of wetting agent Meanwhile, to prevent the complex from settling and to ensure its uniform dispersion in soybean oil, a wetting agent, soybean lecithin, was added. In this experiment, uniformity (30%), fluidity (30%), and sedimentation volume ratio (40%) were used as indicators to investigate the suspending effect of peony extract dry extract: lecithin at ratios of 1:0.03, 1:0.04, 1:0.05, 1:0.06, and 1:0.07.

[0060] Table 3. Investigation of the proportion of soybean lecithin

[0061] Conclusion: The highest overall score was achieved when the ratio of medicinal powder (i.e., dry extract of Paeonia lactiflora extract) to soybean lecithin was 1:0.06, therefore this ratio was selected.

[0062] (4) Selection of surfactants Tween-80 is a commonly used surfactant. In pharmaceuticals, Tween-80 can function as an emulsifier, dispersant, stabilizer, and solubilizer. In the preparation of the soft capsule contents of this invention, Tween-80 is used as the surfactant, which greatly improves the solubility of lipid-soluble extracts such as saikosaponins and total glycosides of paeony, slowing down the diffusion of the contents and thus solving the problem of content migration to the capsule shell. Through literature review, the optimal dosage of Tween-80 was determined to be 1:0.01 for the dry extract of paeony and saeony: Tween-80.

[0063] In summary, the contents are prepared by homogenizing a mixture of soybean oil and peony extract, beeswax, soybean lecithin and surfactant Tween-80 in a mass ratio of 5:1:0.1:0.06:0.01.

[0064] 2.2 Preparation of soft capsule shells The most common formulation for soft capsule shells is glycerin, gelatin, and water. This experiment designed different proportions of glycerin and gelatin, followed by water bath heating, mixing, and cooling. The hardness and elasticity of the soft capsule shells were used as evaluation indicators to determine a suitable soft capsule shell preparation process.

[0065] (1) Selection of preservatives Considering that capsules may become moldy during storage, an appropriate amount of preservative needs to be added to the capsule shell. Ethylparaben is a broad-spectrum antibacterial preservative. 0.2wt% ethylparaben (i.e., ethylparaben content is 0.2% of the total mass of the soft capsule shell) is selected as the preservative for the soft capsule shell. The type of preservative cannot be replaced.

[0066] (2) Selection of glycerin-gelatin ratio This experiment used the hardness and elasticity of soft capsule shells as indicators to investigate the properties of the shells when the gelatin-glycerol ratios were 1:0.3, 1:0.4, 1:0.5, 1:0.6, and 1:0.7, and screened out the optimal gelatin-glycerol ratio.

[0067] Table 4 Results of the study on the elasticity and hardness of the rubber sheet under different gelatin-glycerol ratios

[0068] Conclusion: When the ratio of gelatin to glycerol is 1:0.4, the resulting capsule shell has good elasticity, high strength, and good molding properties.

[0069] (3) Selection of heating temperature This experiment used the hardness and elasticity of the soft capsule shell as indicators to investigate the properties of the shell at water bath temperatures of 50℃, 60℃, 70℃, 80℃, and 90℃, and screened out the optimal heating temperature.

[0070] Table 5 Results of the study on the elasticity of rubber and the solubility of gelatin under different temperature conditions

[0071] Conclusion: Gelatin dissolves well and exhibits high elasticity at temperatures of 80 and 90℃. Considering both experimental simplicity and energy efficiency, a dissolution temperature of 80℃ was chosen.

[0072] In summary, the capsule shell solution is prepared by mixing gelatin, glycerin and water in a mass ratio of 1:0.4:1 and then heating to 80°C and stirring to dissolve.

[0073] 2.3 Preparation of Paeonia lactiflora saponin soft capsules Take glycerin, purified water, preservative, and gelatin according to the proportions shown in the above experiments. First, put the glycerin, purified water, and preservative into a solvent tank, stir and heat, then add gelatin granules, stir, and use a vacuum pump to defoam and degas the solution. Observe the condition of the solution, filter it through an 80-mesh filter, place it in a glue bucket, keep it warm and let it stand, fill the material, press it into capsules, and dry it to obtain Chai Shao soft capsules.

[0074] Specifically, soybean oil, dried extract of *Coptis chinensis*, beeswax, soybean lecithin, and surfactant Tween-80 are taken in a mass ratio of 5:1:0.1:0.06:0.01. The dried extract of *Coptis chinensis*, beeswax, and soybean lecithin are dissolved in soybean oil, sieved, and the solution is obtained.

[0075] Mix gelatin, glycerin, and water in a mass ratio of 1:0.4:1, and add 0.2wt% ethylparaben (preservative). Heat the mixture of glycerin, water, and preservative to 80°C, then add the gelatin to gelatinize and disperse evenly. Degas using a vacuum method at a vacuum level of -0.07 to -0.08 MPa. During vacuuming, carefully observe the state of the adhesive solution until no obvious bubbles are released from the surface and the surface exhibits a honeycomb-like flow pattern, indicating that degassing is complete and operation can be stopped. Under ambient temperatures of 16-18°C and relative humidity ≤50%, preheat the adhesive box to 50°C and the spray nozzle to 40°C. Turn on the air compressor to press the adhesive solution from the insulated container into the adhesive box through the delivery pipe. Turn on the blower and adjust the adhesive thickness by adjusting the gap between the oil shaft and the rollers to spread the adhesive onto the rollers to form a continuous tape. The contents flow through the feeding system to the filling pump, and the nozzle injects and fills the tape. The tape is then rotated and pressed into soft capsules by the roller mold. The prepared soft capsules were transferred to a rotary dryer and dried at 35°C and 30% relative humidity for 4 hours. This yielded the Paeonia lactiflora saponin soft capsules.

[0076] Under the standard formulation, the soft capsules dissolve in the stomach within 1-2 hours after administration, allowing the drug to be absorbed.

[0077] Example 2 Antidepressant effects of peony saponins soft capsules Depressive mood and behavior are the main symptoms of the depression subtype of PMDD liver qi stagnation syndrome. This pharmacodynamic experiment was conducted to verify the antidepressant effect of Chai Shao saponin soft capsules.

[0078] 1.1 Experimental Materials 1.1.1 Medicines and Reagents

[0079] 1.1.2 Experimental Apparatus

[0080] 1.2 Experimental Methods After arriving at the laboratory, Kunming mice underwent a one-week acclimatization period with free access to food and water. A 12h / 12h light / dark cycle was maintained, with day and night reversed. The laboratory temperature was controlled at 23±1℃, humidity at 50%–60%, and noise level ≤60dB. All experimental procedures were conducted under dim red light (<12 lux). All animal experimental procedures followed the 2015 revised "Public Health Service Policy on the Humane Management and Use of Laboratory Animals" issued by the National Institutes of Health (NIH). After the acclimatization period, ungrouped mice were weighed and grouped according to body weight using a uniform grouping method. Each batch of mice consisted of: a high-dose group of Paeonia lactiflora saponin extract, a medium-dose group, a low-dose group, a fluoxetine group, and a blank control group, with 10 mice in each group, housed in separate cages of 5 mice each.

[0081] (1) Animal administration In this experiment, mice were administered the drug at a dose of 0.1 mL / 10 g via gavage. After the acclimatization period, mice were administered the drug via gavage for 7 days, with the last gavage administered 30 minutes before the behavioral experiments. The mice were given different doses of the peony saponin extract: a high-dose group (18 g / kg), a medium-dose group (9 g / kg), a low-dose group (4.5 g / kg), and a fluoxetine group (4.05 mg / kg). The blank control group received an equal volume of pure water.

[0082] (2) Behavioral experiments The forced swimming experiment utilized the Smart 3.0.05 animal behavior video acquisition and analysis system, employing activity level analysis to collect behavioral indicators such as immobility time and immobility latency. The procedure for the forced swimming experiment in mice was as follows: 24 hours prior to the experiment, mice were placed in a transparent glass cylinder (40cm high, 20cm diameter) at a water temperature of 23℃±1℃ and a depth of approximately 30cm, ensuring that the tail tip and hind paws did not touch the bottom of the cylinder, allowing the mice approximately 6 minutes for acclimatization. The actual experiment involved continuous recording for 6 minutes using a camera, following the same method. The water was changed and the cylinder walls were cleaned after each mouse's swim to ensure good light transmission and to avoid the influence of odors or other factors. After swimming, the mice were gently wiped dry with a soft towel and returned to their original cages. Immobility refers to the mouse maintaining buoyancy on the water surface with only minimal movement, ensuring that its nostrils are above water. The immobility time was analyzed after the experiment. The tail suspension test primarily uses behavioral testing software to obtain the relative and absolute resting times of test mice to reflect their depressive state and the effects of antidepressants and stimulants on their current behavioral performance. During the experiment, medical tape is used to fix the mouse's tail to the bottom of the horizontal bar of the test box, with the mouse's head downwards and its nose 30cm above the ground, in an inverted state. Two minutes after the start of the experiment, the software records the mouse's activity over a four-minute period, accumulating the total time and number of times it remains still. Care must be taken to ensure the tape is applied properly to avoid injuring the mouse.

[0083] (3) Statistical analysis All data were analyzed and plotted using GraphadPrism 7.04 software. One-way ANOVA was used for comparisons between multiple groups. Results are expressed as Mean ± SEM (mean standard error of mean). p <0.05 and p A value <0.01 indicates a statistically significant difference.

[0084] 1.3 Experimental Results The FST behavioral characterization was used to verify the intervention effect of Bupleurum extract combined with paeoniflorin on depressive-like mood in mice. The results of three replicates showed that: ① Time spent suspended motionless: such as Figure 3 As shown, in the first batch of experiments, the suspension immobility time of the fluoxetine group and the medium-dose group of peony saponin extract was significantly reduced compared with the blank control group. p <0.05), the suspension time of the high and low dose groups of Paeonia lactiflora saponin extract decreased compared with the blank control group, but there was no significant difference. p>0.05); In the second batch of experiments, the suspension time of the fluoxetine group, the high, medium and low dose groups of peony saponin extract were significantly reduced compared with the blank control group. p <0.01, p <0.0001); In the third batch of experiments, the suspension time of the fluoxetine group, the high- and medium-dose groups of peony saponin extract was significantly reduced compared with the blank control group. p <0.05, p <0.01), the suspension time of the low-dose group of Paeonia lactiflora saponin extract showed a decreasing trend compared with the blank control group, but there was no significant difference. p >0.05); ②Lapse period of immobility: such as Figure 4 As shown, in the first batch of experiments, the low-dose group of Paeonia lactiflora saponin extract showed a significantly increased suspension latency compared to the blank control group. p <0.05), the suspension immobility latency of the fluoxetine group, the high- and medium-dose groups of peony saponin extract showed an increasing trend compared with the blank control group, but there was no significant difference. p >0.05); In the second batch of experiments, the high-dose group of peony saponin extract showed a significantly increased suspension latency compared to the blank control group. p <0.05), the suspension time of the medium-dose group of Paeonia lactiflora saponin extract decreased compared with the blank control group. p >0.05), the suspension immobility latency of the fluoxetine group and the low-dose group of paeoniflorin extract showed an increasing trend compared with the blank control group, but there was no significant difference. p >0.05); In the third experiment, the suspension immobility latency of the fluoxetine group was significantly increased compared with the blank control group ( p <0.05), the suspension immobility latency of the high, medium, and low dose groups of Paeonia lactiflora saponin extract showed an increasing trend compared with the blank control group. p >0.05).

[0085] ③ The tail suspension test evaluates the depressive-like mood and the effect of drug intervention in mice by measuring the time they remain still. The experiment shows that: Figure 5 As shown, in the first batch of experiments, the fluoxetine group and the high-dose group of paeoniflorin extract had significantly reduced immobility time compared to the blank control group. p <0.01), the resting time of the medium-dose group of peony saponin extract was significantly reduced compared with the blank control group. p <0.05), the low-dose group of Paeonia lactiflora saponin extract showed a trend of reduced immobility time compared with the blank control group. p >0.05); In the second batch of experiments, the resting time of the fluoxetine group and the saponin extract of Paeonia lactiflora was significantly reduced compared with the low-dose group and the blank control group. p <0.01,p <0.05), the high-dose group of Paeonia lactiflora saponin extract showed a trend of decreasing immobility time compared with the blank control group, but the difference was not statistically significant. P >0.05); In the third batch of experiments, the resting time of the fluoxetine group and the high and low dose groups of peony saponin extract was significantly reduced compared with the blank control group. p <0.01), the resting time of the medium-dose group of peony saponin extract was significantly reduced compared with the blank control group. p <0.05).

[0086] 1.4 Experimental Conclusions The experimental results showed that, in the three batches of FST experiments, the suspension immobility time of the fluoxetine group and the high, medium, and low dose groups of *Chaiyao* saponin extract showed a decreasing trend compared with the blank control group. Specifically, the fluoxetine group significantly shortened the suspension immobility time in all three batches of experiments, as did the medium dose group of *Chaiyao* saponin extract. The high and medium dose groups of *Chaiyao* saponin extract significantly shortened the suspension immobility time in the second and third batches of experiments, respectively. Regarding the suspension immobility latency, except for the medium dose group of *Chaiyao* saponin extract, which showed a significant shortening compared with the blank control group in the second experiment, all other batches of experiments showed a trend of prolonged suspension immobility latency compared with the blank control group. The fluoxetine group and the high and low dose groups of *Chaiyao* saponin extract significantly prolonged the suspension immobility latency in the third, second, and first batches of experiments, respectively. This suggests that all treatment groups can reduce the suspension time, but the fluoxetine group and the medium-dose group of bupleurum saponin extract showed the best effect in intervening in the depressive-like mood of mice. The antidepressant fluoxetine and the suspension latency in response to the antidepressant state also corroborate that the various treatment groups of bupleurum saponin extract produced an antidepressant effect rather than a neuroexcitatory effect.

[0087] In the TST experiment, the fluoxetine group significantly shortened the immobility time of mice in all three batches of experiments compared to the blank control group. However, the high-dose group of *Chaiyao* saponin extract only significantly reduced the immobility time in the second batch of experiments, while the low-dose group of *Chaiyao* saponin extract significantly reduced the immobility time in the second and third batches of experiments. This suggests that the higher and lower doses of *Chaiyao* saponin extract have better antidepressant effects in the tail suspension experiment.

[0088] The results of interventions for depressive-like mood in the two behavioral experiments were not entirely consistent. For example, the medium-dose group of bupleurum saponin extract significantly shortened the immobility time in the FST experiment, and the effect was better than that of the low-dose group. However, the low-dose group of bupleurum saponin extract was more effective than the medium-dose group in reducing immobility time in the TST experiment, and the latter did not show a significant reduction in the TST experiment. This result may be due to the different sensitivities of the two behavioral methods to the drugs and the endogenous neurotransmitters in mice.

[0089] Example 3 The intervention effect and pharmacological mechanism of bupleuropaeoniflorin soft capsules on a naturally screened rat model of PMDD liver qi stagnation syndrome and depression. The onset of symptoms before menstruation, with symptoms lessening or disappearing during and after menstruation, is a characteristic of PMDD. To examine the pharmacological efficacy of Chai Shao Saponin Soft Capsules in treating the depressive subtype of PMDD with liver qi stagnation, the following experiment was conducted.

[0090] 1. The intervention effect of paeoniflorin saponins soft capsules on the behavioral epigenetics of a naturally screened PMDD rat model with liver qi stagnation syndrome. 1.1 Experimental Materials 1.1.1 Medicines and Reagents

[0091] 1.1.2 Experimental Apparatus

[0092] 1.2 Experimental Methods After initial introduction to the laboratory, experimental rats were acclimatized for 7 days to familiarize themselves with the experimental environment. Rats were randomly assigned to cages of 5. Starting on the second day of acclimatization, grasping manipulation was performed daily to minimize the adverse effects of subsequent human intervention. During this period, rats had free access to water and food, maintaining a 12h / 12h light / dark cycle. A reversed day-night cycle was implemented (lights on at 8:00 PM, lights off at 8:00 PM). The room temperature was maintained at 23±1℃, with dim red light (<25 lux) and noise ≤60dB. All animal experiments complied with the 2015 revised Public Health Service (PHS) Humane Management and Use of Laboratory Animals policy. All behavioral tests and other experimental procedures were conducted during the rats' dark period (8:00-8:00 PM).

[0093] Eight days after arriving at the laboratory, rats were screened based on the regularity of their estrous cycles using a fertility analysis method (vaginal resistance measurement). Vaginal resistance was measured daily between 13:00 and 15:00 using a rat fertility analyzer. The rat estrous cycle is divided into receptive phases (R) and non-receptive phases (NR). The R phase lasts approximately one day and includes the proestrous stage (P) and estrus (E). The NR phase lasts approximately three days and includes one day each of metestrus (M), diestrus 1 (D1), and diestrus 2 (D2). A resistance value greater than 3 Kohms indicated a receptive phase, while a resistance value less than 3 Kohms indicated a non-receptive phase. Subsequent experimental procedures were based on rats with regular estrous cycles.

[0094] Rats exhibiting consistent vaginal electrical resistance values ​​for two or more consecutive estrous cycles were deemed suitable for the PMDD liver qi stagnation rat model. Only rats with regular estrous cycles were included and subjected to subsequent experimental procedures. The PMDD liver qi stagnation model rats should meet the following criteria: regular estrous cycles and recurring premenstrual (R phase) depressive mood, which disappears postmenstrual (NR phase). Utilizing the characteristic that the suspension time of rats in the forced swimming test reflects depressive mood and the degree of depression, this part of the study selected the R and NR phases of the estrous cycle as time points for experiments on rats with regular estrous cycles. Finally, the difference in suspension time between the R and NR phases within the two time points was used to determine the model rats suitable for PMDD liver qi stagnation.

[0095] The specific experimental procedure was as follows: After screening rats with regular estrous cycles using vaginal electrical resistance testing, rats in the D1 phase were selected daily for behavioral testing during the first time period (4 days). The tests included: an elevated cruciate maze test starting at 8:00 AM, followed by the FST test. Smart3.0.05 software was used to collect data on suspension time, number of suspensions, and suspension latency. This was a single-rat experimental procedure, with each rat undergoing the same procedures sequentially. During the second time period (4 days), to avoid frequent stimulation, only rats in the D1 phase were selected for the Splashtest, with video recording of the duration and frequency of the rats' modified behaviors. In the third time period (4 days), rats in the R phase were selected for the EPM and FST tests, with the same data collection indicators as in the D1 phase. In the fourth time period (4 days), test rats in the R phase were selected for water spraying experiment, and the recorded indicators were the same as in the D1 phase. In the fifth time period (4 days), test rats in the NR phase were selected for EPM and FST experiments, and the collected indicators were the same as in the D1 phase.

[0096] By detecting the estrous cycle of 220 rats, 60 rats with regular estrous cycles of two or more consecutive cycles were screened out.

[0097] After the above behavioral experiments were completed, the suspension immobility time collected by the FST experiment was taken as the main analysis index. The difference between the suspension immobility time and the R period was calculated and sorted in descending order. The top 30% of the rats in the ranking met the inclusion criteria for the PMDD liver qi stagnation rat model, namely, exhibiting depressive-like symptoms with prolonged immobility time in the non-acceptance phase and shortened immobility time in the acceptance phase. These rats were randomly grouped according to the difference in immobility time and designated as the PMDD liver qi stagnation rat model group (Model), fluoxetine group (Fluoxetine, 2.7 mg / kg), high-dose ChaiShao saponin group (ChaiShao-H, 0.4 g / kg), medium-dose ChaiShao saponin group (ChaiShao-M, 0.2 g / kg), and low-dose ChaiShao saponin group (ChaiShao-L, 0.1 g / kg). The difference in immobility time between the NR and R phases was close to 0, indicating no difference in depressive-like mood between the two phases, consistent with the behavior of normal rats, and was designated as the normal control group (Control). Rats with a difference of more than 30% and not meeting the criteria for normal rats were excluded.

[0098] After screening and grouping the rats, drug administration began, with a dosing cycle of approximately 12 days (three estrous cycles). Administration was performed via gavage, with 1 mL of drug administered per 100g rat, at 8:30 AM daily.

[0099] 1.3 Experimental Results ① Forced swimming experiment The FST experiment performed before drug administration is one of the main bases for screening PMDD liver qi stagnation syndrome model rats. The results of suspension immobility time and suspension immobility number of rats in each group at different time points showed: ① Figure 6 As shown, the suspension immobility time of the model group rats was significantly higher in both non-acceptance periods than in the acceptance period. p <0.05), while in the normal control group, the suspension immobility time during the two non-acceptance periods did not show a significant increasing or decreasing trend compared to the suspension immobility time during the acceptance period, and there was no significant difference. p >0.05), therefore, it is believed that the characteristic of increased immobility during the non-acceptance period and significantly decreased immobility during the acceptance period in the model rats is consistent with the clinical manifestation of PMDD liver qi stagnation syndrome symptoms appearing before menstruation and disappearing after menstruation. ② As Figure 7 As shown, the number of times the model rats remained suspended in place was consistent with the trend of suspension time, that is, the number of times they remained suspended in place during the non-acceptance period was more than the number of times they remained suspended in place during the acceptance period, and the number of times they remained suspended in place during the second non-acceptance period test was significantly more than the number of times they remained suspended in place during the acceptance period. p <0.05); There was no significant difference in the number of times rats remained suspended motionless during the two non-acceptance periods and the number of times they remained suspended motionless during the acceptance period between the two non-acceptance periods ( ). p >0.05).

[0100] The FST test performed after drug administration is one of the main methods to evaluate the effect of bupleuropaeoniflorin on the PMDD liver qi stagnation rat model. By comparing the suspension immobility time of each group during the acceptance period and the non-acceptance period after drug administration, the test is conducted. Figure 8 It can be observed that the model group's suspension time during the non-acceptance period was still significantly increased compared to the acceptance period after the dosing period. p <0.05), after administration, there were no significant differences in suspension immobility time between the normal control group, fluoxetine group, high-dose saponin group, medium-dose saponin group, and low-dose saponin group during the acceptance and non-acceptance periods. p >0.05).

[0101] ② Elevated Cross Maze Experiment The results of the elevated cruciate maze experiment (before drug administration) during the preparation of the PMDD rat model of liver qi stagnation are as follows: Figure 9 As shown, ①OT%: The test results of the model group during the acceptance period were significantly higher than those of the normal control group during the acceptance period ( p <0.05, the model group's acceptance period test results showed an increasing trend compared to the first non-acceptance period test results, and the second non-acceptance period test results showed a decreasing trend compared to the acceptance period test results, but there was no statistically significant difference. p>0.05); the results of the normal control group during the acceptance period test and the second non-acceptance period test compared with the first acceptance period test all showed a decreasing trend, and there were no significant differences between the groups. p >0.05); ②OE%: There were no significant differences between the model group and the blank control group in each period of testing ( p >0.05), but the OE% test results of the model group showed the same trend as the OT% test results, that is, the test results of the non-acceptance period decreased compared with the acceptance period.

[0102] The OT% results before and after drug administration in PMDD liver qi stagnation model rats are shown below: Figure 10 As shown, there were no significant differences in the test results at each time period among the normal control group, model group, high-dose Chai Shao group, medium-dose Chai Shao group, and low-dose Chai Shao group. p >0.05), the second treatment period results in the fluoxetine-treated group were significantly higher than those in the first treatment period (>0.05). p <0.05).

[0103] ③ Sprinkling experiment The modification time and number of times before and after drug administration were as follows between the normal control group and the model group: Figure 11 As shown: Before drug administration, the modification time of the model group was significantly reduced compared with that of the normal control group. p <0.05), the number of modifications all showed a decreasing trend, but there was no significant difference ( p >0.05). After drug administration, there was no significant trend in modification time and number of modifications between the normal control group and the model group, and there were no statistically significant differences in either. p >0.05).

[0104] Analysis of the modification time and frequency before and after drug administration in each drug administration group and the normal control group revealed that although the modification time in the high-dose group, medium-dose group, low-dose group, and fluoxetine group all showed a decreasing trend compared to the normal control group, there was no significant difference. p >0.05). And as Figure 12 As shown, after administration, the high-dose and low-dose groups of *Chai Shao* showed an increasing trend in the number of modifications compared to other groups, but there was no significant difference. p >0.05).

[0105] 1.4 Experimental Conclusions FST data revealed that the model group rats exhibited a significantly increased suspension immobility time in both non-acceptance phases compared to the acceptance phase during the model preparation stage. In contrast, the normal control group rats, selected based on a near-zero difference in suspension immobility time between the acceptance and non-acceptance phases, showed no significant difference or increasing / decreasing trend in suspension immobility time between the acceptance and non-acceptance phases during the model preparation stage. Therefore, the model group rats' recurrence of depression in the first non-acceptance phase, its disappearance during the acceptance phase, and its re-emergence in the second non-acceptance phase indicates a clear estrous cycle dependence of their depressive state, consistent with the clinical manifestations of PMDD (post-epidural dysregulation) in human females.

[0106] Immobility time was the main criterion for grouping and preparation of the PMDD liver qi stagnation rat model in this study. Analysis of the immobility time of rats in each group before and after drug administration showed that there was no significant change in the immobility time of the normal control group before and after drug administration. The rats in the model group still showed estrus cycle dependence of depression after the drug administration period (gavage with pure water), that is, the immobility time of the non-acceptance period was significantly higher than that of the accepting period. However, there was no significant difference in the increase of the non-acceptance period compared with the accepting period after drug administration in the fluoxetine group and the high, medium and low dose groups of bupleuropeptide. This indicates that fluoxetine and bupleuropeptide can improve depressive mood by reducing immobility time. Among the various dose groups of bupleuropeptide, the high dose group had the treatment effect closest to the normal control group, and the low dose group had the most significant effect on the decrease of immobility time in the non-acceptance period compared with the accepting period.

[0107] The water-spraying test can be used to study rat models by leveraging the rodents' sweet tooth and their tendency to lick their fur. The higher the proportion of time rats spend licking their fur within a specified time, the lower their level of depression. Because the water-spraying test was conducted during two non-acceptance periods before and after drug administration to avoid excessive sugar intake affecting the drug intervention effect, this study only performed a longitudinal comparison before and after drug administration. The results showed that before drug administration, the modification time in the model group was significantly lower than that in the normal control group, while the number of modifications showed a decreasing trend. After drug administration, the modification time and number of modifications in the model group did not show a decreasing trend compared to the normal control group. Furthermore, the modification time in the high-dose and low-dose bupleuropaeoniflorin groups showed a significant increasing trend compared to the blank control group after drug administration, indicating that the PMDD liver qi stagnation model rats exhibited significant depressive symptoms compared to the normal control group, and that treatment with bupleuropaeoniflorin could reverse this depressive state. In conclusion, if the water-spraying test is to be used to detect the depressive-like emotional state in the PMDD liver qi stagnation rat model, further optimization of the experimental protocol and selection of more experimental points are needed for in-depth research.

[0108] The EPM experiment results showed that the OT% in the model group rats and the normal control group rats were significantly increased during the acceptance period, and the OT% in the model group rats during the acceptance period showed an increasing trend compared with the non-acceptance period, but the difference was not statistically significant. The OE% in the model group and the normal control group rats showed no significant difference at any of the three time points, but the overall OE% and OT% in the model group showed the same trend, that is, the OE% was higher during the acceptance period than the non-acceptance period. The EPM experiment is one of the experimental methods for studying anxiety-like behavior in rats and mice. Compared with methods that induce anxiety behavior in rats using electrical stimulation and noise stimulation, EPM has the advantages of simple operation and minimal harm to experimental animals. Because of the hypothesis that anxiety and depression often co-occur, this study used EPM to evaluate the animal model prepared by FST before and after drug administration. However, the OT% and OE% results in the EPM experiment of PMDD liver qi stagnation model rats showed that the model group rats did not exhibit behavioral changes related to the presentation of symptoms during the acceptance / non-acceptance period. Furthermore, the OT% results before and after drug administration in each treatment group indicated that the therapeutic effect of paeoniflorin on depressive-like behavior in PMDD liver qi stagnation model rats was difficult to reflect in the EPM experiment. This result may be because the EPM experiment is more applicable to anxiety behaviors and cannot effectively reflect depressive-like emotions caused by PMDD liver qi stagnation. The differences in the sensitivity of the two behavioral tests to endogenous neurotransmitters in experimental rats and the different sites of drug action may also be important reasons. This suggests that the EPM experiment may not be a good evaluation method for this model.

[0109] 2. Effects of Paeonia lactiflora saponins on the microscopic mechanisms of the brain center and peripheral blood in a naturally screened PMDD rat model with liver qi stagnation syndrome. 2.1 Experimental Materials 2.1.1 Experimental Reagents

[0110] 2.1.2 Experimental Apparatus

[0111] 2.2 Experimental Methods Peripheral blood serum and brain regions (hypothalamic and hippocampal regions) were collected from 60 PMDD rats naturally screened for liver qi stagnation syndrome in the aforementioned study. Six groups were established: model group, normal control group, high-dose bupleuropeptide group, medium-dose bupleuropeptide group, low-dose bupleuropeptide group, and fluoxetine group, with 10 rats in each group. Appropriate techniques were used for measurement.

[0112] All data from this experiment were analyzed and plotted using GraphadPrism 7.04 software. One-way ANOVA was used for comparisons between multiple groups. Results are expressed as Mean ± SEM (mean ± standard error of mean).p <0.05 and p A value <0.01 indicates a statistically significant difference.

[0113] 2.3 Experimental Results (1) Results of detection of ALLO content in peripheral blood serum and hypothalamus and hippocampus of rats Based on the test results ( Figure 13 It can be found that the ALLO content in each group of rats is as follows: ① In peripheral blood serum: the ALLO content in the model group showed a decreasing trend compared with the normal control group, while the ALLO content in the fluoxetine group showed an increasing trend compared with the model group, but there was no significant difference. p >0.05). The ALLO content in the high-dose paeoniflorin group was significantly higher than that in the model group (>0.05). p <0.01), other groups showed an increasing trend compared to the model group, but there was no significant difference. p >0.05); ② In the hippocampus: the ALLO content in the model group was significantly lower than that in the normal control group ( p <0.01), the fluoxetine group showed an increasing trend compared to the model group ( p >0.05), the high, medium, and low dose groups of paeoniflorin showed a significant increase compared to the model group ( p <0.0001, p <0.001, p <0.05). ③ In the hypothalamus: there was no difference or trend change in ALLO content between the model group and the normal control group. p >0.05), the ALLO content in the high-dose group of paeoniflorin was significantly lower than that in the model group. p <0.0001), and there were no significant differences between the other groups and the model group. p >0.05).

[0114] (2) Results of detection of P content in peripheral blood serum, hippocampus, and hypothalamus of rats The test results show that ( Figure 14 The P content among the rat groups was as follows: ① In peripheral blood serum: the P content in the model group was significantly lower than that in the normal control group ( p <0.05), the content of P in both the high- and low-dose groups of paeoniflorin was significantly increased compared with that in the model group (P <0.05). p <0.01, p <0.05); ② In the hippocampus: the P content in the model group was significantly higher than that in the normal control group ( p <0.001), and there was no significant difference between the other groups and the model group, but they showed an increasing trend. p >0.05); ③ In the hypothalamus: the model group showed a decreasing trend compared with the normal control group, while the fluoxetine group and the low-dose bupleurum and peony groups showed an increasing trend compared with the model group, but there was no significant difference.p >0.05).

[0115] (3) Results of E2 content detection in rat peripheral blood serum The changes in E2 levels in peripheral blood serum among different groups of rats are as follows: Figure 15 As shown: The E2 content in the model group was significantly lower than that in the normal control group. p <0.01), the E2 content in the fluoxetine group was significantly higher than that in the model group ( p <0.05), the E2 content in the high-dose group of paeoniflorin was significantly higher than that in the model group ( p <0.05); The E2 content in the medium-dose group of paeoniflorin was also significantly increased compared with that in the model group, and the difference was statistically significant. p <0.001); The E2 content in the low-dose group of paeoniflorin was significantly higher than that in the model group (<0.001); p <0.05).

[0116] 2.4 Experimental Conclusions Clinical studies have shown that fluctuations in progesterone levels, including P, E2, and ALLO, are closely related to the onset of PMDD symptoms. ALLO, a metabolite of P, is particularly relevant to PMDD symptoms due to dysregulation of its regulatory mechanism. International research indicates that mood symptoms appear when serum ALLO concentrations are similar to endogenous luteal phase levels, while the opposite occurs when ALLO concentrations are high or low. Analysis of progesterone levels in the aforementioned naturally selected rat model revealed that the PMDD liver qi stagnation model group showed lower expression of ALLO, P, and E2 in peripheral serum compared to the normal group, consistent with previous studies. This suggests that decreased levels of ALLO, P, and E2 in peripheral serum of PMDD liver qi stagnation patients may be one of the underlying mechanisms of its pathogenesis. Furthermore, changes in ALLO and P levels in the hippocampus and hypothalamus showed a significant decrease in ALLO but a significant increase in P in the hippocampus of the model group rats, suggesting a possible difference in the levels of these two hormones in the hippocampus.

[0117] Treatment with fluoxetine and bupleurum saponins revealed that high doses of bupleurum saponins significantly increased the ALLO content in the peripheral blood serum of model rats. High, medium, and low doses of bupleurum saponins all increased the P content in the peripheral blood serum, while the E2 content in the peripheral blood serum also significantly increased after intervention with high, medium, and low doses of fluoxetine and bupleurum saponins. After administration, the ALLO content in the hippocampus significantly increased under the intervention of high, medium, and low doses of bupleurum saponins. The ALLO content in the hypothalamus significantly decreased after intervention with high doses of bupleurum saponins, but the P content showed an increasing trend after intervention with low doses of bupleurum saponins.

[0118] One of the pathological mechanisms of PMS and PMDD may be the persistently low levels of E2 and P. Analysis of previous behavioral data revealed a strong correlation between the depressive state of PMDD rats with liver qi stagnation and changes in the levels of ALLO, P, and E2 in peripheral serum and the hippocampus. ALLO levels decreased due to the decline in P, and the decrease in serum E2 levels in the model group rats was also a contributing factor to the depressive state. Paeoniflorin can improve the symptoms of PMDD with liver qi stagnation by increasing serum ALLO, P, and E2 levels, as well as ALLO levels in the hippocampus and P levels in the hypothalamus.

[0119] Example 4 The intervention effect and mechanism of action of peony saponin soft capsules on hormone-induced PMDD-PWD rat model with liver qi stagnation syndrome Given the low number of naturally induced PMDD rats with liver qi stagnation syndrome and unstable estrous cycles, the following experiment was conducted using a hormone-induced model.

[0120] 1. The intervention effect of bupleurum saponin soft capsules on the epibehavioral characteristics of a rat model of PMDD with liver qi stagnation induced by hormones. 1.1. Experimental Materials 1.1.1 Medicines and Reagents

[0121] 1.1.2 Experimental Apparatus

[0122] 1.2. Experimental Methods (1) Animal husbandry One hundred rats were transported and placed in the laboratory for one week to acclimatize. They had free access to food and water, and were kept under a 12h / 12h light / dark cycle with their day and night reversed (lights on at 8:00 PM and off at 8:00 PM). The room where the rats were housed was maintained at 23±1℃. Before the formal experiments began, five rats were housed in each cage during the acclimatization period. All experimental procedures had to be performed under dim red light (<25 lux). All animal experimental procedures followed the NIH's "Guidelines for the Care and Use of Laboratory Animals." All behavioral tests were conducted during the dark phase of the rats, from 10:00 AM to 5:00 PM.

[0123] (2) Ovariectomy in rats Ovariectomy was performed on rats after the above-mentioned adaptive feeding. The original feeding conditions were maintained, allowing approximately two weeks for recovery. After the rat wounds had completely healed, two EPM, FST, and Splash Tests were conducted. Parameters such as suspension time, latency, and frequency during interestrus phase 1 and the presumed receptive phase, as well as behavioral evaluation parameters, were recorded for both groups. Specific methods for ovariectomy: All surgical instruments were placed in a surgical instrument sterilizer beforehand. The rats were connected to a small animal anesthesia machine, and after complete unconsciousness, the abdominal hair was shaved with scissors dipped in a small amount of water. The skin at the shaved area was wiped with a cotton swab soaked in a small amount of 75% ethanol. The abdominal cavity was incised (1 cm wide) with a scalpel to expose the abdominal cavity. Under cold light illumination, two hemostatic forceps were used to pull open the opening from both sides. Holding one hemostatic forceps in each hand, the ovaries were located along the midline of the rat's abdomen. After ligating the uterus with surgical sutures, the ovaries were removed. In the sham surgery group, only a portion of the abdominal cavity was opened, some fat was removed, and the wound was sutured; ovariectomy was not performed. After releasing the hemostatic forceps holding the skin, penicillin was instilled, and the rat wound was sutured. A few more drops of penicillin were then instilled around the wound. For three days post-surgery, each rat received an intraperitoneal injection of 20,000 units of penicillin daily to reduce inflammation and prevent infection. The planned design for rat ovariectomy included eight groups: a drug administration group (high-dose, medium-dose, and low-dose), a fluoxetine group, a model group, a sham surgery group, a normal control group, and a natural control group, with 80 rats ovariectomized in total, aiming to ensure a minimum of 10 rats per group.

[0124] (3) Hormone-induced estrous cycle in rats Hormone induction was performed on the ovariectomized rats described above. The normal cycle of the ovariectomized rats was induced using the method of Hoi-PorHo et al. At 12:00 noon (0h), each rat was subcutaneously injected with 0.5 mg / 0.1 ml DMSO of estradiol benzoate. At 20:00 on the second day (32h), each rat was subcutaneously injected with 0.5 mg / 0.1 ml DMSO of estradiol. At 8:00 on the third day (44h), each rat was subcutaneously injected with 0.5 mg / 0.1 ml DMSO of progesterone. Vaginal smear examination of vaginal cells showed that this hormone injection method resulted in cell levels consistent with those of normal rats in the accepting phase on days 2 and 3, and consistent with those of normal rats in the non-accepting phase on day 5. Therefore, day 3 was selected for the accepting phase experiment, and day 5 was selected for the non-accepting phase (interphase 1) experiment. In the experiment on the intervention effect of Paeonia lactiflora saponin soft capsules on a naturally screened rat model of PMDD with liver qi stagnation, the rats underwent EPM, FST, and Splash Test once each during the accepting phase (R phase) and the interphase 1 (NR phase) (twice). Parameters such as suspension immobility time, latency, and frequency were recorded. Rats were continuously injected with estrogen during forced swimming. Behavioral testing: The rats underwent EPM, FST, and Splash Tests under hormone induction. Based on the FST suspension immobility time data, data analysis was performed to show that suspension immobility time was estrus cycle-dependent (i.e., suspension immobility time in the non-accepting phase minus suspension immobility time in the accepting phase). Specifically, suspension immobility was significantly prolonged in the non-accepting phase than in the accepting phase, simulating the premenstrual symptoms of clinical PMDD with liver qi stagnation, which subside or disappear after menstruation. The results of the FST, EPM, and Splash Tests were used for evaluation. Grouping: The model group (Model) consisted of rats whose FST (French estrus cycle) suspension immobility time was dependent on the estrous cycle, meaning they had the longest suspension immobility time during the non-receptive phase and the shortest or no suspension immobility time during the estrous phase. The control group consisted of rats with very short suspension immobility time during the non-receptive phase and the shortest or no suspension immobility time during the receptive phase. After grouping, any remaining rats that were not grouped were discarded.

[0125] (4) Intervention with paeoniflorin saponins After screening and grouping the rats, drug administration began, with a dosing cycle of approximately 12 days (three estrous cycles). Administration was by gavage, with 1 mL of drug administered per 100g rat, performed daily at 8:30 AM. Dosage was as follows:

[0126] In the last cycle of gavage, the experimental rats underwent behavioral tests once each in the NR and R phases to evaluate the therapeutic effect of paeoniflorin.

[0127] (5)Acquisition of materials After all behavioral indicators were collected, the rats were euthanized by decapitation. Approximately 5 ml of peripheral blood was collected, allowed to stand for 30 minutes, and then centrifuged at 3000 rpm for 15 minutes. The serum was then aliquoted and stored at -80°C for later analysis. The entire brain of the rat was dissected as quickly as possible and carefully performed on ice to prevent the brain tissue from melting. The prefrontal cortex, amygdala, hypothalamus, and hippocampus were then isolated separately on ice.

[0128] (6) Statistical analysis All data were analyzed and plotted using GraphadPrism 7.04 software. One-way ANOVA was used for comparisons among multiple groups. Results are expressed as Mean ± SEM (mean ± standard error of mean). p <0.05 and p A value <0.01 indicates a statistically significant difference.

[0129] 1.3. Experimental Results (1) Figure 16 An experimental protocol was established for the PMDD-PWD rat model of liver qi stagnation and depression subtype. Figure 17 The results of the forced swimming test in rats treated with fluoxetine after hormone induction showed that the PMDD-PWD liver qi stagnation depression subtype rat model effectively simulated the core symptoms of PMDD liver qi stagnation syndrome, namely, the symptoms appeared during the non-acceptance phase of the estrous cycle and disappeared during the acceptance phase; the depressive-like symptoms disappeared after ovariectomy; and reappeared after artificial cycles induced by exogenous hormones. Furthermore, the first-line anti-PMDD drug fluoxetine could reverse the depressive behavioral symptoms in this model rats. Significant changes were also observed in the serum estrogen and progesterone levels, the levels of mood-related neurotransmitters serotonin, norepinephrine, and γ-aminobutyric acid in the hippocampus of the model rats, and the expression of the GABAA receptor 4α subunit was significantly increased. These abnormal changes could all be effectively reversed by fluoxetine intervention.

[0130] (2) First, the suspension immobility time of rats in each group before and after drug administration was compared in terms of FST behavior. The results are as follows: like Figure 18 As shown, the suspension immobility time of rats in the non-acceptance period model group and each drug-treated group showed a significant increasing trend compared with the normal control group, and the suspension immobility time of the high-dose bupleuron saponin group was significantly increased compared with the normal control group. p<0.05); Before administration, there was no significant difference in suspension time among the groups, and the trend of suspension time differences among the groups was not obvious; After administration, in the non-acceptance period, the suspension time of the normal control group, fluoxetine group, medium-dose bupleurum saponin group, and low-dose bupleurum saponin group showed a relatively significant decreasing trend compared with the model group, but no significant difference was observed. p >0.05); there was no significant difference in the suspension immobility time among the rats in each group during the post-drug administration period. p >0.05).

[0131] (3) The suspension immobility time of the model group rats in each stage was compared, and the results are as follows: like Figure 19 As shown, the immobile suspension time of the experimental rats during the non-acceptance period in the first testing cycle tended to increase compared to the acceptance period. In the second testing cycle, the immobile suspension time of the experimental rats during the non-acceptance period was significantly increased compared to the acceptance period. p <0.05), consistent with the characteristic of increased immobility time during the non-acceptance period compared to the acceptance period in the PMDD rat model of liver qi stagnation.

[0132] (4) The suspension immobility time of rats in the normal control group at each stage was compared, and the results are as follows: like Figure 20 As shown, the suspension immobility time of rats in the normal control group did not increase significantly compared to the acceptance period during the non-acceptance period, and the suspension immobility time during the non-acceptance period and the acceptance period showed a similar trend in the first detection cycle, which is consistent with the variation characteristics of suspension immobility time in rats in the normal control group.

[0133] (5) The suspension immobility time of rats in each drug administration group was compared. like Figure 21 As shown, there was a significant difference in the suspension immobility time of rats in the fluoxetine group before and after drug administration between the acceptance and non-acceptance periods. p <0.05), compared with before administration, the trend of difference in suspension immobility time in the non-acceptance period compared with the acceptance period after administration was no longer significant, indicating that fluoxetine showed a good therapeutic effect on model rats; the rats in the high-dose group of bupleuropaeoniflorin still showed a trend of increased suspension immobility time in the non-acceptance period compared with the acceptance period after administration, so no improvement effect was observed; although the medium-dose group and the low-dose group of bupleuropaeoniflorin showed better improvement in suspension immobility time than the high-dose group of bupleuropaeoniflorin, and the suspension immobility time in the non-acceptance period after administration was reduced compared with before administration, no significant difference was observed. p >0.05).

[0134] (6) Note: In other behavioral experiments, the main test indicators of the rats in different estrous cycles showed little significant difference and the trend was not obvious, which is not very indicative.

[0135] 1.4 Experimental Conclusions The model group rats exhibited the same hovering immobility time in each estrous cycle as the PMDD liver qi stagnation rat model, i.e., the hovering immobility time was significantly higher in the non-acceptance phase than in the acceptance phase. Compared with the normal control group, the model group rats showed the same pattern of hovering immobility time variation as the main manifestations of the PMDD liver qi stagnation rat model.

[0136] Fluoxetine, a positive control drug, showed a significant therapeutic effect on the model rats after administration, specifically improving the significantly increased suspension immobility time during the non-acceptance phase compared to the acceptance phase. While the low- and medium-dose groups of bupleuropaeoniflorin showed a more significant improvement in suspension immobility time during the non-acceptance phase compared to the higher-dose group, no significant difference was observed. Therefore, it is concluded that bupleuropaeoniflorin has a limited effect on improving the PMDD liver qi stagnation model rats screened by hormone-induced estrus cycle combined with forced swimming. The specific reasons for this require further investigation through microscopic mechanisms.

[0137] 2. Effects of Paeonia lactiflora saponins on the microscopic mechanisms of the brain and peripheral blood in a rat model of hormone-induced PMDD and liver qi stagnation. 2.1 Experimental Materials 2.1.1 Experimental Reagents

[0138] 2.1.2 Experimental Apparatus

[0139] 2.2 Experimental Methods Serum samples were thawed and centrifuged. Brain tissue samples were ground, precipitated, and the supernatant was extracted and incubated overnight. The pH was then adjusted to slightly acidic, and the samples were randomized and analyzed. The column oven temperature was 35°C, the sample tray temperature was 4°C, and the injection volume was 5µl.

[0140] Mobile phase conditions: Phase A of liquid chromatography: 10 mM ammonium formate / 0.1% formic acid aqueous solution.

[0141] Phase B of liquid chromatography: acetonitrile.

[0142] The mobile phase chromatographic gradient is as follows:

[0143] Mass spectrometry conditions: Before performing UHPLC-MS / MS analysis, standard solutions of the target compounds were introduced into the mass spectrometer. For each target compound, several parent ion-daughter ion pairs with the highest signal intensity were selected for quantitative and qualitative analysis.

[0144] Qualitative and quantitative ion counts of the target compound

[0145] 2.3 Experimental Results The levels of NE, GABA, 5-HT, and Glu in the left hippocampus of rats in each group are as follows: Figure 22 As shown, the levels of NE in the left hippocampus of the normal control group, fluoxetine group, and high- and medium-dose bupleurum and peony root saponins showed an increasing trend compared to the model group. The low-dose bupleurum and peony root saponins group showed a significantly higher NE content compared to the model group. p <0.0001); In the left hippocampus, the GABA content in the normal control group, fluoxetine group, and high- and medium-dose bupleurum and peony saponin groups showed an increasing trend compared with the model group, and the GABA content in the low-dose bupleurum and peony saponin group was significantly increased compared with the model group. p <0.01); In the left hippocampus, the 5-HT content in the normal control group, fluoxetine group, and medium-dose bupleurum and peony root groups showed an increasing trend compared with the model group, and the 5-HT content in the high-dose and low-dose bupleurum and peony root saponin groups was significantly higher than that in the model group. p <0.001, p <0.01); In the left hippocampus, the Glu content in the normal control group, fluoxetine group, and high-dose bupleurum and peony root saponin group showed an increasing trend compared with the model group, and the Glu content in the low-dose bupleurum and peony root saponin group was significantly higher than that in the model group. p <0.001).

[0146] The levels of NE, GABA, 5-HT, and Glu in the right hippocampus of rats in each group are as follows: Figure 3 As shown, the NE content in the right hippocampus was significantly higher in the natural control group compared to the model group. p <0.0001), while no upward trend was observed in other groups ( p >0.05); and the same trend was observed in the right hippocampus, i.e., the 5-HT content was significantly higher in the natural control group compared to the model group ( p <0.0001); In the right hippocampus, the low-dose group of paeoniflorin showed an increasing trend in GABA content compared to the model group, but the difference was not statistically significant. However, the Glu index showed a significant increase in content in the natural control group compared to the model group. p <0.05), the normal control group showed a decreasing trend compared to the model group, while the levels of paeoniflorin were high, and the medium-dose group showed the same trend. p >0.05).

[0147] The levels of GABA and Glu in the hypothalamus of rats in each group are as follows: Figure 24 As shown, the GABA content in the hypothalamus was significantly lower in the fluoxetine group and the medium-dose peony saponin group compared to the model group. p <0.05), the GABA content in the sham surgery group and the natural control group was also significantly lower than that in the model group. p<0.01), the GABA content in the normal control group, the low-dose bupleurum saponin group, and the model group showed a decreasing trend. It is noteworthy that 5-HT content was not detected in the hypothalamus of rats in any group; the fluoxetine group and the medium-dose bupleurum saponin group ( p <0.01, p <0.05), and the Glu content in the sham-operated group and the natural control group was significantly lower than that in the model group ( p <0.001, p <0.01).

[0148] The levels of NE, GABA, 5-HT, and Glu in the peripheral serum of rats in each group are as follows: Figure 25 As shown, the NE content in peripheral serum of the high-dose paeoniflorin group was significantly higher than that of the model group. p <0.001), the NE content in the medium-dose group of paeoniflorin was significantly higher than that in the model group. p <0.05); The high-dose group of paeoniflorin showed a significantly higher level of GABA in peripheral serum compared to the model group. p <0.0001); 5-HT levels in peripheral serum of the fluoxetine group and the low-dose bupleurum and peony root group showed a decreasing trend, but the difference was not statistically significant. p >0.05); the Glu content in the sham surgery group was significantly higher than that in the model group ( p <0.05).

[0149] 2.4 Experimental Conclusions Microscopic analysis of brain regions in a rat model of PMDD (post-traumatic brain injury) induced by hormone-induced estrous cycles combined with forced swimming revealed diametrically opposed changes in NE (norepinephrine) and 5-HT (5-HT) levels in the left and right hippocampal regions. The levels of various neurotransmitters in the left hippocampus were significantly higher than in the right hippocampus, and almost no NE or 5-HT was detected in the right hippocampus of the model rats and rats in each drug-treated group. This study indicates that different hippocampal regions, as part of the limbic system, each play a unique role in information processing. The hippocampus is primarily responsible for long-term memory storage, conversion, and orientation. Neurotransmitters are chemical substances that transmit information between neurons or between neurons and effector cells such as muscle cells and glandular cells. The different expressions of NE, GABA, and 5-HT in the left and right hippocampal regions, as representatives of neurotransmitters, suggest a close correlation between their levels and the manifestations of this syndrome.

[0150] The hypothalamus, an important component of the diencephalon, receives numerous nerve impulses and serves as the center of the endocrine and nervous systems. It regulates anterior pituitary function, synthesizes neurohypophyseal hormones, and controls the autonomic and vegetative nervous systems. In a rat model of PMDD with liver qi stagnation induced by hormones, the GABA content in the hypothalamus of the fluoxetine group and the medium-dose bupleurum saponin group was significantly lower than that in the model group after administration. GABA, as an inhibitory neurotransmitter, can affect the physiological functions of the pituitary gland and gonads through the hypothalamic-pituitary-gonadal axis, thereby influencing hormone secretion regulation. Fluctuations in hormone levels are closely related to the symptoms of PMDD with liver qi stagnation. In the left hippocampus, low-dose bupleurum saponin significantly increased GABA content, while high-dose bupleurum saponin significantly increased the levels of NE and GABA in the peripheral serum of rats. Furthermore, the fluoxetine group and the low-dose bupleurum saponin group significantly reduced the 5-HT content in the peripheral serum, suggesting that bupleurum saponin achieves its intervention effect by improving the levels of this indicator in the peripheral serum.

[0151] Example 5 Clinical trial protocol for the treatment of PMDD liver qi stagnation syndrome depressive subtype with bupleuropaeoniflorin soft capsules Providing a clinical trial protocol for drugs treating PMDD liver qi stagnation syndrome depression subtype is essential information for the registration application of new traditional Chinese medicine. Therefore, the following protocol is proposed.

[0152] 1. Diagnostic criteria for PMDD liver qi stagnation syndrome / depression subtype 1.1 PMDD Diagnostic Criteria This was formulated with reference to the Premenstrual Dysphoric Disorder Diagnostic Criteria in the Diagnostic and Statistical Manual of Mental Disorders (DSM-5).

[0153] A. In most menstrual cycles, women of reproductive age must experience at least five symptoms that appear in the last week before menstruation begins, begin to improve within a few days after menstruation begins, and become mild or disappear within a week after menstruation.

[0154] B. One or more of the following symptoms must be present: 1) Obvious emotional instability (e.g., mood swings: suddenly feeling sad or crying) 2) Significantly irritable, angry, or experiencing interpersonal conflict.

[0155] 3) Obvious depressive mood, feelings of hopelessness, or self-deprecating thoughts.

[0156] 4) Significant anxiety, tension, and / or feeling tense or nervous.

[0157] C. One or more of the following symptoms must be added in combination with the symptoms in criterion B above to reach a total of five symptoms.

[0158] 1) Decreased interest in daily activities (such as work, school, friends, hobbies).

[0159] 2) Subjectively, it is difficult to concentrate.

[0160] 3) Drowsiness, fatigue, or obvious lack of energy.

[0161] 4) A significant change in appetite; overeating; or a specific food craving.

[0162] 5) Hypersomnia or insomnia.

[0163] 6) A feeling of being conquered or out of control.

[0164] 7) Physical symptoms such as breast tenderness or swelling, joint or muscle pain, a feeling of "bloating", or weight gain.

[0165] D. These symptoms are associated with clinically significant distress or disruption to work, school, normal social activities, or relationships with others.

[0166] E. This disorder is not merely an exacerbation of symptoms of another illness, such as major depressive disorder, panic disorder, persistent depression (dysthymia), or personality disorder (although it may occur concurrently with any of these illnesses).

[0167] F. Criterion A should be confirmed using a prospective Daily Rating Scale (DRSP) over at least two symptom cycles. (Note: A diagnosis may be made provisionally prior to this confirmation.) Diagnosis: Based on the four criteria of ABCD, a provisional diagnosis was made by retrospective questioning and scoring using the DRSP for two menstrual cycles; subsequently, a definitive diagnosis was made by prospective daily scoring for two symptom cycles.

[0168] 1.2 PMDD Diagnosis of Liver Qi Stagnation Syndrome in Traditional Chinese Medicine The TCM syndrome differentiation and diagnostic criteria were formulated with reference to the People's Republic of China TCM industry standard "Standards for Diagnosis and Efficacy of TCM Diseases and Syndromes" and the preliminary research results of the invention team.

[0169] Liver Qi Stagnation Syndrome: This refers to a syndrome caused by liver qi deficiency and inadequate qi circulation, as diagnosed by PMDD in Traditional Chinese Medicine.

[0170] Main symptoms: Premenstrual mood swings, depression, irritability, and a tendency to cry; Secondary symptoms: (1) breast tenderness; (2) Headache and heaviness in the head; (3) Decline in work and household management abilities; (4) Chest tightness and sighing; (5) Lower abdominal distension and pain; (6) Poor sleep and many dreams; (7) Slow appetite; (8) Weight gain, or even edema; (9) Fear of difficulties; (10) Laziness, or even reluctance to see people; (11) Delayed menstruation; (12) Low libido; (13) The pulse is deep and thready, or thready and wiry; the tongue is pale, and the tongue coating is pale white or yellowish-white; Diagnosis: Meets the diagnostic criteria for PMDD; has two or more of the main and secondary symptoms of liver qi stagnation syndrome.

[0171] 1.3 PMDD Western Medicine Diagnosis of Depression Subtypes The depression subtype refers to individuals whose scores on the Daily Recording of Symptom Severity (DRSP) items are primarily based on the subscale scores of "depressed mood, depression, and avoidance or reduction of social activities".

[0172] Diagnosis: Meets the diagnostic criteria for PMDD; has the above scores on the DRSP subscale as the primary indicator.

[0173] 2. Trial grouping and inclusion and exclusion criteria 2.1 Experimental Grouping This study employed a prospective, randomized, controlled trial for selection, using a parallel-controlled randomization method. The ratio of cases in the control group to the treatment group was no less than 1:3, with a total number of cases in the treatment group of no less than 300 and in the control group of no less than 100.

[0174] Randomized controlled trials: The inclusion and exclusion criteria for controls and cases are the same; the inclusion time for controls and cases is the same; the interventions for controls and cases are administered concurrently; and, except for the experimental intervention factor, other factors are distributed as similarly as possible between the two groups. Based on the intervention received by the control group, it can be further divided into placebo controls and positive drug controls.

[0175] Based on the inclusion and exclusion criteria, women with normal menstrual cycles were selected as the control group, while PMDD patients whose syndrome type matched the liver qi stagnation syndrome and whose subtype matched the depression subtype were selected as the study subjects.

[0176] 2.2 Inclusion Criteria (1) Those diagnosed with PMDD by Western medicine; diagnosed with liver qi stagnation syndrome by traditional Chinese medicine; and classified as depression subtype by Western medicine; and those who meet the diagnostic criteria for this disease and whose condition is moderate or above, accounting for no less than 2 / 3.

[0177] (2) Female patients aged 18-45 years.

[0178] 2.3 Exclusion Criteria (1) Women under 18 years of age or over 45 years of age who are experiencing menstrual disorders during puberty or menopause, or who have allergies or drug allergies.

[0179] (2) Patients with hypertension, visual impairment, vestibular dysfunction (Meniere's syndrome), breast tumors, lobular hyperplasia of the breast, primary dysmenorrhea, pelvic inflammatory disease, endometriosis, uterine tumors, chronic colitis, intestinal tumors, etc. could not be ruled out.

[0180] (3) Patients with serious primary diseases such as cardiovascular, cerebrovascular, liver, kidney and hematopoietic system diseases, or mental illness.

[0181] (4) History of drug abuse, including taking medication for PMDD within the past three months.

[0182] (5) Pregnant or breastfeeding women.

[0183] (6) Those who have aphasia, impaired consciousness, dementia or other conditions that prevent them from cooperating with the examination.

[0184] (7) Those who have undergone unilateral oophorectomy or miscarriage within the past six months.

[0185] (8) Those who are subject to observation but whose data are incomplete, thus affecting the judgment of efficacy and safety.

[0186] (9) Patients who discontinue medication midway through the course of treatment for reasons other than poor efficacy or adverse reactions.

[0187] (10) Those who switch to or add other Chinese or Western medicines and treatments related to the treatment of diseases.

[0188] 3. Medication and Treatment Course 3.1 Test Drug and Positive Control Drug The experimental drug, paeoniflorin saponins, was taken twice daily, 3 capsules each time. The positive control drug is fluoxetine hydrochloride capsules, an international first-line treatment drug, taken twice a day, 2 capsules each time.

[0189] 3.2 Medication and Treatment Course Start taking it 10-7 days before menstruation, and continue taking it until menstruation begins and symptoms lessen or disappear.

[0190] 4. Therapeutic effects and evaluation 4.1 Judgment of therapeutic effect evaluation Clinical cure: Clinical symptoms disappear, basal body temperature or endocrine tests are basically normal, and there is no recurrence after 3 menstrual cycles of observation after stopping medication.

[0191] Significant effect: Clinical symptoms are significantly improved, and the total score after treatment is reduced by ≥2 / 3 compared with the total score before treatment; basal body temperature or endocrine measurement is close to normal, and there is no worsening of symptoms during the 3 menstrual cycles observed after the end of the treatment course.

[0192] Effective: Clinical symptoms improved, the total score after treatment decreased by ≥1 / 3 compared with the total score before treatment, and although the symptoms recurred after stopping the medication, they were better than before treatment.

[0193] Ineffective: No improvement or worsening of various clinical symptoms.

[0194] 4.2 Statistical Methods The significance of efficacy is tested using the chi-square test; clinical grading is compared using the Ridit test; semi-quantitative data are analyzed using the rank-sum test; continuous data are analyzed using the t-test; correlation between variables is assessed using linear correlation or linear regression; if there is heterogeneity in the condition between the treatment group and the control group, a stratified t-test is used for comparison. Traditional Chinese medicine syndrome differentiation can be assessed using a comparative efficacy index between groups, where the efficacy index = difference in scores before and after treatment ÷ score before treatment × 100%.

[0195] 4.3 Observation of adverse reactions Adverse reactions or unexpected toxic side effects should be carefully observed and monitored. Adverse reactions caused by taking the investigational drug... Any adverse reactions, such as gastrointestinal reactions, should be recorded accurately, and the time it takes for the adverse reactions to disappear after discontinuation should be observed.

[0196] 4.4 Precautions (1) Medical staff participating in the trial should personally observe patients taking the medication as prescribed, so as to objectively reduce the possibility of patients not adhering.

[0197] (2) All participating units should test the subjects according to the prescribed indicators.

[0198] (3) All cases must be explained without exception. Cases that can be evaluated for efficacy should be evaluated according to the Level 4 standard, and cases that cannot be evaluated should be explained.

[0199] (4) Recording terminology and observation methods should be standardized, and observation units should meet the standards.

[0200] (5) The observation and recording times should be consistent.

[0201] (6) The observation time must be reasonable and follow the principle of simultaneous observation with the control group.

[0202] (7) All observation indicators (including symptoms, signs, laboratory tests, etc.) must have their own previous records.

[0203] (8) Carefully fill out each case observation form and verify that the relevant data is accurate. (9) After the experiment, medical records must not be altered at will, and all data must be statistically processed.

[0204] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A soft capsule containing paeoniflorin saponins, characterized in that, The soft capsule consists of contents and a shell; the contents are made from peony root extract, soybean oil, beeswax, soybean lecithin, and Tween-80, in the following weight ratios: soybean oil:peony root extract:soy lecithin = 5~7:1:0.03~0.07; peony root extract:beeswax = 10:1 or 15:1; Tween-80 is used as a surfactant in the contents. The dried extract of Paeonia lactiflora is composed of saikosaponins and total peony glycosides; the mass ratio of saikosaponins to total peony glycosides is 0.05~0.075:0.04~0.

06. The bupleurum saponin soft capsules are used to treat premenstrual irritability syndrome with liver qi stagnation and depression.

2. The peony saponin soft capsule according to claim 1, characterized in that, The capsule is composed of gelatin, glycerin, water, and preservatives.

3. The peony saponin soft capsule according to claim 2, characterized in that, The ratio of gelatin, glycerin, and water is 1:0.4:

1.

4. The peony saponin soft capsule according to claim 2, characterized in that, The preservative is ethylparaben.

5. The method for preparing the peony saponin soft capsules according to any one of claims 1-4, characterized in that, The steps include the following: The dry extract of Paeonia lactiflora, beeswax, and soybean lecithin were dissolved in soybean oil, sieved, and the resulting liquid was obtained. Glycerin, water, and preservatives are heated to a certain temperature, gelatin is added to gelatinize, the mixture is dispersed evenly, degassed, and filtered to obtain a gel solution. The medicinal liquid and the gel were used to prepare soft capsules by compression, and then dried to obtain the peony saponin soft capsules.

6. The preparation method according to claim 5, characterized in that, The temperature of the gelling agent is 80~90℃.

7. The preparation method according to claim 5, characterized in that, Degassing is performed using a vacuum method with a vacuum level of -0.07 to -0.08 MPa. Degassing continues until no obvious bubbles are released from the surface of the adhesive, and the surface exhibits a honeycomb-like flow pattern.

8. The preparation method according to claim 5, characterized in that, The pressing process is as follows: under ambient temperature of 16~18℃ and relative humidity ≤50%, the temperature of the glue box is preheated to 48~52℃ and the temperature of the spray body is preheated to 38~42℃. The air compressor is turned on to press the glue liquid into the glue box through the glue delivery pipe. The blower is turned on to adjust the thickness of the rubber sheet and make a continuous tape. The medicine liquid is filled into the tape and pressed by rotating the roller mold to obtain soft capsules.

9. The preparation method according to claim 5, characterized in that, The drying process is carried out at 33~37℃ and relative humidity of 28%~32% for 3.5~4.5 hours.

10. The use of the paeoniflorin saponin soft capsules according to any one of claims 1-4 in the preparation of a drug for treating a subtype of premenstrual irritability disorder, characterized in that, The aforementioned disease subtype is premenstrual irritability disorder with liver qi stagnation and depression.

11. The application according to claim 10, characterized in that, The applications include a rat model of premenstrual irritability disorder with liver qi stagnation syndrome and depression subtype established by a forced swimming experiment on rats with regular estrous cycles through natural selection, and a clinical trial protocol for drugs to treat this subtype of the disorder.

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