Use of a kind of ringing grass in the preparation of treating perimenopausal depression drug

Abstract

The application discloses an application of Campanula medium in treatment of perimenopausal depression, and a method is as follows: Campanula medium is added into 10 times of distilled water in weight, decocted for 1 h and filtered, residues are added with 8 times of distilled water, and then decocted for 1 h and filtered, and filtrates are combined, and concentrated into 1 g / mL of medicinal liquid according to medicinal material, so that Campanula medium medicinal liquid is obtained. According to the experimental result, Campanula medium can improve the depression and anxiety behavior of PMD model mice. Campanula medium can improve the hippocampus tissue morphology and structure of the PMD model mice, increase the neuron Nissl body abundance of the hippocampus, promote the ER-BDNF-TrkB path, improve the monoamine neurotransmitter level, correct the HPA axis hyperactivity, and thus improve the depression behavior.

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to the application of *Rhododendron molle* in a drug for treating perimenopausal depression. Background Technology

[0002] Depression is a chronic and even life-threatening mental illness. Its most common core symptoms are anxiety, low mood, and slowed thinking, accompanied by negative physical symptoms such as fatigue and weakness. Severe cases can lead to suicidal tendencies. It affects more than 300 million people worldwide and is showing a year-on-year upward trend. During the perimenopausal period, women experience a gradual decline in ovarian function and significant fluctuations in sex hormones, which greatly increases their risk of developing depression.

[0003] Modern research suggests that perimenopausal depression (PMD) is related to estrogen deficiency. The decline in estrogen induces a deficiency of monoamine neurotransmitters, hyperactivity of the HPA axis, and a reduction in neurotrophic factors, which may contribute to PMD. PMD typically occurs during the menopausal transition and within one year after the last menstrual period, a specific time when estrogen levels experience a sharp drop. This sudden withdrawal of estrogen can cause mood disturbances and easily trigger depression. The main clinical symptoms of PMD are extremely similar to those of menopausal syndrome, leading to a very low recognition rate and hindering timely prevention and treatment. PMD adds significant suffering to women; statistics show that more than half of patients in my country have had suicidal thoughts, causing a significant negative impact on their families. Therefore, researching the pathogenesis and treatment of PMD is of great importance.

[0004] Although there are various drugs available for treating perimenopausal depression, their use and efficacy are not satisfactory due to various reasons. Therefore, the improvement and innovation of drugs for treating perimenopausal depression has been a technical problem that those skilled in the art have been researching and solving.

[0005] *Crotalaria ferruginea* Grah. or *C. linifolia* Lf., belonging to the legume family, is the root or whole plant of these plants. It is also known as wild peanut, dog bell, and yellow wild lily. It is a perennial herb densely covered with pale yellow silky hairs. According to *Chinese Dai Medicine Records*, it is used in traditional Chinese medicine to nourish the liver and kidneys, relieve coughs and asthma, and promote diuresis. *Chinese Materia Medica* states that it functions to astringe lung qi, nourish the spleen and kidneys, promote urination, reduce swelling and toxicity, nourish the liver and kidneys, and regulate menstruation. *Crotalaria ferruginea* has a long history of medicinal use and significant efficacy, and is one of the commonly used traditional medicines in Yunnan. Currently, there are very few reports on this Chinese herb. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of *Rhododendron simsii* in the treatment of perimenopausal depression, which can effectively improve the problem of medication for the treatment of perimenopausal depression.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The application of a type of bellflower in the treatment of perimenopausal depression.

[0009] Preferably, the bellflower can be prepared as a solid bellflower preparation, a bellflower herbal liquid, or a bellflower extract.

[0010] Preferably, the preparation method of the herbal decoction of *Callicarpa japonica* is as follows: add 10 times the weight of distilled water to *Callicarpa japonica*, decoct for 1 hour and filter. Add 8 times the amount of distilled water to the residue and continue to decoct for 1 hour and filter. Combine the filtrates and concentrate them into a decoction of 1 g / mL based on the amount of raw herb to obtain the herbal decoction of *Callicarpa japonica*.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) *Callicarpa japonica* significantly improved depressive-like behavior in PMD model mice. *Callicarpa japonica* may exert its neuroprotective effect by modulating the plasma metabolic network of PMD model mice, mainly modulating 19 differential metabolites, involving multiple metabolic pathways such as phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, linoleic acid metabolism, taurine and hypotaurine metabolism, arachidonic acid metabolism, tryptophan metabolism, and tyrosine metabolism. Through analysis of the changes in differential metabolites and metabolic pathways, it was found that the mechanism of *Callicarpa japonica* in PMD may be related to neurotransmitter levels, HPA axis function, and neurotrophic factors.

[0013] (2) Bellflower can improve depression-anxiety-like behavior in PMD model mice. Bellflower can improve depression-like behavior by improving the tissue morphology and structure of the hippocampus in PMD model mice, increasing the abundance of Nissl bodies in hippocampal neurons, promoting the ER-BDNF-TrkB pathway, improving the level of monoamine neurotransmitters, and correcting HPA axis hyperactivity. Attached Figure Description

[0014] Figure 1 Images of vaginal exfoliated cell smears (HE staining, ×400) from rats at different stages of blood component analysis of the liquid containing the herbal medicine *Rhodiola rosea*.

[0015] Figure 2 Plasma BPC diagrams under positive and negative ion modes;

[0016] Figure 3 Image of vaginal exfoliated cell smears (HE staining, ×200) in mice at different stages of plasma metabolism analysis to improve depressive behavior in a PMD model using *Cephalotaxus fortunei*. A: Proestrus; B: Estrous stage; C: Mesentery; D: Interestrus.

[0017] Figure 4This is a graph showing the plasma PCA analysis of mice in each group under positive ion mode;

[0018] Figure 5 This is a graph showing the analysis of OPLS-DA in mouse plasma under positive ion mode;

[0019] Figure 6 For the displacement test in positive ion mode, R² = (0.0, 0.696), Q² = (0.0, -0.47);

[0020] Figure 7 This is a graph showing the principal component analysis of plasma from mice in each group under negative ion mode;

[0021] Figure 8 Analysis of mouse plasma OPLS-DA under negative ion mode;

[0022] Figure 9 For the displacement test in negative ion mode, R² = (0.0, 0.998), Q² = (0.0, -0.102);

[0023] Figure 10 Analysis of MetPA as a potential biomarker in PMD model mice;

[0024] Figure 11 The OFT trajectory diagrams of mice in each group during the mechanism analysis of how *Callicarpa japonica* improves depressive behavior in a PMD model mouse are shown. A: Group S; B: Group M; C: Group E; D: Group X; E: Group Z.

[0025] Figure 12 The following are the EPM test trajectory diagrams for each group of mice: A: Group S; B: Group M; C: Group E; D: Group X; E: Group Z;

[0026] Figure 13 Figure 1 shows the effect of XLC on hippocampal neurons in PMD mice (×200). A: Group S; B: Group M; C: Group E; D: Group X; E: Group Z.

[0027] Figure 14 Figure 1 shows the effect of XLC on Nissl bodies in the hippocampus of PMD mice (×200). A: Group S; B: Group M; C: Group E; D: Group X; E: Group Z.

[0028] Figure 15 The expression of BDNF and TrkB proteins in the hippocampus of mice was measured (x±S, n=3). A: S group; B: M group; C: E group; D: X group; E: Z group. Detailed Implementation

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Embodiment

[0031] Application of Crotalaria pallida Ait. in drugs for treating perimenopausal depression. The Crotalaria pallida Ait. is made into a medicinal liquid of Crotalaria pallida Ait., and the preparation method is as follows: Add Crotalaria pallida Ait. to 10 times the weight of distilled water, decoct for 1 h and filter. Add 8 times the amount of distilled water to the residue and continue to decoct for 1 h and filter. Combine the filtrates and concentrate them into a medicinal liquid with a crude drug amount of 1 g / mL to obtain the medicinal liquid of Crotalaria pallida Ait.

[0032] Evaluate the pharmacodynamic effect of the medicinal liquid of Crotalaria pallida Ait. prepared in this embodiment.

[0033] I. Analysis of the components entering the blood in vivo of the medicinal liquid of Crotalaria pallida Ait.

[0034] 1.1 Experimental animals

[0035] In this part of the experiment, 24 SPF - level healthy SD female rats at 3 - 4 months old, with a body weight of 230 ± 20 g, were used. They were provided by Beijing SPF Biotechnology Co., Ltd., and the certificate number was SCXK(Beijing)2019 - 0010. All animals were raised under the conditions of a temperature of 25 ± 1 °C and a humidity of 55 ± 5%. The experimental procedures followed the regulations of the Yunnan Experimental Animal Management Committee for the management and protection of experimental animals.

[0036] 1.2 Establishment of the PMD model

[0037] The SD female rats were randomly divided into a sham - operation group and a model group, with 12 rats in each group. The rats in the model group underwent OVX surgery; the rats in the sham - operation group were treated in the same way as the model group, but without removing the bilateral ovaries.

[0038] 1.3 Bilateral ovariectomy (OVX)

[0039] The rats in the model group were anesthetized with isoflurane. The rats were placed in a supine position and the extremities were fixed. The hair was cut at the intersection of the extension line of the thigh root and the mid - back line, and disinfected in the conventional method; the skin was incised about 1 cm, and the lumbar muscle layer was dissected. The cauliflower - like ovaries on both sides could be seen. After removal, sprinkle some penicillin sodium powder on the wound, and then sew up the lumbar muscle and skin in sequence. The other ovary was removed in the same way. The postoperative rats were placed on a warming pad until they were awake and could move freely. The sham - operation group was the same as above. After anesthesia, the abdomen was opened, the ovaries were found but not removed, and only the same - sized fat masses near them were removed, and then sewn up in the same way.

[0040] 1.4 Observation of the estrous cycle in rats

[0041] Vaginal secretions from rats in each group were collected at fixed times daily from day 4 to 7 post-surgery. 30 μL of physiological saline was repeatedly aspirated into the rat vagina using a Papanicolaou dropper 3-5 times. The extracted secretions were smeared onto glass slides, allowed to air dry, and then stored for later use. The vaginal mucus was treated with hematoxylin and eosin (HE) staining, and changes in the estrous cycle were observed based on cell shedding to determine the success of the OVX procedure.

[0042] 1.5 Chronic Unpredictable Mild Stimuli (CUMS)

[0043] On the 8th day post-surgery, CUMS modeling was established in rats of each group. Stimuli included: fasting for 24 hours, water deprivation for 24 hours, moist bedding for 24 hours, tilting the cage for 12 hours, shaking the cage for 15 minutes, day-night reversal, and ultrasound stimulation for 1 hour. Different stimulation methods were selected for modeling within 7 days, for a total of 21 days.

[0044] 1.6 Model Validation

[0045] 1.6.1 HE staining of vaginal exfoliated cytology smears

[0046] Take the dried slide from section 1.4 and perform the following steps: Immerse in 95% ethanol for 15 minutes for fixation; then immerse sequentially in hematoxylin solution for 10 minutes, rinse with running water, acid-alcohol solution for 30 seconds, rinse with running water, dissolve eosin in alcohol for 1 minute, rinse with running water, and stain; then immerse sequentially in 95% ethanol I for 15 seconds, 95% ethanol II for 15 seconds, 100% ethanol for 1 minute, and xylene for 5 minutes for dehydration and clearing; allow to air dry slightly, mount with neutral resin, and after drying, observe and photograph under a microscope. The photographic results are as follows. Figure 1 .like Figure 1 As shown, the vaginal epithelial cells of the sham-operated rats exhibited typical periodic changes during proestrus, estrus, metestrus, and estrus. Proestrus (Figure A) was characterized by intact epithelial cells; estrus (Figure B) was characterized by densely packed keratinocytes; metestrus (Figure C) was characterized by predominantly leukocytes and an increase in keratinocytes; and estrus was characterized by a small number of epithelial cells. The periodic changes in vaginal cells in the model group rats disappeared, and the condition remained in the estrus phase (Figure D).

[0047] 1.6.2 General Condition Observation

[0048] Before the behavioral experiment, the general condition of the rats in each group was observed, including food intake, activity level, responsiveness, and coat luster. The results are shown in Table 1 below:

[0049] Table 1

[0050]

[0051] 1.6.3 Behavioral Validation Model

[0052] The model was replicated using the OVX combined with CUMS method. After modeling, rats in the sham-operated group and the model group were tested in open field to validate the model.

[0053] The Open Field Test (OFT) was conducted in a square enclosure with a side length of 150 cm and a height of 50 cm. The bottom and inner walls were black. The experiment was performed in a quiet environment. Rats were placed in the center of the enclosure's bottom surface, and video recording was started. Recording was stopped after 5 minutes. After each rat's experiment, the inner walls and bottom were wiped with alcohol to ensure the accuracy of the results for the next rat. The total distance traveled (cm) and the central distance traveled (cm) for each rat were analyzed after the experiment. A decrease in total distance reflected reduced activity, while a decrease in central distance reflected reduced curiosity about the new environment. The results are shown in Table 2.

[0054] Table 2

[0055]

[0056] Note: Compared to Group S ** P<0.01

[0057] Compared with the sham-operated group, the total distance and central activity distance of the rats in the model group were reduced, indicating that the PMD model rats were successfully established.

[0058] 1.6.4 Collection and processing of plasma samples

[0059] Rats whose model was successfully validated were divided into a model group and a model drug treatment group. Rats in the model group were administered an equal volume of distilled water by gavage. Rats in the model drug treatment group were administered a single dose of *Rhizophora stylosa* extract by gavage, at a dose of 2.7 g / kg (based on crude drug weight), according to the human-to-rat dosage conversion ratio. Blood samples were collected seven days after administration. Rats were fasted for 12 hours before blood collection, but allowed normal water intake. After the last administration, blood was collected from the orbital sinus at 30, 60, 90, and 120 minutes, placed in heparinized centrifuge tubes, and centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was then stored at -80°C.

[0060] Pre-sample preparation: Take a centrifuge tube, add 300 μL of plasma sample and 1200 μL of cold methanol-acetonitrile (1:1), vortex to mix, let stand at -20℃ for 10 min, centrifuge at 13000g, 4℃ for 15 min, collect the supernatant, and purge with nitrogen. Then redissolve with 200 μL of 75% methanol solution, vortex to mix for 2 min, filter through a 0.22 μm microporous membrane, and prepare for sample injection.

[0061] UPLC-Q-TOF-MS technology was used to perform qualitative analysis on the plasma of rats in the *Rhizoma Cymbidium* test solution, model group, and model drug administration group under positive and negative ion modes. BPC chromatograms are shown below. Figure 2 POS: A is the *Callicarpa japonica* test sample; B is the plasma of the model group; and D is the plasma of the model drug-treated group. NEG: D is the *Callicarpa japonica* test sample; E is the plasma of the model group; and F is the plasma of the model drug-treated group. First, the ion chromatograms of the model group plasma and the model drug-treated group were compared, and then compared with the ion chromatograms of the *Callicarpa japonica* in vitro sample solution to obtain the original blood-entry components of *Callicarpa japonica*. Based on chromatographic retention time, molecular fragment peaks, and database analysis, seven original blood-entry components were identified, all of which are flavonoids. See Table 3:

[0062] Table 3

[0063]

[0064] As shown in Table 3 above, seven components were detected in rats after administration of *Heliotropium indicum*. Literature review revealed that all seven components were flavonoids. While these compounds each have their own characteristics, most are phytoestrogens, and some have modern pharmacological evidence regarding their ability to improve depressive behaviors. Among them, kaempferol, luteolin, quercetin, and apigenin can cross the blood-brain barrier to exert neuroprotective effects and improve neurological disorders.

[0065] Soy isoflavones can improve depressive-like behavior in chronically stressed rats, increase BDNF expression in the hippocampal dentate gyrus, and enhance nervous system plasticity. Luteolin, through its antioxidant properties, improves hippocampal neuronal development, increases hippocampal neuronal plasticity, and thus plays a role in improving depression. The antidepressant activity of quercetin may be achieved by increasing the expression of 5-HT and BDNF in the hippocampus and reducing the expression of inflammatory factors. Quercetin can improve BDNF expression in the hippocampus of mice, improve depressive-like behavior in depressed mice, and exert an antidepressant effect. Farnesin can inhibit the degeneration of dopamine neurons in the substantia nigra and striatum of the brain and the depletion of dopamine levels. Kaempferol can reduce the expression of apoptosis genes in the hippocampus, reduce the expression of inflammatory factors, improve the antioxidant capacity of cells, and increase the expression of BDNF and NGF in hippocampal tissue, exhibiting good neuroprotective effects. Kaempferol can increase the content of monoamine neurotransmitters in the prefrontal cortex and improve depressive-like behavior in depressed rats. Apigenin activates signaling pathways through estrogen receptors, promoting neuronal differentiation and enhancing neuronal plasticity, thereby protecting cells. Genistein promotes ERK and CREB phosphorylation and increases BDNF expression in the rat hippocampus and prefrontal cortex. BDNF plays an important role in neuronal development, promoting the repair of damaged neurons, improving neuronal pathological states, and its increased expression is beneficial in alleviating depressive symptoms.

[0066] The components of Crotalaria ferruginea that enter the blood are mostly estrogen components, and they can exert neuroprotective effects through estrogen receptors. Daidzein, genistein, luteolin, apigenin, and quercetin can increase the expression of BDNF in the brain and enhance the synaptic plasticity of neuronal cells. Quercetin, genistein, and acacetin can improve the synthesis of monoamine neurotransmitters in the brain. Thus, the components in Crotalaria ferruginea play a certain role in neuroprotection and other aspects.

[0067] II. Plasma Metabolic Analysis of Crotalaria ferruginea in Improving Depressive Behavior in PMD Model

[0068] 2.1 Experimental Animals

[0069] In this part of the experiment, 24 3-month-old SPF-grade healthy KM female mice, weighing 30±5 g, were provided by Spf (Beijing) Biotechnology Co., Ltd., with the certificate number SCXK (Beijing) 2019-0010. The animals were all raised under the conditions of a temperature of 25±1°C and a humidity of 55±5%. The experimental procedures followed the regulations of the Yunnan Experimental Animal Management Committee on the management and protection of experimental animals.

[0070] 2.2 Classification of Experimental Animals and Administration Methods

[0071] Twenty-four female Kunming mice were randomly divided into a sham operation (S) group, a model (M) group, and a Crotalaria ferruginea (X) group, with 8 mice in each group. The M group and the X group underwent OVX surgery; the S group removed the same volume of fat and retained the ovaries, and the operation method was the same as that in 2.2.1 of Chapter 2. Four to seven days after the operation, vaginal exfoliated cells were taken for HE staining. Starting from the 8th day after the operation, CUMS stimulation was carried out for a total of 21 days. The S group and the M group were intragastrically administered the same volume of normal saline, and the X group was intragastrically administered 3.9 g / kg of Crotalaria ferruginea medicinal liquid. The administration and stimulation were carried out simultaneously. After 28 days, an animal behavior experiment was conducted. After the behavior experiment was completed, plasma samples were collected.

[0072] 2.3 Establishment of PMD Model

[0073] The OVX combined with CUMS method was used to establish the model, and the operation method was the same as that in 1.3.

[0074] 2.4 Model Verification

[0075] 2.4.1 HE staining of vaginal exfoliated cell smears, the method was the same as that in 1.6.1, and the test results were as Figure 3 . As Figure 3 shown, the results of vaginal smears showed that the cyclic changes of vaginal cells in OVX mice disappeared and they remained in the estrous interphase ( Figure 3 -D), indicating the success of the OVX surgery.

[0076] 2.4.2 Open Field Test (OFT)

[0077] The mouse open field test chamber was a square with sides of 50 cm and a height of 50 cm, with the bottom and inner walls black. The experiment was conducted in a quiet environment. Mice were placed in the center of the bottom of the chamber, and video recording was started. Recording was stopped after 5 minutes of observation. After each mouse's experiment, the inner walls and bottom were wiped with alcohol to ensure the accuracy of the results for the next mouse. The central movement distance (cm) and central movement time (s) of each mouse were analyzed after the experiment. The test results are shown in Table 4.

[0078] Table 4

[0079]

[0080] Note: Compared to S, ** indicates P < 0.01; compared to M, # This indicates that P < 0.05;

[0081] As shown in Table 4, compared with group S, the central movement distance and time of mice in group M were significantly reduced (P < 0.01); compared with mice in group M, both indicators of mice in group X were significantly improved (P < 0.05).

[0082] 2.4.3 Elevated Cross Maze Experiment (EPM)

[0083] The EPM consists of two open arms (50m long and 10m wide), a closed arm (50m long and 10m wide), and a central platform. At the start of the experiment, mice were placed on the central platform with their heads facing one of the open arms. The activity of the mice was recorded, and the percentage of time spent in the open arm and the percentage of times the mice entered the open arm within 5 minutes were calculated. After each mouse was tested, the EPM device was wiped with 75% alcohol to ensure the accuracy of the results. The test results are shown in Table 5.

[0084] Table 5

[0085]

[0086] Note: Compared to S, ** indicates P < 0.01; compared to M, # This indicates that P < 0.05.

[0087] As shown in Table 5 above, compared with group S, the percentage of open arm retention time and the percentage of open arm insertion times of mice in group M were significantly reduced (P<0.01); compared with mice in group M, both indicators in group X were significantly improved (P<0.05).

[0088] 2.4.4 Forced Swimming Test (FST)

[0089] Mice were placed in a cylindrical container with a diameter of 10 cm and a height of approximately 20 cm, at a water temperature of 20-25℃. The water level was approximately 12 cm. Video recordings were made for 6 minutes after the mice entered the water. After the experiment, the mice were removed, wiped clean, and clean warm water was added to the container before continuing the experiment. The time spent at rest within the last 4 minutes was recorded. The test results are shown in Table 6.

[0090] Table 6

[0091]

[0092] Note: Compared to S, ** indicates P < 0.01; compared to M, ## This indicates that P < 0.01

[0093] As shown in Table 6 above, compared with group S, the resting time of mice in group M was significantly increased (P<0.01); compared with group M, the resting time of mice in group X was significantly decreased (P<0.01).

[0094] 2.4.5 Tail Suspension Test (TST)

[0095] Mice were suspended upside down on the TST device, with their tails (1 cm from the tip) secured with tape and their heads 50 cm off the ground. The surrounding environment was kept quiet throughout the experiment. Video recordings were taken of the mice 6 minutes after suspension, and the resting time within the first 4 minutes was analyzed. The test results are shown in Table 7 below:

[0096] Table 7

[0097]

[0098] Note: Compared to S, ** indicates P < 0.01; compared to M, ## This indicates that P < 0.01

[0099] As shown in Table 7 above, compared with group S, the resting time of mice in group M was significantly increased in the TST test (P<0.01); compared with group M, the resting time of mice in group X was significantly decreased (P<0.01).

[0100] According to the results of OFT, EPM, FST, and TST tests, senna has an improving effect on PMD.

[0101] 2.4.6 Collection and preparation of plasma samples

[0102] Following the animal behavioral experiments, blood was collected from mice by enucleation. The blood was placed in heparin sodium centrifuge tubes and allowed to stand for 30 minutes before centrifugation at 4°C, 3500 rpm, and 10 minutes. Sample pretreatment was the same as in section 2.4. 5 μL of the sample was analyzed by UPLC-Q-TOF / MS. The QC sample was prepared by mixing 10 μL of the plasma test solutions from each group. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to identify metabolites between different groups. Differential metabolites were screened using VIP≥1 and T-TEST 0.05. Differential metabolites were identified using the HMDB and PubChem databases. MetaboAnalyst 5.0 was used to analyze the differential metabolites and determine the metabolic pathways. The test results are as follows: Figures 4-9 .

[0103] Depend on Figures 4-9 It was found that groups S and M were clearly separated. From a macroscopic PCA perspective, significant differential metabolic changes had occurred in the plasma of normal mice and PMD-depressed mice. After administration of the decoction of *Callicarpa japonica*, a significant regression trend was observed in the plasma metabolic profile of mice in group X, demonstrating that *Callicarpa japonica* has a certain neuroprotective effect on PMD model mice.

[0104] Preliminary identification results were obtained through direct matching with the database. Further screening and verification were conducted based on the mass spectrometry fragmentation patterns of the compound structures. A total of 19 differentially metabolites in the plasma of PMD model mice were identified, including PC (16:0 / 0:0), Phosphocholine, L-Methionine, Oxypurinol, Guanine, Inosine, Hypoxanthine, Corticosterone, Propionylcarnitine, Tyrosine, Tryptophan, Taurine, Phenylalanine, Linoleic acid, Arachidonic acid, D-Glucose, 3-Hydroxybutyric acid, Hexanoylglycine, and Stearic acid. After intervention with senna, 19 metabolites showed varying degrees of regression.

[0105] The aforementioned biomarkers were used for enrichment analysis on the MetPA open-source website to obtain pathway information affecting metabolic abnormalities in PMD model mice. The results are as follows: Figure 9The main pathways include: phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, linoleic acid metabolism, taurine and hypotaurine metabolism, arachidonic acid metabolism, tryptophan metabolism, and tyrosine metabolism.

[0106] Linoleic acid and arachidonic acid are both long-chain unsaturated fatty acids (LCPUFAs) and play an important role in the biosynthesis of unsaturated fatty acids. As neurotrophic factors, LCPUFAs can effectively promote BDNF synthesis in hippocampal neurons, activate synaptic proteins, and enhance synaptic plasticity, thus playing a crucial role in neural development. Studies have found that the levels of differentially expressed metabolites (linoleic acid and arachidonic acid) in the plasma of PMD model mice were significantly decreased, while *Heliotropium indicum* could reverse the expression trend of these differentially expressed metabolites, promote unsaturated fatty acid synthesis, increase BDNF synthesis in the hippocampus, enhance synaptic plasticity, and exert a neuroprotective effect.

[0107] Tyrosine is an essential amino acid for specific functions of the central nervous system and can cross the blood-brain barrier to exert its effects. As a precursor to neurotransmitters such as dopamine and norepinephrine, neurotransmitters are closely related to the pathogenesis of depression. Supplementing with tyrosine during external stress can reduce the depletion of norepinephrine (NE) in patients, maintain neurotransmitter levels, and thus treat depression. Phenylalanine is an essential amino acid widely found in mammals. It is not only a precursor to tyrosine synthesis but also to substances such as dopamine and norepinephrine, and can produce catecholamine effects. Tryptophan is one of the essential amino acids in mammals. As a precursor to 5-HT, it can also be used in protein synthesis. Tyrosine, phenylalanine, and tryptophan are closely related to neurotransmitters and participate directly or indirectly in their synthesis. In this part of the study, we found abnormal changes in the levels of tyrosine, phenylalanine, and tryptophan in the plasma of PMD mice. These changes can affect the synthesis of neurotransmitters to some extent. After treatment with *Callicarpa japonica*, the levels of these substances in the plasma showed a significant rebound, and the depressive-like behavior of PMD mice also showed a significant improvement. This indicates that *Callicarpa japonica* can treat PMD by regulating the metabolic processes of tyrosine, phenylalanine, and tryptophan in life activities and maintaining the dynamic balance of neurotransmitters.

[0108] Taurine is a semi-essential amino acid in the body, widely present in the brain, kidneys, and heart, and plays a crucial role in regulating the endocrine system. Depression is the most prominent feature of taurine deficiency, accompanied by symptoms such as sleep disorders. Studies have shown that the taurine content in the body of patients with depression is basically insufficient. Increasing the taurine content can improve depressive symptoms. The reduction of taurine leads to an increase in ACTH and CORT in the serum, and supplementing taurine will reverse this situation. It is inferred that taurine intervention can improve the dysfunction of the HPA axis. Research has found that the taurine content in the plasma of PMD model mice decreased significantly. After treatment with Crotalaria ferruginea, the taurine level increased, and the metabolic pathways of taurine and hypotaurine were adjusted back, indicating that the antidepressant effect of Crotalaria ferruginea may be due to promoting the synthesis of taurine and inhibiting the overactivity of the HPA axis, thereby regulating depression.

[0109] Crotalaria ferruginea may regulate these differential metabolites to varying degrees, affect the related indicators of PMD, such as 5-HT, NE, BDNF, HPA axis, etc., and play a neuroprotective role in the depressive behavior of PMD model mice. These potential differential metabolites and their changing trends are closely related to the development of PMD and the physiological and biochemical processes such as the synthesis of neurotransmitters, the function of the HPA axis, and synaptic function.

[0110] III. Mechanism analysis of the improvement of depressive behavior in PMD model mice by Crotalaria ferruginea

[0111] 3.1 Experimental animals

[0112] In this part of the experiment, 75 3-month-old SPF-grade healthy KM female mice with a body weight of 30±5 g were used. They were provided by Spf (Beijing) Biotechnology Co., Ltd., and the certificate number was SCXK (Beijing) 2019-0010. All animals were housed under the conditions of a temperature of 25±1°C and a humidity of 55±5%. The experimental procedures followed the regulations of the Yunnan Provincial Laboratory Animal Management Committee for the management and protection of experimental animals.

[0113] 3.2 Grouping of experimental animals and drug administration methods

[0114] Seventy-five female Kunming mice were randomly divided into four groups: sham surgery (S), model (M), estradiol (E), *Rhododendron simsii* (X), and blocker (Z), with 15 mice in each group. In the S group, an equal volume of fat was removed, while the ovaries were preserved. The other groups underwent ovarian endoscopic vaginoplasty (OVX). Four to seven days post-surgery, vaginal exfoliated cells were collected for HE staining to determine the success of the model. In mice with successful OVX models, CUMS stimulation began on day 8 post-surgery and continued for 21 days. The S and M groups were administered the same volume of physiological saline by gavage; the X group was administered 3.9 g / kg (based on crude drug weight) of *Rhododendron simsii* extract by gavage at a dose of 0.1 mL / 10 g; the E group was administered 0.013 mg / kg of E2 at a dose of 0.1 mL / 10 g; and the Z group received 100 μg / kg of the drug, followed by an intraperitoneal injection of the nonspecific blocker ICI182780 every other day. Drug administration and stimulation were performed concurrently. After 28 days, behavioral experiments were conducted. The treatment methods for each group are shown in Table 8:

[0115] Table 8

[0116]

[0117] 3.3 Replicating the PMD Model

[0118] The OVX combined with CUMS method replicates the PMD model, and the operation method is the same as in 1.3.

[0119] 3.4 Model Validation

[0120] 3.4.1 Open Field Experiment (OFT)

[0121] The method is the same as in 2.4.2, and the test results are shown in Table 9:

[0122] Table 9

[0123]

[0124]

[0125] Note: Compared to S, ** indicates P < 0.01; compared to M, # This indicates that P < 0.05. ## This indicates that P < 0.01; compared to X, &

[0126] This indicates that P < 0.05. && This indicates that P < 0.01

[0127] As shown in Table 9 and Figure 11As shown, compared with group S, the central movement distance and time of mice in group M were significantly reduced (P < 0.01); compared with mice in group M, both indicators of mice in groups X and E were significantly improved (P < 0.01, P < 0.05); there was no significant difference between groups X and E (P > 0.05); compared with group X, both indicators of mice in group Z were significantly reduced (P < 0.01).

[0128] 3.4.2 Elevated Cross Maze Experiment (EPM)

[0129] The method is the same as in 2.4.3, and the test results are shown in Table 10:

[0130] Table 10

[0131]

[0132] Note: Compared to S, ** indicates P < 0.01; compared to M, ## This indicates that P < 0.01; # This indicates that P < 0.05; compared to X, && This indicates that P < 0.01. & This indicates that P < 0.05.

[0133] As shown in Table 10 and Figure 12 As shown, compared with group S, the percentage of open arm retention time and the percentage of open arm insertion times were significantly reduced in group M mice (P<0.01); compared with group M mice, both indicators were significantly improved in groups X and E (P<0.01, P<0.05); there was no significant difference between groups X and E (P>0.05); compared with group X mice, both indicators were significantly reduced in group Z mice (P<0.01, P<0.05).

[0134] 3.4.3 Forced Swimming Test (FST)

[0135] The method is the same as in 2.4.4, and the test results are shown in Table 11:

[0136] Table 11

[0137]

[0138] Note: Compared to S, ** indicates P < 0.01; compared to M, ## This indicates that P < 0.01; compared to X, && This indicates that P < 0.01

[0139] As shown in Table 11, compared with group S, the resting time of mice in group M was significantly increased (P<0.01); compared with group M, the resting time of mice in groups X and E was significantly decreased; there was no significant difference between groups X and E (P>0.05); compared with group X, the resting time of mice in group Z was significantly increased (P<0.01).

[0140] 3.4.4 Tail Suspension Test (TST)

[0141] The method is the same as in 2.4.5, and the test results are shown in Table 12:

[0142] Table 12

[0143]

[0144] Note: Compared to S, ** indicates P < 0.01; compared to M, ## This indicates that P < 0.01; compared to X, && This indicates that P < 0.01

[0145] As shown in Table 12, compared with group S, the resting time of mice in group M was significantly increased (P<0.01); compared with group M, the resting time of mice in groups X and E was significantly decreased (P<0.01); there was no significant difference between groups X and E (P>0.05); compared with group X, the resting time of mice in group Z was significantly increased (P<0.01).

[0146] Sampling methods: After animal behavior studies, blood was collected from the eyes of mice in each group. After standing for 30 minutes, the blood was centrifuged at 12,000 rpm for 30 minutes, and the supernatant serum was collected and stored at -80℃. After blood collection, the thoracic cavity of the mice was cut open to expose the heart. A small incision was made at the right atrial appendage, and a round-tipped needle was inserted through the interventricular region to perform perfusion. The perfusion fluid flowed from the right atrial appendage to the liver and turned white. Brain tissue sample collection methods: ① Six mice were randomly selected from each group. The heart was perfused with 4% paraformaldehyde until the liver turned grayish-white. The mice were then quickly decapitated, and the skull was separated on an ice tray to remove the brain. The olfactory bulb and cerebellum were quickly discarded, and the brain was sagittally divided along the midline of the brain to divide it into left and right hemispheres. The brain was fixed in 4% paraformaldehyde for 48 hours for HE staining and Nissl staining. ②The other 9 mice were decapitated directly, and the skull was separated and the brain was dissected on an ice tray. The olfactory bulb and cerebellum were quickly discarded, and a sagittal cut was made along the midline of the brain. After the cortex was dissected, the hippocampus was removed, placed in a cryovial, and quickly placed in a liquid nitrogen tank and stored at -80°C for use in kits, RT-qPCR experiments, and WB experiments.

[0147] 3.5 Pathological staining

[0148] 3.5.1 HE staining

[0149] Brain tissue was divided into left and right hemispheres and fixed separately in 4% neutral methanol solution for 24-48 hours. After removal, the tissue was placed in an embedding cassette and rinsed with water for several hours until odorless. The rinsed brain tissue was then dehydrated and successively immersed in ethanol solutions of 70%, 80%, 90%, 95%, 95%, 100%, and 100% ethanol for 1 hour each; followed by clearing treatment, and then successively immersed in n-butanol solution for 30 minutes, xylene I solution for 10 minutes, and xylene II solution for 10 minutes; finally, it was immersed in paraffin at 54-56℃ and 56-58℃ for 1 hour each, and then in paraffin at 60-62℃ overnight. The tissue was removed and fixed in an embedding cassette with paraffin. After the paraffin block cooled, the tissue embedding was complete. The tissue was cut into 5μm sections using an automatic microtome, flattened in distilled water at approximately 38℃, adhered to a glass slide to prevent detachment, and placed in a 37℃ oven overnight. After cooling the paraffin slides to room temperature, they were sequentially immersed in xylene I for 5 min, xylene II for 5 min, 100% ethanol for 5 min, 95% ethanol for 5 min, and distilled water for 2 min for dewaxing and rehydration. Then, they were sequentially immersed in hematoxylin solution for 10 min, rinsed with running water, acid-alcohol for 30 s, rinsed with running water, dissolved in eosin in alcohol for 1 min, rinsed with running water, and stained. Next, they were sequentially immersed in 95% ethanol I for 15 s, 95% ethanol II for 15 s, 100% ethanol for 1 min, and xylene for 5 min for dehydration and clearing. After air-drying, they were mounted with neutral resin, dried, and observed and photographed under a microscope to observe neurons in the mouse hippocampus. The results are as follows: Figure 13 .

[0150] like Figure 13 The results showed that group S mice had a higher number of hippocampal neurons, a denser distribution, and better morphology. Compared to group S, group M mice had fewer hippocampal neurons, a sparser distribution, and obvious nuclear pyknosis. Compared to group M, groups X and E mice had a denser distribution and a greater number of hippocampal neurons, with less obvious nuclear pyknosis. Compared to group X, group Z mice had a less dense and fewer hippocampal neurons, with more obvious nuclear pyknosis.

[0151] 3.5.2 Nissl staining

[0152] The embedded paraffin slides were cut into 5 μm sections using an automatic microtome. These sections were then flattened in distilled water at approximately 38°C, adhered to a glass slide to prevent detachment, and placed in a 37°C oven overnight. After cooling to room temperature, the slides were sequentially immersed in xylene I for 10 min, xylene II for 10 min, xylene III for 10 min, 100% ethanol for 5 min, and 90% ethanol for 2 min, followed by rinsing with distilled water for 2 min for dewaxing and rehydration. Subsequently, they were sequentially immersed in Nissl staining solution for 10 min, rinsed with running water, and stained. Then, they were sequentially immersed in 95% ethanol I for 2 min, 95% ethanol II for 2 min, xylene I for 5 min, and xylene II for 5 min for dehydration and clearing. After slight drying, they were mounted with neutral resin and observed and photographed under a microscope to observe the morphology and number of Nissl bodies in the mouse hippocampus. Results are as follows: Figure 14 .

[0153] like Figure 14 The results showed that the Nissl bodies in the hippocampus of mice in group S were darker, more structurally complete, and more numerous; the Nissl bodies in the hippocampus of mice in group M were lighter, partially atrophied and deformed, and fewer in number; the Nissl bodies in the hippocampus of mice in groups X and E were darker, more structurally complete, and more numerous; and the Nissl bodies in the hippocampus of mice in group Z were lighter, less structurally complete, and fewer in number.

[0154] 3.6 Western blot detection

[0155] 3.6.1 Extraction of total protein from tissues

[0156] The hippocampus was placed in a centrifuge tube, shredded, and the protein extraction was performed on ice. Ten volumes of RIPA lysis buffer (containing 1% PMSF) were added, and the mixture was homogenized in a homogenizer to ensure complete disruption of the hippocampal tissue. The mixture was sonicated on ice for 30 minutes, followed by centrifugation at 1000 rpm for 40 minutes at 4°C. The supernatant was collected, and its volume was recorded. The supernatant was stored at -20°C for later use.

[0157] 3.6.2 BCA method for protein concentration determination and preparation of the test protein

[0158] Add 0.8 mL of protein standard preparation solution to the protein standard (20 mg BSA), dissolve thoroughly to prepare a 25 mg / mL protein standard solution, and then dilute to a final concentration of 0.5 mg / mL. Prepare the BCA working solution by mixing reagent A and B solutions (50:1). Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of standard solution to the wells of a 96-well plate, and dilute to a final volume of 20 μL, equivalent to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Pipette 20 μL of the supernatant from each group into the sample wells of the 96-well plate. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min. Remove the plate and measure the absorbance of the sample at 562 nm using a microplate reader. Calculate the protein concentration of each sample based on the standard curve. After quantifying the protein samples, add 4×bromophenol blue loading buffer (3:1). After denaturing the proteins in a water bath at 100℃ for 5 min, aliquot the samples at low temperature and store them at -80℃ for later use.

[0159] 3.6.3 SDS-PAGE electrophoresis

[0160] Prepare the separating and stacking gels according to the method in Table 13. Drop the separating gel to approximately 1.5 cm from the top edge, seal with deionized water, and let stand at room temperature for 60 minutes to allow it to solidify. Discard the top layer of deionized water, add the stacking gel, insert the comb, and let stand at room temperature for approximately 30 minutes. Load the samples using a pipette, adding the samples and pre-stained molecular weight protein standards. Set up the electrophoresis apparatus, connect the power supply, and electrophores the stacking gel at 80V for 30 minutes. Change the voltage, and electrophores the separating gel at 120V for 90 minutes, until the gel reaches the bottom of the plate. Cut gel pieces of appropriate size according to the desired protein molecular weight.

[0161] Table 13

[0162]

[0163]

[0164] 3.6.4 Wet Conversion

[0165] Forty minutes before the end of electrophoresis, prepare filter paper and a PVDF membrane. Soak the PVDF membrane in anhydrous methanol for 5 minutes, deionized water for 2 minutes, and transfer buffer for 30 minutes. After electrophoresis, cut the gel to the desired molecular weight, then soak the labeled gel in transfer buffer for 10 minutes, and soak the thick filter paper in transfer buffer for 10 minutes. Finally, arrange the above transfer materials in a specific order, fix the transfer clamp in the electrophoresis tank, connect the apparatus, place the electrophoresis tank in crushed ice, and transfer at a steady current of 200 mA for 90 minutes.

[0166] 3.6.5 Closed

[0167] After the transfer was completed, the PVDF membrane was marked with the front and back sides, placed in the blocking solution, and blocked on a horizontal shaker at room temperature for 2 hours.

[0168] 3.6.6 Antibody incubation

[0169] After sealing, the PVDF membrane was placed in a hybridization bag, and 1 mL of the target antibody diluent (refer to the instructions) was added to the front side of the membrane. The membrane was then sealed and incubated overnight at 4°C. After primary antibody incubation, the PVDF membrane was removed and immersed in TBST solution on a shaker, washing four times for 5 minutes each time. The PVDF membrane was then placed in a hybridization bag, and secondary antibody diluent (1:5000 dilution) was added. The membrane was incubated on a shaker at room temperature for 1 hour, followed by four washes with TBST for 5 minutes each time. Finally, ECL imaging was performed. The antibody dilution ratios are shown in Table 14.

[0170] Table 14

[0171]

[0172] The test results are shown in Table 15 and Figure 15 :

[0173] Table 15

[0174]

[0175]

[0176] Note: Compared with group S, ** indicates P < 0.01; compared with group M, ## This indicates that P < 0.01; compared to X, && This indicates that P < 0.01

[0177] As shown in Table 15 and Figure 15 As shown, compared with group S, the levels of BDNF and TrkB proteins in the hippocampus of mice in group M were significantly decreased (P<0.01); compared with group M, the levels of BDNF and TrkB proteins in the hippocampus of mice in groups E and X were significantly increased (P<0.01); there was no significant difference in protein expression between groups E and X (P>0.05); compared with group X, the levels of BDNF and TrkB proteins in the hippocampus of mice in group Z were significantly decreased (P<0.01).

[0178] 3.7 RT-qPCR detection

[0179] 3.7.1 Total RNA extraction from tissues

[0180] Weigh 100 mg of fresh hippocampal tissue, cut it into small pieces, add two ice-cold steel balls, and then add 1 mL of Trizol lysis buffer. Mix well in a homogenizer, centrifuge to remove foam, remove the steel balls, transfer to a new centrifuge tube, sonicate for 5 min, shake by hand, let stand at room temperature for 5 min, then add 200 mL of chloroform solution, shake vigorously for 15 s, incubate at room temperature for 3 min, centrifuge at 12000 rpm at 4°C for 15 min, take the clear liquid containing RNA from the middle layer, add 500 μL of isopropanol, shake, incubate at room temperature for 10 min, centrifuge under the same conditions for 10 min, discard the supernatant, add 1 mL of 75% ice-cold ethanol, mix well, and suspend the precipitate. Centrifuge at 7500 rpm at 4°C for 5 min, discard the supernatant. Air dry at room temperature to obtain RNA. Note that the entire process should be performed on ice to prevent RNA degradation. Dissolve RNA in 30 μL of enzyme-free ddH2O. Determine the RNA concentration directly using a Nano-100 micro spectrophotometer. Before the experiment, calibrate with RNase-ddH2O to ensure accurate concentration measurement. The OD 260 / OD 280 ratio should be between 1.8 and 2.0. A ratio closer to 2.0 indicates higher RNA purity.

[0181] 3.7.2 cDNA Synthesis

[0182] cDNA was synthesized using a cDNA first-strand synthesis kit to serve as a template for PCR amplification. Based on the measured total RNA concentration for each group, the total RNA volume was adjusted to 200 ng. The reaction was performed on ice. The calculated RNA volume was transferred to a centrifuge tube, and 1 μL of Oligo(dT)18 (0.5 μg / μL) was added. Water was added to a final volume of 13 μL, and the tube was incubated at 70°C for 5 min, followed immediately by incubation on ice. Then, 5 μL of RT-Buffer containing dNTPs was added, and the tube was incubated at 37°C for 5 min. Next, 2 μL of MMLV reverse transcriptase was added, bringing the final reaction volume to 20 μL. The tube was incubated at 42°C for 60 min, and then at 70°C for 10 min to terminate the reaction. The tube was then placed on ice for later use.

[0183] 3.7.3 RT-qPCR reaction

[0184] Amplification reactions were performed using the SGExcel UltraSYBR Mixture (with ROX) kit on an Mx3000P real-time PCR instrument. Primer sequences were designed and synthesized by Shanghai Sangon Biotech Co., Ltd., and are shown in Table 16 below.

[0185] Table 16

[0186]

[0187] Preheat the Mx3000P machine and computer, prepare the reaction solution, and make three replicates and one negative control for each gene, as shown in Table 17.

[0188] Table 17

[0189]

[0190] Add 19 μL of reaction solution to each well of the eight-tube strip, then add 0.8 μL of cDNA. Replace the negative control well with ddH2O. The PCR reaction procedure is shown in Table 18 below.

[0191] Table 18

[0192]

[0193] Based on the Ct values ​​provided by the software, the relative expression level of mRNA is calculated. The Ct difference ratio formula is: ΔCt = Intervention group (Ct target gene - Ct internal reference gene) - Control group (Ct target gene - Ct internal reference gene). Relative expression level = 2 -ΔΔCt .

[0194] The test results are shown in Table 19:

[0195] Table 19

[0196]

[0197] Note: Compared to S, ** indicates P < 0.01; compared to M, ## This indicates that P < 0.01; compared to X, && As shown in Table 19, compared with group S, the mRNA expression levels of BDNF and TrkB in the hippocampus of mice in group M were significantly decreased (P<0.01); compared with group M, the mRNA expression levels of BDNF and TrkB in the hippocampus of mice in group X were significantly increased (P<0.01); there was no significant difference between groups E and X (P>0.05); compared with group X, the mRNA expression levels of BDNF and TrkB in the hippocampus of mice in group Z were significantly decreased (P<0.01).

[0198] 3.8 Reagent Kit Detection

[0199] Enzyme-linked immunosorbent assay (ELISA) was used to test the levels of CRH, ACTH, and CORT in serum, and the levels of 5-HT, DA, and NA in the hippocampus.

[0200] Three mice were selected for each group, along with their hippocampus and serum. A certain amount of PBS solution was added to each solution according to the required ratio, and the mixture was homogenized. The homogenate was then centrifuged at 3000 rpm for 20 minutes at 4°C. The supernatant was collected, and the mixture was centrifuged again under the same conditions. The supernatant was then placed on ice for testing. The remaining steps were performed according to the ELISA kit instructions. The VIP optical density (OD) value was measured using a microplate reader. The test results for 5-HT, DA, and NA in mouse serum are shown in Table 20.

[0201] Table 20

[0202]

[0203] Note: Compared to S, ** indicates P < 0.01; compared to M, ## This indicates that P < 0.01; compared to X, && This indicates that P < 0.01

[0204] As shown in Table 20, compared with group S, the levels of 5-HT, DA, and NE in the hippocampus of mice in group M were significantly decreased (P<0.01); compared with group M, the levels of 5-HT, DA, and NE in the hippocampus of mice in groups X and E were significantly increased (P<0.01), while there was no statistically significant difference between groups X and E (P>0.05). Compared with group X, the levels of 5-HT, DA, and NE in the hippocampus of mice in group Z were significantly decreased (P<0.01).

[0205] The results of the CRH, ACTH, and CORT levels in mouse serum are shown in Table 21:

[0206] Table 21

[0207]

[0208] Note: Compared to S, ** indicates P < 0.01; compared to M, ## This indicates that P < 0.01; compared to X, && This indicates that P < 0.01

[0209] As shown in Table 21, compared with group S, the serum levels of CRH, ACTH, and CORT in group M mice increased (P<0.01); compared with group M, the serum levels of CRH, ACTH, and CORT in groups E and X mice decreased (P<0.01); however, there was no significant difference between groups E and X (P>0.05); compared with group X, the serum levels of CRH, ACTH, and CORT in group Z mice increased (P<0.01).

[0210] The experimental results above indicate that *Callicarpa japonica* can improve depressive behavior in PMD model mice by improving the morphology and number of hippocampal neurons and Nissl bodies. Furthermore, *Callicarpa japonica* can improve depressive-like behavior by improving the tissue morphology and structure of the hippocampus in PMD model mice, increasing the abundance of Nissl bodies in hippocampal neurons, promoting the ER-BDNF-TrkB pathway, improving monoamine neurotransmitter levels, and correcting HPA axis hyperactivity.

[0211] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of a type of herb called "Rhododendron" in the preparation of a drug for treating perimenopausal depression.

2. The application according to claim 1, characterized in that, The bellflower can be prepared as a solid bellflower preparation, a bellflower herbal liquid, or a bellflower extract.

3. The application according to claim 2, characterized in that, The preparation method of the herbal decoction of *Callicarpa japonica* is as follows: add 10 times the weight of distilled water to *Callicarpa japonica*, decoct for 1 hour and filter. Add 8 times the amount of distilled water to the residue and continue to decoct for 1 hour and filter. Combine the filtrates and concentrate them into a decoction of 1 g / mL based on the amount of raw herb, thus obtaining the herbal decoction of *Callicarpa japonica*.

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