Traditional Chinese medicine composition for dispelling melancholy and clearing collaterals as well as preparation, pharmaceutical composition and application of traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for relieving depression and clearing collaterals and a preparation, a pharmaceutical composition and application thereof. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 6-12 parts of radix bupleuri, 9-15 parts of radix curcumae, 6-12 parts of rhizoma acori graminei, 3-9 parts of fructus gardeniae, 3-9 parts of polygala tenuifolia, 9-15 parts of radix paeoniae rubra and 9-20 parts of cortex albiziae. The traditional Chinese medicine composition provided by the invention can be used for remarkably improving depression-like behaviors of mice with depression, reversing synaptic injury and improving the expression of synaptic associated proteins SYP and PSD-95. The anti-depression mechanism of the traditional Chinese medicine composition is that the traditional Chinese medicine composition has the effects of synergistically inhibiting activation of microglial cells and reducing the level of proinflammatory cytokines such as Tnf alpha, Il1beta and Il6, so that neuroinflammation is remarkably inhibited, and the traditional Chinese medicine composition has the advantages of novel formula, definite mechanism and definite curative effect for treating depression, particularly patients with anxiety and dysphoria symptoms.

Description

Technical Field

[0001] This application belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for relieving depression and clearing collaterals, its preparations, pharmaceutical compositions and applications. Background Art

[0002] Depression is a common mental disorder with persistent low mood and reduced interest as its core symptoms, often accompanied by anxiety, cognitive impairment and sleep problems. Modern medicine mainly uses antidepressants such as selective serotonin reuptake inhibitors (SSRIs), but there are problems such as slow onset, many side effects and insufficient response rate.

[0003] In traditional Chinese medicine theory, depression mostly belongs to the category of "depressive syndrome". Traditional treatments mostly start from the liver and take soothing the liver and regulating qi as the main principle. Representative prescriptions include Chaihu Shugan San, Xiaoyao San, etc. These prescriptions have significant effects on the syndrome of liver qi stagnation in the initial stage and pure excess without deficiency. However, clinical observations have found that in the early and middle stages of depression, the pathogenesis is often not simply qi stagnation, but often evolves into a complex state of "liver qi stagnation, phlegm turbidity obstructing the interior, and transformation into heat due to depression". The qi mechanism is not smooth, the distribution of body fluids is abnormal, and phlegm accumulates; the combination of phlegm and qi obstructs the flow, and over time, it turns into heat, disturbing the mind, forming a pathological situation of the interweaving of "phlegm, qi, and fire". If the simple prescription of soothing the liver and regulating qi is still used, it is difficult to resolve phlegm turbidity and clear away depressive heat, and the curative effect is limited. For example, in the patent with publication number CN106421668A, a traditional Chinese medicine prescription for treating depression is mentioned. This prescription consists of a liver-soothing and qi-regulating composition (Bupleurum chinense, Red Paeony Root, White Peony Root, Perilla Leaf, Tangerine Peel, Immature Bitter Orange, Aucklandia lappa, Cyperus rotundus, Curcuma aromatica, Albizia julibrissin), a healthy qi-tonifying composition (Prepared Rehmannia Root, Rhizoma Chuanxiong, Angelica sinensis, Atractylodes macrocephala, Poria cocos, Astragalus membranaceus, Codonopsis pilosula, Cornus officinalis, Morus alba, Lycium barbarum, Polygonatum odoratum, Glehnia littoralis, Polygonatum sibiricum, Eucommia ulmoides, Morinda officinalis, Amomum villosum, Alpinia katsumadai), and a heart-soothing and mind-calming composition (Oyster, Dragon Bone, Concha Margaritifera Usta, Magnetitum, Semen Ziziphi Spinosae, Lotus Seed, Polygala tenuifolia, Alpinia oxyphylla, Fructus Tritici Levis, Gardenia jasminoides, Salvia miltiorrhiza, Persica vulgaris, Carthamus tinctorius, Acorus tatarinowii, Pinellia ternata, Licorice Root, Chinese Date), although it shows the curative effect of treating depression through specific cases, the composition of this prescription is complex and its mechanism of action is unclear.

[0004] Research in modern psychiatry has shown that chronic stress can activate microglia in the central nervous system and release Tnf α , Il1β and <U+ Il6 and other pro-inflammatory cytokines, triggering "neuroinflammation", which in turn leads to decreased synaptic plasticity and neuronal damage, providing a modern biological interpretation for the pathogenesis of "phlegm-heat stasis-toxin" blocking the brain collaterals in traditional Chinese medicine. At present, there is still a blank for traditional Chinese medicine compounds that can follow the essence of syndrome differentiation and treatment in traditional Chinese medicine and precisely intervene in the neuroinflammatory pathway. Summary of the Invention

[0005] To address the aforementioned issues, this application, based on traditional methods of soothing the liver and relieving depression, incorporates the principles of "resolving phlegm and unblocking the meridians, clearing the heart and calming the mind," providing a traditional Chinese medicine composition that simultaneously regulates Qi, clears phlegm and heat, inhibits neuroinflammation, and repairs synaptic damage. This composition aims to meet the clinical needs for treating depression, particularly those accompanied by anxiety and irritability. The specific technical solution is as follows: In the first aspect, this application proposes a traditional Chinese medicine composition for relieving depression and clearing the meridians, which is composed of the following raw materials in parts by weight: Bupleurum chinense 6-12 parts, Curcuma longa 9-15 parts, Acorus tatarinowii 6-12 parts, Gardenia jasminoides 3-9 parts, Polygala tenuifolia 3-9 parts, Paeonia lactiflora 9-15 parts, and Albizia julibrissin bark 9-20 parts.

[0006] Furthermore, the traditional Chinese medicine composition consists of the following raw materials in parts by weight: Bupleurum chinense 9 parts, Curcuma longa 12 parts, Acorus tatarinowii 9 parts, Gardenia jasminoides 6 parts, Polygala tenuifolia 6 parts, Paeonia lactiflora 12 parts, and Albizia julibrissin 15 parts.

[0007] Secondly, this application proposes a traditional Chinese medicine preparation containing the aforementioned traditional Chinese medicine composition.

[0008] Furthermore, the formulation types of the traditional Chinese medicine preparations include tablets, capsules, granules, pills, decoctions, or powders.

[0009] Thirdly, this application proposes a pharmaceutical composition in which at least one of the traditional Chinese medicine compositions is used as an active pharmaceutical ingredient.

[0010] Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable excipients, including one or more of fillers, binders, disintegrants, lubricants, and solubilizers.

[0011] Fourthly, this application proposes the use of the described traditional Chinese medicine composition, or the described traditional Chinese medicine preparation, or the described pharmaceutical composition in the preparation of medicaments for treating and / or preventing depression.

[0012] Fifthly, this application proposes the use of the aforementioned traditional Chinese medicine composition, or the aforementioned traditional Chinese medicine preparation, or the aforementioned pharmaceutical composition in the preparation of a drug that inhibits microglial cell activation.

[0013] Sixthly, this application proposes the use of the aforementioned traditional Chinese medicine composition, or the aforementioned traditional Chinese medicine preparation, or the aforementioned pharmaceutical composition in the preparation of a drug that reduces the expression of pro-inflammatory factors, wherein the pro-inflammatory factors include Tnfα , Il1β , Il6。

[0014] Seventhly, this application proposes the use of the aforementioned traditional Chinese medicine composition, or the aforementioned traditional Chinese medicine preparation, or the aforementioned pharmaceutical composition in the preparation of a drug that protects the structure and function of hippocampal synapses, wherein the drug can enhance the expression levels of SYP and PSD-95 proteins.

[0015] Compared with the prior art, this application has the following advantages: The traditional Chinese medicine composition for relieving depression and clearing the meridians proposed in this application can significantly improve depression-like behaviors in mice with chronic mild unpredictable stress (CUMS)-induced depression. Specifically, it increases sucrose preference rate (improving anhedonia), shortens novelty inhibition feeding latency (improving anxiety-like behaviors), and shortens immobility time in tail suspension and forced swimming tests (improving behavioral despair). Simultaneously, it significantly increases the expression of hippocampal synaptic proteins SYP and PSD-95, and increases dendritic spine density and dendritic branch number, effectively reversing stress-induced synaptic damage and protecting synaptic structure and function. It also synergistically inhibits microglia activation and reduces hippocampal depression. Tnfα , Il1β and Il6 The effects of pro-inflammatory cytokines significantly inhibit neuroinflammation, providing a modern scientific basis for the traditional Chinese medicine treatment principle of "resolving phlegm, clearing the meridians, calming the mind, and soothing the nerves."

[0016] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The behavioral test results of mice in each experimental group in this application embodiment are shown; where A is the result of the sucrose preference test, showing the sucrose preference rate of mice in each group; B is the result of the novelty inhibition feeding test, showing the feeding latency of mice in each group; C and D are the results of the tail suspension test and forced swimming test, respectively, showing the immobility time of mice in each experimental group; all data are expressed as mean ± standard deviation (n=10). Compared with the normal group, ### P <0.001; compared with the model group, P <0.01, P <0.001; Figure 2The results of the sucrose preference experiment (A) and forced swimming experiment (B) of each group of mice in the comparative experiment of this application embodiment are shown; all data are expressed as mean ± standard deviation (n=10); compared with the model group, ### P <0.001; compared with the whole formula group, P <0.05, P <0.01; Figure 3 The results of the effects of each experimental group on the expression of synapse-related proteins in mice are shown in the embodiments of this application; where A is the representative band of SYP and PSD-95 synapse-related proteins in hippocampal tissue detected by Western Blot; B is the semi-quantitative statistical analysis results of SYP and PSD-95 protein expression; all data are expressed as mean ± standard deviation (n=3); compared with the normal group, ### P <0.001; compared with the model group, P <0.01, P <0.001; Figure 4 The following are the results of the detection of hippocampal pro-inflammatory factor mRNA expression levels in mice of each experimental group using real-time quantitative PCR in the embodiments of this application; wherein, A, B, and C are respectively Tnfα , Il1β and Il6 Relative expression levels of mRNA; all data are expressed as mean ± standard deviation (n=6); compared with the normal group, ### P <0.001; compared with the model group, P <0.01, P <0.001; Figure 5The results of immunofluorescence staining and quantitative analysis of mice in different experimental groups are shown. A represents a representative image of microglia (Iba-1, red) in the CA1 region of the hippocampus, displayed by double immunofluorescence staining, with cell nuclei labeled by DAPI (blue); scale bar: 200 μm, 50 μm; B shows the quantitative analysis results of the number of Iba-1 positive cells and their fluorescence intensity per unit area; C shows representative three-dimensional reconstructed images of microglia in each experimental group; D shows the results of quantitative analysis of microglia processes in the CA1 region of the hippocampus in each experimental group using Sholl analysis; E shows the total process length of Iba-1 positive microglia in each experimental group; F shows the total number of process branches per Iba-1 cell in each experimental group; G shows the size of Iba-1 cells in each experimental group. All data are expressed as mean ± standard deviation (n=6). Compared with the normal group, ## P <0.01, ### P <0.001; compared with the model group, P <0.05, P <0.01; Figure 6 The following diagram illustrates the Golgi staining and quantitative analysis results of hippocampal tissue from mice in each experimental group in this application embodiment; where A is a representative image of hippocampal neuron dendritic branches displayed by Golgi staining (scale bar: 50 µM); B is the result of quantitative statistical analysis of dendritic branch complexity by Sholl analysis; C is a representative image of hippocampal neuron dendritic spines displayed by Golgi staining (scale bar: 5 µM); D is the result of quantitative statistical analysis of dendritic spine density; all data are expressed as mean ± standard deviation (n=3); compared with normal, ### P <0.001; compared with the model group, P <0.05, P <0.01, P <0.001; Figure 7 The accompanying diagram illustrates the Golgi staining and quantitative analysis results of hippocampal tissue from mice in each group during the comparative experiment of this application. A represents a representative image of hippocampal neuron dendritic branches displayed by Golgi staining (scale bar: 50 µM); B represents the quantitative statistical results of dendritic branch complexity obtained through Sholl analysis; C represents a representative image of hippocampal neuron dendritic spines displayed by Golgi staining (scale bar: 5 µM); and D represents the quantitative statistical analysis results of dendritic spine density. All data are expressed as mean ± standard deviation (n=3). Compared with the model group, ###P <0.001; compared with the whole formula group, P <0.05, P <0.01. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] For the early and middle stages of depression, with liver qi stagnation, phlegm obstruction, and initial signs of stagnant heat as the core pathogenesis, and based on the guiding principle of "soothing the liver and relieving depression, resolving phlegm and unblocking the meridians, and clearing the heart and calming the mind," this embodiment proposes a Chinese herbal composition consisting of seven herbs: Bupleurum, Curcuma longa, Acorus tatarinowii, Gardenia jasminoides, Polygala tenuifolia, Paeonia lactiflora, and Albizia julibrissin. This treatment method is highly consistent with the pathological mechanisms of neuroendocrine-immune network disorder, neuroinflammation, and impaired synaptic plasticity under the background of chronic stress in depression.

[0021] The proposed herbal composition for relieving depression and clearing the meridians uses Bupleurum as the chief ingredient to soothe the liver and address the root cause; Curcuma and Acorus tatarinowii as assistant ingredients to treat qi, blood, and phlegm stagnation; Gardenia jasminoides to clear heat, Polygala tenuifolia to resolve phlegm, and Paeonia lactiflora to cool the blood and unblock the meridians; and Albizia julibrissin as the guiding ingredient to calm the mind and soothe the spirit. The entire formula integrates "soothing, resolving, clearing, unblocking, and calming," promoting the smooth flow of qi, resolving phlegm, clearing stagnant heat, and harmonizing the meridians, thus bringing peace of mind. This precisely addresses the core pathogenesis of depression, namely liver stagnation and phlegm obstruction, and stagnant heat disturbing the mind. Specifically, it consists of the following raw materials in parts by weight: Astragalus membranaceus 15-30 parts, Ginseng 3-9 parts, Atractylodes macrocephala (fried) 9-15 parts, Angelica sinensis 9-15 parts, Poria cocos 12-20 parts, Longan aril 6-12 parts, Curcuma longa 6-12 parts, and Glycyrrhiza uralensis (prepared) 3-9 parts.

[0022] Principal drug: Astragalus: Bitter and pungent in taste, slightly cold in nature, and enters the liver and gallbladder meridians. It excels at soothing the liver and relieving stagnation, regulating liver qi, and eliminating stagnation at its root, making it an essential medicine for treating liver qi stagnation.

[0023] Minister's Medicine: Turmeric: It has a pungent and bitter taste, is cold in nature, and enters the liver, heart, and lung meridians. It can both regulate qi and relieve depression, assisting Bupleurum in relieving liver stagnation, and also invigorate blood, cool the blood, clear the heart and open the orifices, targeting the mechanism of prolonged stagnation transforming into heat and internal generation of blood stasis.

[0024] Acorus tatarinowii: pungent and bitter in taste, warm in nature, and enters the heart and stomach meridians. It excels at clearing phlegm and opening the orifices, resolving dampness and harmonizing the middle jiao, calming the mind and soothing the nerves, and effectively removing pathogenic factors that obscure the mind with phlegm. Together with the principal herb, they serve as assistant herbs, assisting the principal herb in enhancing its effects of relieving depression, resolving phlegm, and opening the orifices.

[0025] Adjuvant: Gardenia: Bitter in taste, cold in nature, and enters the heart, lung, and triple burner meridians. It clears and drains stagnant fire in the triple burner, guides heat downward, and allows the heat evil to be expelled through urination, targeting stagnant heat disturbing the mind.

[0026] Polygala tenuifolia: Bitter and pungent in taste, warm in nature, and enters the heart, kidney, and lung meridians. It eliminates phlegm, opens the orifices, and harmonizes the heart and kidneys. When used in combination with Acorus tatarinowii, it enhances the effects of resolving phlegm and calming the mind.

[0027] Red peony root: bitter in taste, slightly cold in nature, and enters the liver meridian. It clears heat and cools the blood, invigorates blood circulation and removes blood stasis, unblocks the meridians, and improves the state of "microscopic blood stasis and toxins".

[0028] Used medicine: Albizia bark: sweet in taste, neutral in nature, and enters the heart, liver, and lung meridians. It is effective in relieving depression and calming the mind, promoting blood circulation and reducing swelling. Its effect of "bringing joy and freedom from worry" directly targets the main symptoms of depression. At the same time, its blood-activating and anti-inflammatory effects are consistent with the overall principle of "unblocking the meridians" in the formula, and it also harmonizes the effects of other herbs.

[0029] Example 1 In this embodiment, the composition of the traditional Chinese medicine for relieving depression and clearing the meridians is as follows: Bupleurum chinense 9g, Curcuma longa 12g, Acorus tatarinowii 9g, Gardenia jasminoides 6g, Polygala tenuifolia 6g, Paeonia lactiflora 12g, and Albizia julibrissin bark 15g.

[0030] The preparation method (decoction) is as follows: Take the above seven medicinal slices and place them in a decoction container. First, add water to cover the herbs by about 3-5 cm and soak for 30 minutes. Bring to a boil over high heat, then simmer over low heat for 30 minutes. Filter the liquid. Second, add water to cover the herbs, bring to a boil over high heat, then simmer over low heat for 20 minutes. Filter the liquid. Combine the two decoctions and concentrate to an appropriate volume (e.g., 300-400 ml) to obtain the decoction (named Jieyu Qingluo Decoction).

[0031] If preparing powders, tablets, capsules, granules, or pills, the combined decoction is extracted using dynamic circulation, low-temperature concentration, and spray drying techniques to produce a dry extract. Pharmaceutical excipients are then added, and the corresponding dosage form is prepared according to conventional pharmaceutical processes.

[0032] Example 2 Pharmacodynamic studies were conducted on the Jieyu Qingluo Decoction prepared in Example 1: 2.1 Test drugs and reagents.

[0033] Jieyu Qingluo Decoction: Prepared in Example 1.

[0034] Fluoxetine: a selective serotonin reuptake inhibitor, purchased from Sigma, prepared with sterile saline.

[0035] Main antibodies: anti-SYP, anti-PSD-95, etc., purchased from CST.

[0036] 2.2 Experimental animals, grouping, and model establishment.

[0037] Healthy SPF-grade male C57BL / 6J mice, weighing 22-25g and aged 6-8 weeks, were provided by Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. [License No.: SCXK (Zhejiang) 2019-0004] and housed at the Experimental Animal Center of the Institute of Neurology, Anhui University of Traditional Chinese Medicine. All mice were acclimatized for one week under standard conditions (12 / 12-hour light / dark cycle, temperature 22±2℃, free access to food and water).

[0038] 2.2.1 Grouping of research on main mechanisms.

[0039] Normal group: C57BL / 6J mice.

[0040] Model group (CUMS): The model was established using a chronic, mild, and unpredictable stress method. CUMS mice were housed individually and randomly subjected to 2-3 different low-intensity stress stimuli daily (such as 24-hour fasting, 24-hour water deprivation, overnight light exposure, tilted cages, moist bedding, swimming in 4°C cold water, and physical restraint), for 6 weeks. Specific stress methods are shown in Table 1.

[0041] Table 1 CUMS Model Preparation

[0042] Jieyu Qingluo Decoction Group: Starting from the 3rd week of modeling, Jieyu Qingluo Decoction decoction (equivalent raw drug dose) was administered by gavage daily.

[0043] Fluoxetine group: positive control group, starting from the 3rd week of modeling, was given fluoxetine solution (10mg / kg) by gavage daily.

[0044] 2.2.2 Comparative experimental grouping.

[0045] Another group of CUMS model mice was randomly divided into 4 control groups (n=10): The complete formula group (Jieyu Qingluo Decoction Group): The formula is the same as that of Jieyu Qingluo Decoction Group (Bupleurum 9g, Curcuma 12g, Acorus tatarinowii 9g, Gardenia 6g, Polygala tenuifolia 6g, Paeonia lactiflora 12g, Albizia julibrissin 15g).

[0046] The formula for soothing the liver, relieving depression, and calming the mind consists of: Bupleurum chinense 9g, Curcuma longa 12g, Acorus tatarinowii 9g, Gardenia jasminoides 6g, Polygala tenuifolia 6g, Albizia julibrissin bark 15g (excluding Paeonia lactiflora).

[0047] Low-dose Paeonia lactiflora group: Bupleurum chinense 9g, Curcuma longa 12g, Acorus tatarinowii 9g, Gardenia jasminoides 6g, Polygala tenuifolia 6g, Paeonia lactiflora 6g, Albizia julibrissin bark 15g.

[0048] High-dose Paeonia lactiflora group: Bupleurum chinense 9g, Curcuma longa 12g, Acorus tatarinowii 9g, Gardenia jasminoides 6g, Polygala tenuifolia 6g, Paeonia lactiflora 18g, Albizia julibrissin bark 15g.

[0049] Danshen Alternative Group: Bupleurum chinense 9g, Curcuma longa 12g, Acorus tatarinowii 9g, Gardenia jasminoides 6g, Polygala tenuifolia 6g, Danshen 12g, Albizia julibrissin bark 15g.

[0050] The turmeric-led group: turmeric is increased to 15g, bupleurum is reduced to 6g, and the other herbs and dosages remain the same as the whole formula group (i.e., the formula is bupleurum 6g, turmeric 15g, acorus tatarinowii 9g, gardenia 6g, polygala tenuifolia 6g, red peony root 12g, and albizia bark 15g).

[0051] Bupleurum Reduction Group: Bupleurum dosage reduced to 6g, while the other herbs and dosages remain unchanged (i.e., the formula is: Bupleurum 6g, Curcuma 15g, Acorus tatarinowii 9g, Gardenia 6g, Polygala tenuifolia 6g, Paeonia lactiflora 12g, Albizia julibrissin 15g).

[0052] All CUMS mice underwent a 6-week chronic unpredictable mild stress model. Starting from week 3 of modeling, each treatment group was administered the corresponding drug solution (equivalent dose of raw drug) daily by gavage, while the normal group and the model group were administered an equal volume of physiological saline by gavage. The intervention continued until the end of week 6.

[0053] 2.3 Behavioral tests.

[0054] A series of behavioral tests were conducted at the end of week 6 to assess the mice’s depressive-like behavior.

[0055] 2.3.1 Sucrose Preference Test (SPT): Used to assess anhedonia. Mice were trained to adapt to sucrose water before the experiment. During the actual experiment, after a 24-hour period of fasting but not food, each mouse was given one pre-weighed bottle of 1% sucrose water and one bottle of pure water. One hour later, these were removed and the mice were weighed again. Sucrose preference rate = (Sucrose water consumption / (Sucrose water consumption + Pure water consumption)) × 100%.

[0056] The results are as follows Figure 1 As shown in Figure A, it can be seen that compared with the normal group, the sucrose preference rate of the model group mice was significantly reduced ( P <0.01). Compared with the model group, the sucrose preference rate of mice in both the Jieyu Qingluo Decoction group and the fluoxetine group was significantly increased ( P <0.01).

[0057] 2.3.2 Novelty Inhibition Feeding Test (NSF): Used to assess anxiety-like behavior. Mice that have been fasted for 24 hours are placed in the center of an unfamiliar open area with a small amount of food particles placed in the center. The latency period from placement to the start of feeding (feeding latency) is recorded. The total amount of food consumed within 5 minutes after the test is completed is measured.

[0058] The results are as follows Figure 1 As shown in Figure B, the feeding latency in the model group mice was significantly prolonged ( P<0.01). Compared with the model group, the feeding latency of mice in the Jieyu Qingluo Decoction group and the Fluoxetine group was significantly shortened ( P <0.01).

[0059] 2.3.3 Tail Suspension Test (TST) and Forced Swimming Test (FST): Used to assess behavioral despair. In the tail suspension test, the mouse's tail is fixed and it is suspended upside down in a box. The immobility time in the last 4 minutes within a 6-minute period is recorded. In the forced swimming test, the mouse is placed in a transparent cylindrical container (25 cm high, 10 cm in diameter), with a water depth of 10 cm and a water temperature of 23-25℃. The immobility time in the last 4 minutes within a 6-minute period is recorded.

[0060] The results are as follows Figure 1 As shown in C and D, compared with the normal group, the immobility time of mice in the model group was significantly prolonged in both the tail suspension and forced swimming tests. P <0.001). Compared with the model group, the immobility time of mice in both the Jieyu Qingluo Decoction group and the Fluoxetine group was significantly shortened ( P <0.001).

[0061] The above experiments show that Jieyu Qingluo Decoction can effectively improve CUMS-induced anhedonia, anxiety-like behavior, and behavioral despair in mice.

[0062] The results of the sucrose preference test and forced swimming test for mice in each control group are as follows: Figure 2 As shown in Figures A and B, the overall effect of this application in improving anhedonia and behavioral despair was significantly better than that of the Liver-Soothing, Depression-Relieving, and Mind-Calming Formula group, the low-dose Paeonia lactiflora group, the high-dose Paeonia lactiflora group, the Salvia miltiorrhiza substitution group, the Curcuma longa as the principal ingredient group, and the Bupleurum chinense reduced dosage group. P <0.05). Among them, the group without red peony root showed improvement compared with the model group, but its effect was not as good as the group with the whole formula in this application, indicating that simply soothing the liver, relieving depression and resolving phlegm has limitations in improving certain core behavioral symptoms.

[0063] 2.4 Sample collection and molecular biological detection.

[0064] After the behavioral tests were completed, the mice were euthanized, and the hippocampus was quickly dissected for subsequent molecular biological testing. 2.4.1 Western Blot; Total protein was extracted from the hippocampus, and after the concentration was determined, SDS-PAGE electrophoresis, membrane transfer, and blocking were performed. SYP, PSD-95, NLRP3, ASC, Caspase-1, IL-1β, and internal control GAPDH primary antibody were added, and the mixture was incubated overnight at 4°C. The next day, the mixture was incubated with HRP-labeled secondary antibody, and the membrane was developed by ECL chemiluminescence. The gray values ​​of the bands were analyzed using ImageJ software.

[0065] Effects on synapse-related protein expression: Western blot results showed that, compared with the normal group, the expression levels of SYP and PSD-95 proteins in the hippocampus of the model group mice were significantly downregulated. P <0.001, such as Figure 3 (A and B). Intervention with Jieyu Qingluo Decoction can significantly reverse this trend ( P <0.001, such as Figure 3 The effects of the A and B groups were comparable to those of the fluoxetine group.

[0066] 2.4.2 Real-time quantitative PCR (qRT-PCR); Total RNA was extracted from the hippocampus and reverse transcribed into cDNA. Detection was performed using the SYBR Green assay with real-time quantitative PCR. Tnfα , Il1β , Il6 mRNA expression levels. Using GAPDH as an internal control, 2... ΔΔCt The relative expression level is calculated using this method.

[0067] qRT-PCR results are as follows Figure 4 As shown in the AC diagram, compared with the normal group, the model group mice had significantly higher levels of pro-inflammatory factors in their hippocampal tissue. Tnfα , Il1β and Il6 The mRNA expression levels were significantly increased ( P <0.001). The intervention with Jieyu Qingluo Decoction significantly inhibited the overexpression of these pro-inflammatory factors ( P <0.001).

[0068] 2.4.3 Immunofluorescence staining; Frozen sections of mouse brain tissue were prepared. After antigen retrieval, immunofluorescence staining was performed using anti-Iba-1 (a microglia marker), and cell nuclei were counterstained with DAPI. The number of Iba-1 positive cells in the CA1 region of the hippocampus was observed and counted under a confocal microscope, and microglia were reconstructed and quantitatively analyzed using Imaris.

[0069] Immunofluorescence results as follows Figure 5 As shown in the AG, compared with the normal group, the number of Iba1-positive microglia and the expression intensity of Iba1 in the CA1 region of the hippocampus of the model group mice were significantly increased. After intervention with Jieyu Qingluo Decoction, the number of microglia and the expression intensity of Iba1 were significantly decreased (P<0.01, as shown in the AG). Figure 5 (A and B). Morphological analysis of microglia showed that the processes of microglia in the model group mice were significantly shortened, the number of branches was reduced, and the cell body area was significantly increased. After intervention with Jieyu Qingluo Decoction, the activation of microglia was significantly inhibited ( P <0.01, such as Figure 5Medium CG).

[0070] 2.4.4 Golgi staining; Fresh hippocampal tissue was collected and processed according to the instructions of the Golgi staining kit. After imaging the stained neurons, the dendritic spine density and dendritic branching complexity (using Sholl analysis) were reconstructed and quantitatively analyzed using Neurolucida software.

[0071] Golgi staining results as follows Figure 6 As shown in the AD diagram, compared with the normal group, the model group mice showed a significant decrease in dendritic spine density and dendritic branch complexity in hippocampal neurons (Sholl analysis showed a reduction in the number of crossover points). P <0.001). The dendritic spine density and dendritic branching complexity of mice in the Jieyu Qingluo Decoction group were significantly improved. P <0.001). (Combined) Figure 3 It can be seen that Jieyu Qingluo Decoction can effectively protect the structure and function of hippocampal synapses and reverse synaptic damage caused by CUMS.

[0072] The results of Golgi staining in the comparative experiment are as follows: Figure 7 As shown in AD, the entire formula of this application also shows a significant advantage in terms of dendritic spine density and dendritic branch complexity. P <0.05).

[0073] The above experiments demonstrate that the Jieyu Qingluo Decoction proposed in this application can significantly improve depressive-like behavior in CUMS mice. Its mechanism of action involves inhibiting microglial cell activation, thereby reducing downstream pro-inflammatory factors (such as...). Tnfα , Il1β and Il6 The release of SYP / PSD-95 ultimately protects the structure and function of hippocampal synapses (increasing SYP / PSD-95 expression and dendritic spine density and complexity), thereby exerting an antidepressant effect. The efficacy of this formula is comparable to that of the positive control drug fluoxetine, and its scientific connotation of "soothing the liver and relieving depression, resolving phlegm and unblocking the meridians, and calming the mind and soothing the nerves" has been verified from the perspective of modern pharmacology.

[0074] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A traditional Chinese medicine composition for relieving depression and clearing the meridians, characterized in that, It is composed of the following raw materials in parts by weight: Bupleurum chinense 6-12 parts, Curcuma longa 9-15 parts, Acorus tatarinowii 6-12 parts, Gardenia jasminoides 3-9 parts, Polygala tenuifolia 3-9 parts, Paeonia lactiflora 9-15 parts, and Albizia julibrissin bark 9-20 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, It is composed of the following raw materials in parts by weight: Bupleurum chinense 9 parts, Curcuma longa 12 parts, Acorus tatarinowii 9 parts, Gardenia jasminoides 6 parts, Polygala tenuifolia 6 parts, Paeonia lactiflora 12 parts, and Albizia julibrissin 15 parts.

3. A traditional Chinese medicine preparation, characterized in that, The traditional Chinese medicine composition contains the one described in claim 1 or 2.

4. The traditional Chinese medicine preparation according to claim 3, characterized in that, The types of traditional Chinese medicine preparations include tablets, capsules, granules, pills, decoctions, or powders.

5. A pharmaceutical composition, characterized in that, At least one of the active pharmaceutical ingredients is the traditional Chinese medicine composition as described in claim 1 or 2.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition further contains pharmaceutically acceptable excipients, including one or more of fillers, binders, disintegrants, lubricants, and solubilizers.

7. The use of the traditional Chinese medicine composition of claim 1 or 2, or the traditional Chinese medicine preparation of claim 3 or 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of a medicament for treating and / or preventing depression.

8. The use of the traditional Chinese medicine composition of claim 1 or 2, or the traditional Chinese medicine preparation of claim 3 or 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of a drug for inhibiting microglial cell activation.

9. The use of the traditional Chinese medicine composition of claim 1 or 2, or the traditional Chinese medicine preparation of claim 3 or 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of a drug that reduces the expression of pro-inflammatory factors, wherein the pro-inflammatory factors include Tnfα , Il1β and Il6。 10. The use of the traditional Chinese medicine composition of claim 1 or 2, or the traditional Chinese medicine preparation of claim 3 or 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of a drug that protects the structure and function of hippocampal synapses, wherein the drug can enhance the expression levels of SYP and PSD-95 proteins.

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