Traditional Chinese medicine composition for preventing and / or treating depression accompanied by insomnia and hormone level reduction, preparation method and application

Abstract

The invention discloses a traditional Chinese medicine composition for preventing and / or treating depression accompanied with insomnia and hormone level reduction, a preparation method and application. The traditional Chinese medicine composition for preventing and / or treating depression accompanied by insomnia and hormone level reduction comprises the following components in parts by weight: 15-24 parts of radix pseudostellariae, 10-16 parts of radix puerariae, 5-8 parts of herba epimedii and 8-12 parts of traditional Chinese medicine A. The traditional Chinese medicine A comprises one or more of Chinese dates, spina date seeds, polygala tenuifolia, poria cocos, cistanche and ginseng. According to the traditional Chinese medicine composition provided by the invention, all the components are mutually matched, the compatibility is reasonable, multi-link, multi-target and multi-path regulation and control can be realized on the basis of overall regulation and treatment based on syndrome differentiation, and the traditional Chinese medicine composition has the characteristics of treating both symptoms and root causes and obvious curative effect, and can safely and effectively relieve depression and accompanying sleep disorder and hormone level reduction thereof.

Description

Technical Field

[0001] This application belongs to the field of traditional Chinese medicine technology, and relates to a traditional Chinese medicine composition, preparation method and application for the prevention and / or treatment of depression accompanied by insomnia and low hormone levels. Background Technology

[0002] Depression profoundly erodes an individual's quality of life, with insomnia not only being a symptom but also an independent risk factor for various mental disorders, even constituting a periodic and progressive disease state. Approximately 80% of patients with depression suffer from severe sleep problems, and clinical observations further reveal that sleep disturbances often precede depressive moods and are closely associated with an increased risk of developing depression. Although the underlying mechanisms of insomnia and depression comorbidity are not fully understood, their clinical manifestations and risk factors highly overlap, suggesting that they are both closely intertwined and possess unique characteristics. Studies indicate that insomnia may contribute to depression by interfering with synaptic plasticity-related signaling pathways; simultaneously, hippocampal dysfunction is also considered an important pathway by which insomnia triggers depression and cognitive deficits. Circadian rhythm disruption and accompanying melatonin abnormalities constitute a significant feature of depression. In this complex regulatory network, the serotonin (5-HT) system plays a central role in regulating mood and the sleep-wake cycle; its dysfunction is closely related to both depression and insomnia. GABA, as the brain's main inhibitory neurotransmitter, directly leads to difficulty maintaining sleep and anxiety-like behaviors when its activity is insufficient. An imbalance in the excitability of the glutamate (GLU) system may exacerbate mood disorders and disrupt sleep structure through neurotoxicity and synaptic dysfunction. Orexin secreted by the hypothalamus participates in the regulation of the sleep-wake cycle, making its receptor antagonists a research focus in the treatment of insomnia. Agonists of melatonin 1a and melatonin 1b receptors, such as agomelatine, offer new insights into the treatment of depression accompanied by insomnia. Currently, the pathological mechanisms of depression accompanied by insomnia are not fully understood, and commonly used Western medicine regimens, such as venlafaxine combined with diazepam, not only easily induce patient resistance but also often have unsatisfactory efficacy. Current medications often require multiple drugs to address depression accompanied by decreased hormone levels and disrupted sleep structure, and issues such as side effects and drug tolerance remain unresolved. Against this backdrop, Traditional Chinese Medicine (TCM) demonstrates unique advantages. TCM emphasizes the harmony between humanity and nature and the balance of Yin and Yang, incorporating insomnia and depression into categories such as "depression syndrome" and "insomnia," addressing the issue holistically rather than solely targeting symptoms. TCM compound formulas, through the synergistic effects of multiple components and targets, can calm the mind, soothe the liver, and relieve depression, while also harmonizing the body's vital energy and balancing circadian rhythms. This improves sleep quality while deeply addressing the root causes of emotional distress, emphasizing the harmonious interaction between the heart and kidneys and the overall homeostasis of liver Qi. Therefore, it exhibits sustained and gentle efficacy in alleviating the comorbid state of depression and insomnia, minimizing drug dependence and adverse reactions. Summary of the Invention

[0003] Therefore, it is necessary to provide a traditional Chinese medicine composition, preparation method, and application for the prevention and / or treatment of depression accompanied by insomnia and decreased hormone levels.

[0004] In some embodiments, a traditional Chinese medicine composition is provided for the prevention and / or treatment of depression accompanied by insomnia and low hormone levels, comprising the following components in parts by weight: 15-24 parts of Codonopsis pilosula, 10-16 parts of Pueraria lobata, 5-8 parts of Epimedium brevicornu, and 8-12 parts of herb A.

[0005] The Chinese herbal medicine A includes one or more of the following: jujube, sour jujube seed, polygala root, poria cocos, cistanche deserticola, and ginseng.

[0006] In some embodiments, the provided traditional Chinese medicine composition includes the following components in parts by weight: 16 to 20 parts of Codonopsis pilosula, 12 to 16 parts of Pueraria lobata, 6 to 8 parts of Epimedium brevicornu, and 10 to 12 parts of traditional Chinese medicine A.

[0007] In some embodiments, the provided traditional Chinese medicine composition comprises the following components by weight percentage: 31%~55% Codonopsis pilosula, 20%~39% Pueraria lobata, 9%~21% Epimedium brevicornu, and 15%~25% Traditional Chinese Medicine A.

[0008] In some embodiments, a method for preparing the traditional Chinese medicine composition is provided, comprising the steps of separately preparing extract A1 and extract A2, and the step of mixing extract A1 and extract A2; wherein,

[0009] The steps for preparing extract A1 include: mixing the Codonopsis pilosula, Chinese herbal medicine A with water, performing a first extraction, collecting the aqueous extract, concentrating the aqueous extract to prepare a concentrated aqueous extract, precipitating the concentrated aqueous extract with ethanol, collecting the precipitate, and preparing total polysaccharides of Codonopsis pilosula and Chinese herbal medicine A.

[0010] The steps for preparing extract A2 include: mixing the kudzu root and epimedium with an ethanol aqueous solution for a second extraction, collecting the ethanol extract, concentrating the ethanol extract for a second time to prepare a concentrated ethanol extract, allowing the concentrated ethanol extract to stand, collecting the supernatant, and separating and preparing the total flavonoids of kudzu root and epimedium from the supernatant.

[0011] In some embodiments, in the step of preparing extract A1, the ratio of the total mass of Codonopsis pilosula and traditional Chinese medicine A to the mass of water in the provided method for preparing traditional Chinese medicine composition is 1:(8~12).

[0012] In some embodiments, in the step of preparing extract A1 of the provided method for preparing traditional Chinese medicine composition, the temperature of the first extraction is 85℃~95℃ and the extraction time is 3h~9h.

[0013] In some embodiments, in the step of preparing extract A2 of the provided method for preparing traditional Chinese medicine composition, the ratio of the total mass of kudzu root and epimedium to the mass of the ethanol aqueous solution is 1:(10~15).

[0014] In some embodiments, in the step of preparing extract A2 in the provided method for preparing traditional Chinese medicine composition, the temperature of the second extraction is 70℃~90℃ and the extraction time is 1h~3h.

[0015] In some embodiments, the provided method for preparing the traditional Chinese medicine composition satisfies one or more of the following conditions:

[0016] (1) In the step of preparing extract A1, the temperature of the first concentration is 65℃~85℃;

[0017] (2) In the step of preparing extract A1, in the step of precipitating the concentrated water extract with ethanol solution, the volume percentage of ethanol in the total mixture system is 75%~85%;

[0018] (3) In the step of preparing extract A2, the volume percentage of ethanol in the ethanol aqueous solution is 75%~85%;

[0019] (4) In the step of preparing extract A2, the temperature of the second concentration is 60℃~80℃;

[0020] (5) In the step of preparing extract A2, the concentrated alcohol extract is allowed to stand at a temperature of 10°C or lower for at least 40 hours; alternatively, the concentrated alcohol extract is allowed to stand at a temperature of 4°C to 10°C for 40 to 50 hours.

[0021] (6) In the step of preparing extract A2, the total flavonoids of Epimedium puerarin in the supernatant are obtained by separation using macroporous adsorption resin.

[0022] In some embodiments, the traditional Chinese medicine composition described herein or the traditional Chinese medicine composition prepared by the described preparation method is provided for use in the preparation of products for the prevention and / or treatment of depression accompanied by insomnia and decreased hormone levels.

[0023] The traditional Chinese medicine composition provided in this application has components that work synergistically and are rationally combined. The calming effect of Codonopsis pilosula combined with the blood-nourishing and calming effect of traditional Chinese medicine A can more effectively alleviate sleep disorders in patients with depression. The effects of Pueraria lobata in promoting cerebral blood flow and improving microcirculation complement the effects of Epimedium in improving endocrine system function, helping to improve the overall physiological state of patients with depression. Based on holistic regulation and syndrome differentiation and treatment, it can achieve multi-link, multi-target, and multi-pathway regulation, with the characteristics of treating both the symptoms and the root cause, and significant efficacy. It can safely and effectively alleviate depression and its accompanying sleep disorders and decreased hormone levels. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0025] Figure 1 H&E pathological sections of mouse hippocampus (n=6), scale bar 100 µm;

[0026] Figure 2 Nissl stained sections of mouse hippocampus (n=6), scale bar 100 µm;

[0027] Figure 3 The expression of melatonin receptor MTNR1A in brain tissue (n=4), scale bar 100 µm;

[0028] Figure 4 The expression of melatonin receptor MTNR1B in brain tissue (n=4), scale bar 100 µm. Detailed Implementation

[0029] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0030] It should be noted that when a component is said to be "fixed to" another component, it can be directly attached to the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0033] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0034] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0035] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0036] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0037] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0038] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0039] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0040] In this invention, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0041] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0042] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0043] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0044] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0045] In this application, "room temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0046] In some embodiments, a traditional Chinese medicine composition for preventing and / or treating depression accompanied by insomnia and low hormone levels is provided, comprising the following components in parts by weight: 15-24 parts of Codonopsis pilosula, 10-16 parts of Pueraria lobata, 5-8 parts of Epimedium brevicornu, and 8-12 parts of traditional Chinese medicine A.

[0047] The Chinese herbal medicine A includes one or more of the following: jujube, sour jujube seed, polygala root, poria cocos, cistanche deserticola, and ginseng.

[0048] The provided traditional Chinese medicine composition is based on holistic regulation and syndrome differentiation and treatment, which can achieve multi-link, multi-target and multi-pathway regulation. It has the characteristics of treating both the symptoms and the root cause and has obvious curative effect. It can safely and effectively relieve depression and its accompanying sleep disorders and decreased hormone levels.

[0049] Pseudostellaria heterophylla (Mig.) Paxex Pax et Hoffm is the dried tuberous root of a plant in the Caryophyllaceae family. It has the effects of replenishing qi and promoting body fluid production, strengthening the spleen and benefiting the lungs.

[0050] Jujube, also known as red date, comes from the dried, ripe fruit of the jujube plant (Ziziphus jujuba Mill.) in the Rhamnaceae family. It has the effects of nourishing blood and calming the mind, strengthening the spleen and stomach, and enhancing immunity.

[0051] Kudzu root is the dried root of a legume (Pueraria lobata (Wild.) Ohwi); kudzu root has the effects of relieving muscle tension and fever, promoting rash eruption, generating fluids and quenching thirst, and raising yang and stopping diarrhea.

[0052] Epimedium is the dried aerial part of a plant in the Berberidaceae family (Epimedium brevicornum Maxim). It has the effects of tonifying the kidneys and strengthening yang, strengthening muscles and bones, and dispelling wind and dampness.

[0053] In some embodiments, the provided traditional Chinese medicine composition includes the following components in parts by weight: 16 to 20 parts of Codonopsis pilosula, 12 to 16 parts of Pueraria lobata, 6 to 8 parts of Epimedium brevicornu, and 10 to 12 parts of traditional Chinese medicine A.

[0054] Understandably, in the provided Chinese herbal composition, the weight parts of Codonopsis pilosula can be 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, etc., or can be any range of the two aforementioned values.

[0055] Understandably, in the provided traditional Chinese medicine composition, the weight parts of kudzu root can be 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, etc., or can be any range of the two aforementioned values.

[0056] Understandably, the weight of Epimedium in the provided traditional Chinese medicine composition can be 6 parts, 7 parts, 8 parts, etc., or it can be any of the two aforementioned values.

[0057] Understandably, in the provided traditional Chinese medicine composition, the weight parts of traditional Chinese medicine A can be 10 parts, 11 parts, 12 parts, etc., or can be any range of the two aforementioned values.

[0058] In some embodiments, the provided traditional Chinese medicine composition comprises the following components by weight percentage: 31%~55% Codonopsis pilosula, 20%~39% Pueraria lobata, 9%~21% Epimedium brevicornu, and 15%~25% Traditional Chinese Medicine A.

[0059] Understandably, the weight percentage of Codonopsis pilosula in the provided traditional Chinese medicine composition can be 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, etc., or it can be a range of any two of the aforementioned values.

[0060] Understandably, the weight percentage of kudzu root in the provided traditional Chinese medicine composition can be 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, etc., or it can be a range of any two of the aforementioned values.

[0061] Understandably, the weight percentage of Epimedium in the provided traditional Chinese medicine composition can be 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, etc., or it can be a range of any two of the aforementioned values.

[0062] Understandably, in the provided traditional Chinese medicine composition, the weight percentage of traditional Chinese medicine A can be 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, etc., or it can be a range of any two of the aforementioned values.

[0063] In some embodiments, the traditional Chinese medicine A is jujube.

[0064] In some embodiments, a method for preparing the traditional Chinese medicine composition is provided, comprising the steps of separately preparing extract A1 and extract A2, and the step of mixing extract A1 and extract A2; wherein,

[0065] The steps for preparing extract A1 include: mixing the Codonopsis pilosula, Chinese herbal medicine A with water, performing a first extraction, collecting the aqueous extract, concentrating the aqueous extract to prepare a concentrated aqueous extract, precipitating the concentrated aqueous extract with ethanol, collecting the precipitate, and preparing total polysaccharides of Codonopsis pilosula and Chinese herbal medicine A.

[0066] The steps for preparing extract A2 include: mixing the kudzu root and epimedium with an ethanol aqueous solution for a second extraction, collecting the ethanol extract, concentrating the ethanol extract for a second time to prepare a concentrated ethanol extract, allowing the concentrated ethanol extract to stand, collecting the supernatant, and separating and preparing the total flavonoids of kudzu root and epimedium from the supernatant.

[0067] In some embodiments, in the step of preparing extract A1, the ratio of the total mass of Codonopsis pilosula and Chinese medicine A to the mass of water is 1:(8~12). For example, the ratio of the total mass of Codonopsis pilosula and Chinese medicine A to the mass of water can be 1:8, 1:9, 1:10, 1:11, 1:12, etc., or it can be any range of the two ratios mentioned above.

[0068] In some embodiments, in the step of preparing extract A1 of the provided method for preparing traditional Chinese medicine composition, the temperature of the first extraction is 85℃~95℃, and the time of the first extraction is 3h~9h. For example, the temperature of the first extraction can be 85℃, 90℃, 95℃, etc., or it can be a range of any two of the aforementioned values; the time of the first extraction can be 3h, 4h, 5h, 6h, 7h, 8h, 9h, etc., or it can be a range of any two of the aforementioned values.

[0069] In some embodiments, in the step of preparing extract A2 of the provided method for preparing traditional Chinese medicine composition, the ratio of the total mass of kudzu root and epimedium to the mass of the ethanol aqueous solution is 1:(10~15). For example, the ratio of the total mass of kudzu root and epimedium to the mass of the ethanol aqueous solution can be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc., or it can be any range of the two ratios mentioned above.

[0070] In some embodiments, in the step of preparing extract A2 in the provided method for preparing traditional Chinese medicine composition, the temperature of the second extraction is 70℃~90℃, and the time of the second extraction is 1h~3h. For example, the temperature of the second extraction can be 70℃, 75℃, 80℃, 85℃, 90℃, etc., or it can be a range of any two of the aforementioned values; the time of the second extraction can be 1h, 2h, 3h, etc., or it can be a range of any two of the aforementioned values.

[0071] In some embodiments, in the step of preparing extract A1 of the provided method for preparing traditional Chinese medicine composition, the temperature of the first concentration is 65°C to 85°C. For example, the temperature of the first concentration can be 65°C, 70°C, 75°C, 80°C, 85°C, etc., or it can be a range of any two of the aforementioned values.

[0072] In some embodiments, in the step of preparing extract A1 of the provided method for preparing traditional Chinese medicine composition, the volume percentage of ethanol in the total mixture system is 75% to 85%, for example, it can be 75%, 80%, 85%, etc., or it can be any combination of the two aforementioned values.

[0073] In some embodiments, in the step of preparing extract A2 of the provided method for preparing traditional Chinese medicine composition, the volume percentage of ethanol in the aqueous ethanol solution is 75% to 85%, for example, it can be 75%, 80%, 85%, etc., or it can be any range of the two values ​​mentioned above.

[0074] In some embodiments, in the step of preparing extract A2 in the provided method for preparing traditional Chinese medicine composition, the temperature of the second concentration is 60°C to 80°C. For example, the temperature of the second concentration can be 60°C, 65°C, 70°C, 75°C, 80°C, etc., or it can be a range of any two of the aforementioned values.

[0075] In some embodiments, in the step of preparing extract A2 in the provided method for preparing traditional Chinese medicine composition, the concentrated alcohol extract is allowed to stand at a temperature of less than or equal to 10°C for more than 40 hours. Optionally, the concentrated alcohol extract is allowed to stand at a temperature of 4°C to 10°C for 40 to 50 hours.

[0076] In some embodiments, in the step of preparing extract A2 of the provided method for preparing traditional Chinese medicine composition, total flavonoids of Pueraria lobata and Epimedium are obtained from the supernatant by separating them with macroporous adsorption resin.

[0077] In some embodiments, the traditional Chinese medicine composition described herein or the traditional Chinese medicine composition prepared by the described preparation method is provided for use in the preparation of products for the prevention and / or treatment of depression accompanied by insomnia and decreased hormone levels.

[0078] The following are specific embodiments. They are intended to provide a more detailed description of this application to help those skilled in the art and researchers better understand it. The technical conditions described do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims.

[0079] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0080] The reagents, instruments, experimental animals, experimental animal feed, and animal laboratory conditions used in the examples are as follows.

[0081] Reagents:

[0082] Sodium pentobarbital, batch number: P3761, Sigma, USA; 4-Chloro-DL-phenylalanine (PCPA), batch number: A12IS208585, Shanghai Yuanye Bio-Technology Co., Ltd.; 4% Paraformaldehyde fixative, batch number: DF0135, Biosharp; Endogenous peroxidase scavenger, batch number: SI25-01, Sevan Innovation Reagent Co., Ltd.; Goat serum, batch number: SL038, Beijing Solarbio Science & Technology Co., Ltd.; Hematoxylin staining, batch number: BL702B, Biosharp; MTNR1A antibody, batch number: 3D1DAA24, Merck Biotechnology Co., Ltd.; MTNR1B antibody, batch number: ab203346, Abcam; Horseradish peroxidase-labeled secondary antibody, batch number: SOO1, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Immunohistochemistry pen, batch number: BC003, Biosharp; DAB chromogenic agent, batch number: ZIL-0917, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Neutral gum, batch number: BL702A, Biosharp. BCA Protein Concentration Assay Kit, WU1004626113; 5-Hydroxytryptamine (5-HT) ELISA Kit, batch numbers: WU06FH667168, WU0766689981, γ-Aminobutyric acid (GABA) Colorimetric Assay Kit, batch number: WU114TPP8459, Glutamic acid (GLU) Colorimetric Assay Kit, batch numbers: WU142J663774, WU13PN840804, all purchased from Wuhan Eclite Biotechnology Co., Ltd.

[0083] Experimental animals:

[0084] ICR mice, male, provided by Beijing Huafukang Bio-Technology Co., Ltd., production license number: NO.110322251100607823, the animals were housed in the Animal Center of Xinjiang Uygur Autonomous Region Institute of Materia Medica, use license number: SYXJ (Xin) 2022-0002, this experiment has been approved by the Animal Ethics Review Committee of Xinjiang Uygur Autonomous Region Institute of Materia Medica, ethical approval number: XJIMM-20250103.

[0085] Instruments and equipment:

[0086] LT1000B Electronic Balance, Changshu Tianliang Instrument Co., Ltd.; BS323S Electronic Balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; Open Field Test Analysis System for Mice and Rats, Shanghai Xinruan Information Technology Co., Ltd.; Animal Ethology Video Analysis System, ZS Type Tail Suspension Hardware, Beijing Zhongshi Dichuang Technology Development Co., Ltd.; JXFSTPRP-CL Automatic Sample Freezing Grinder, Shanghai Jingxin Industrial Development Co., Ltd.; H1650R Refrigerated High-Speed Centrifuge, Shanghai LUXIANGYI Centrifuge Instrument Co., Ltd.; Cytation3 Multifunctional Microplate Reader, BioTek Instruments, Inc.; LEICA RM2245 Microtome, Leica; DM4000 Optical Microscope, Leica; SC082132 Ultra-Pure Water Instrument, Sichuan沃特 Experimental Instrument Equipment.

[0087] Experimental animal feed:

[0088] Mouse feed (SPF level), purchased from Beijing Keao Xieli Feed Co., Ltd. (Production License No.: SCXK(Beijing)2019-0003).

[0089] Animal laboratory conditions:

[0090] Humidity 30 - 50%, temperature control 20 - 26°C. 12h:12h light-dark alternating illumination; keep the cage environment clean and dry; add feed and water to animals daily; keep animals moving freely.

[0091] Example 1

[0092] A traditional Chinese medicine composition for relieving depression and its accompanying sleep disorders and reduced hormone levels, the active ingredients of which are prepared from the following raw materials by weight:

[0093] Radix Pseudostellariae 180g, Pueraria lobata 120g, Epimedium brevicornu 60g, and Chinese date 100g.

[0094] The preparation method of the traditional Chinese medicine composition is as follows:

[0095] (1) Pick out impurities from Radix Pseudostellariae; pick out impurities from Pueraria lobata and cut into slices; pick out impurities from Epimedium brevicornu and cut into sections; crush Chinese date. Weigh Radix Pseudostellariae, Pueraria lobata, Epimedium brevicornu, and Chinese date according to the above weights.

[0096] (2) Take Codonopsis pilosula and jujube, add water at 8 times the total mass of Codonopsis pilosula and jujube to the extraction tank and extract three times at 90°C for 3 hours each time. Filter the extract through a 40-mesh sieve. Combine the Codonopsis pilosula and jujube water extracts after the three extractions and concentrate under reduced pressure at 65°C. Concentrate and set aside. Place the Codonopsis pilosula and jujube concentrate in an alcohol precipitation tank, turn on the stirring device and stir. Add ethanol while stirring to make the alcohol content reach 80%. Stir evenly, let stand, take the precipitate, dissolve it in water, add ethanol again, take the precipitate, add an appropriate amount of ethanol to wash the precipitate, discard the supernatant and take the precipitate to obtain the total polysaccharides of Codonopsis pilosula and jujube.

[0097] (3) Place the sliced ​​kudzu root and epimedium in an extraction tank and reflux at 90℃ for three extractions. Each time, add 12 times the mass of 80% ethanol of the sliced ​​kudzu root and epimedium. Each extraction lasts for 1 hour. Filter the extract through a 40-mesh sieve. Combine the kudzu root and epimedium ethanol extracts after the three extractions and filters. Concentrate the extract at 60℃ and filter for later use. Let the concentrated kudzu root and epimedium extract stand at 10℃ for 40 hours. Take the supernatant and pass it through an AB-8 macroporous adsorption resin column at a flow rate of about 1 BV / h. First, wash away impurities with 4 times the weight of the resin with water at a flow rate of 1.5~3 BV / h. Discard the water. The flavonoids were then desorbed using 80% ethanol at 8 times the weight of the resin. The eluent was collected, and the vacuum degree was controlled at -0.05 to -0.09 MPa and the temperature was controlled at 60 to 80°C. The ethanol eluent was concentrated under reduced pressure to a relative density of about 1.08 to 1.15 (65 to 75°C) to obtain total flavonoids from Epimedium pueraria.

[0098] (4) Place the codonopsis pilosula and jujube polysaccharides into a vacuum drying oven, set the vacuum, and dry at a temperature below 50°C for about 26 hours. Crush the dry extract, pass it through a 60-mesh sieve, and put it into a two-dimensional mixer. After mixing, put it into a pharmaceutical low-density polyethylene bag and store it in a barrel for later use.

[0099] (5) Place the total flavonoids of Pueraria lobata and Epimedium into a vacuum drying oven, set the vacuum, and dry at a temperature below 60°C for about 26 hours. Crush the dry extract, pass it through a 60-mesh sieve, and put it into a two-dimensional mixer. After mixing, put it into a pharmaceutical low-density polyethylene bag and store it in a barrel for later use.

[0100] (6) Total mixing: Take Codonopsis pilosula extract, jujube polysaccharide extract, kudzu root and epimedium total flavonoid extract and appropriate amount of silica (passed through 60 mesh sieve) and place them in a two-dimensional motion mixer to obtain the Chinese medicine composition.

[0101] (7) Preparation: The mixture of Chinese medicines is encapsulated or compressed into tablets.

[0102] Example 2

[0103] A traditional Chinese medicine composition for relieving depression and its associated sleep disorders and decreased hormone levels, wherein the active ingredients are prepared from the following raw materials by weight:

[0104] 150g of Codonopsis pilosula, 160g of Pueraria lobata, 50g of Epimedium brevicornu, and 120g of jujube.

[0105] The preparation method of the traditional Chinese medicine composition is the same as that in Example 1.

[0106] Example 3

[0107] A traditional Chinese medicine composition for relieving depression and its associated sleep disorders and decreased hormone levels, wherein the active ingredients are prepared from the following raw materials by weight:

[0108] 240g of Codonopsis pilosula, 100g of Pueraria lobata, 80g of Epimedium brevicornu, and 80g of jujube.

[0109] The preparation method of the traditional Chinese medicine composition is the same as that in Example 1.

[0110] Example 4

[0111] A traditional Chinese medicine composition for relieving depression and its associated sleep disorders and decreased hormone levels, wherein the active ingredients are prepared from the following raw materials by weight:

[0112] 200g of Codonopsis pilosula, 150g of Pueraria lobata, 60g of Epimedium, 60g of jujube, and 60g of Ziziphus jujuba var. spinosa.

[0113] The preparation method of the Chinese herbal medicine composition is similar to that of Example 1, except that in step (2), Codonopsis pilosula, jujube and jujube seed are taken and water with a total mass of 8 times that of Codonopsis pilosula, jujube and jujube seed is added and extracted in an extraction tank at 90°C.

[0114] Example 5

[0115] A traditional Chinese medicine composition for relieving depression and its associated sleep disorders and decreased hormone levels, wherein the active ingredients are prepared from the following raw materials by weight:

[0116] 200g of Codonopsis pilosula, 150g of Pueraria lobata, 60g of Epimedium brevicornu, 80g of jujube, 10g of Poria cocos, and 10g of Cistanche deserticola.

[0117] The preparation method of the traditional Chinese medicine composition is similar to that of Example 1, except that in step (2), Codonopsis pilosula, jujube, Poria cocos and Cistanche deserticola are taken and water with a total mass of 8 times that of Codonopsis pilosula, jujube, Poria cocos and Cistanche deserticola is added and extracted in an extraction tank at 90°C.

[0118] Comparative Example 1

[0119] A traditional Chinese medicine composition, the effective components of which are prepared from the following raw materials by weight: 180g of Codonopsis pilosula, 120g of Pueraria lobata and 60g of Epimedium.

[0120] The preparation method of the traditional Chinese medicine composition is similar to that in Example 1, except that the above-mentioned raw materials are used for extraction.

[0121] Comparative Example 2

[0122] A traditional Chinese medicine composition, the effective components of which are prepared from the following raw materials by weight: 100g of jujubes.

[0123] The preparation method of the traditional Chinese medicine composition is similar to that in Example 1, except that the above-mentioned raw materials are used for extraction.

[0124] Pharmacological testing

[0125] 1. Drug grouping

[0126] The chemical drug positive drug group (venlafaxine hydrochloride, compound diazepam tablets), the low, medium and high dose groups of the traditional Chinese medicine composition of Example 1, the traditional Chinese medicine control group 1 (comparative example 1), and the traditional Chinese medicine control group 2 (comparative example 2).

[0127] 2. Model Building Methods

[0128] (1) Methods for establishing the depression model (CUMS)

[0129] ① Fasting for 24 hours; ② No water for 24 hours; ③ Ice water bath at 4℃ for 2 minutes; ④ Tilting the cage at 45° for 24 hours; ⑤ Noise for 4 hours; ⑥ Damp bedding for 24 hours; ⑦ Restraint; ⑧ Running for 5 minutes. Two types of stimulation are used each day, and the same type of stimulation cannot be used consecutively.

[0130] (2) Methods for establishing the PCPA (Potentially Specific Ambient Pain) model

[0131] On day 15 of the experiment, mice were injected intraperitoneally with 4-chloro-DL-phenylalanine (PCPA).

[0132] 3. Grouping and administration

[0133] One hundred male ICR mice, weighing 18–20 g, were used in this study. They were housed in a specific pathogen-free (SPF) laboratory animal center at a room temperature of (25±2)℃, relative humidity of (40%–50%), and a light / dark cycle of (12h / 12h). After 7 days of acclimatization, the mice were randomly divided into 10 groups (n=10): control group, CUMS group, PCPA group, CUMS + PCPA group, positive control group (venlafaxine hydrochloride + diazepam, 11 mg / kg + 0.4 mg / kg), low, medium, and high dose groups of the traditional Chinese medicine combination (200, 400, and 800 mg / kg, respectively), traditional Chinese medicine control group 1 (comparative example 1, 400 mg / kg), and traditional Chinese medicine control group 2 (comparative example 2, 400 mg / kg).

[0134] Mice in the CUMS group, CUMS + PCPA group, positive control group, low-, medium-, and high-dose groups of traditional Chinese medicine composition, and control groups 1 and 2 of traditional Chinese medicine were subjected to CUMS stimulation for 7 consecutive days. Ten mice in the control group and ten mice in the PCPA group were normally fed. On day 8, the positive control group, low-, medium-, and high-dose groups of traditional Chinese medicine composition, control groups 1 and 2 of traditional Chinese medicine were given the corresponding drug treatments. The CUMS group and CUMS + PCPA group were given the same volume of distilled water by gavage until the end of the experiment. CUMS stimulation continued throughout the drug administration phase. On day 15, except for the control group and CUMS group, mice in other groups were intraperitoneally injected with PCPA (400 mg / kg) to induce the CUMS model. The control group and CUMS group were intraperitoneally injected with the same volume of olive oil once daily for 2 consecutive days. Mice were treated after the open field test on day 21.

[0135] 4. Indicator Testing

[0136] 4.1 Behavioral testing of mice

[0137] (1) Sucrose Preference Test (SPT)

[0138] Due to the large number of animals, the mice were divided into two groups. SPT (Sugar Preference Test) was conducted from day 15 to 18. On day 15, a 1% sucrose solution was placed on both sides of the mouse cage. On day 16, a 1% sucrose solution and pure water were placed on both sides of the mouse cage, with the water bottle positions rotated periodically. After a 24-hour fast, the mice were individually housed on day 17, with a 1% sucrose solution and water placed on both sides of the cage, and the weight of each level was recorded. The weight of the water bottle was recorded again after 24 hours, and the sugar preference index was calculated. The second group of animals underwent the same procedure from day 16 to 19.

[0139] .

[0140] (2) Mouse tail suspension test (TST)

[0141] Due to the large number of animals, the mice were divided into two batches and tested on days 17 and 18. A mouse tail suspension device was used for the test. The mouse tail was fixed to the suspension bar, so that the mouse was suspended head upside down. The camera lens was adjusted so that it was horizontally aligned with the mouse's body position. After 2 minutes of adaptation, the video recording was started, and the number of times the mouse did not move within 4 minutes was observed.

[0142] (3) Forced swimming test (FST)

[0143] FST was performed on day 19. The mice were placed in a forced swimming tub (12cm in diameter) filled with water at a depth of 30cm and a temperature of 23-25℃. The cumulative time the mice remained still in the water within 5 minutes was recorded.

[0144] (4) Sodium pentobarbital test (PST)

[0145] PST was performed on day 20. Mice were fasted for 12 hours but allowed free access to water. Each mouse was injected intraperitoneally with sodium pentobarbital (45 mg / kg), and the duration of sleep in each group was observed and recorded. The formula for calculating sleep duration is: Sleep duration = Wake time - Sleep onset time.

[0146] (5) Open field test

[0147] The open field test was conducted on day 21. During the test, the laboratory was kept quiet, the room temperature was constant, and the lighting was uniform. Mice were placed in the center of the open field box, and the total distance traveled, the time spent in the central area, and the number of times the mice entered the central area were observed and recorded within 5 minutes. Between the two experiments, mouse feces and urine were removed, and odors were eliminated by spraying with 75% alcohol.

[0148] (6) Pathological examination

[0149] Six brain samples were randomly selected from each group for HE and Nissl staining, sectioning, light microscopy, and pathological examination. Based on histopathological changes, the degree of brain injury was graded using indicators such as disordered and loose neuronal cell arrangement, irregular cell morphology, nuclear pyknosis and deep staining, Nissl body rupture and reduction, neuronal loss, and vacuolar degeneration.

[0150] "—" indicates that the neurons and Nissl bodies have normal structural morphology.

[0151] "+" indicates a small number of neuronal and Nissl body structures showing pathological changes (lesion area <25%).

[0152] "++" indicates pathological changes in some neurons and Nissl bodies (lesion range >25% & <50%).

[0153] "++" indicates pathological changes in most neurons and Nissl bodies (lesion extent >50%).

[0154] (7) Neurotransmitter detection

[0155] The brain was dissected, rinsed with saline, dried with filter paper, weighed, and homogenized in PBS. Homogenization was performed according to the kit instructions, and the levels of 5-HT, GLU, and GABA were measured.

[0156] (8) Immunohistochemical detection

[0157] Immunohistochemical staining was used to detect the protein expression levels of melatonin receptor (MTNR1A) and melatonin receptor (MTNR1B) in brain tissue.

[0158] Four mouse brain tissues fixed with 4% paraformaldehyde were randomly selected from each group, paraffin sections were prepared, and placed in a 60℃ oven for 90 min to ensure tight adhesion. Dewaxing was performed sequentially with xylene I and II for 10 min each. The sections were then dehydrated sequentially by immersion in 100% ethanol I (10 min), 100% ethanol II (10 min), 95% ethanol (5 min), 80% ethanol (5 min), and 70% ethanol (5 min), followed by rinsing three times with PBS buffer for 5 min each time. The sections were then microwaved on high with antigen retrieval solution and boiled for 15 min, cooled to room temperature, and rinsed three times for antigen retrieval. The sections were then dehydrated by spin drying, and an immunohistochemical pen was used to draw circles around the tissue. Endogenous peroxidase was added, and the sections were incubated at room temperature for 20 min. The sections were then rinsed three times with PBS buffer for 5 min each time. The tissue edges were dried with absorbent paper, and 5% goat serum was added. The sections were then blocked at 37℃ for 30 min. After drying, blot the tissue edges with absorbent paper, add primary antibody (MTNR1A diluted 1:100, MTNR1B diluted 1:200), and incubate overnight at 4°C. The next day, remove and allow to warm to room temperature, then rinse three times with PBS buffer for 5 minutes each time. After drying, blot the tissue edges with absorbent paper, add secondary antibody, and incubate at 37°C for 30 minutes, then rinse three times with PBS buffer for 5 minutes each time. Develop with DBA for 3-5 minutes, then immediately place the sections in pure water to stop the staining. Perform hematoxylin staining and rinse with tap water. Differentiate with 1% hydrochloric acid ethanol solution, then incubate in tap water to achieve a blue inversion. Dehydrate sequentially by immersing in 70%, 80%, 90%, and 95% ethanol solutions for 5 minutes each, and 100% I and 100% II solutions for 10 minutes each. Clear the tissue sections by immersion in xylene I and II for 10 minutes each. Mount with neutral resin and air dry for 24 hours. Microscopic observation was performed, and the average optical density value (IOD / Area) was analyzed and calculated using ImageJ software.

[0159] (9) Data statistics

[0160] Measurement data adopted Description. Data comparison: Analysis of variance was used for comparisons among multiple groups; nonparametric tests were used for non-normally distributed continuous data (significance level α = 0.05). T-tests were used for analysis of two independent samples (significance level α = 0.05). All data were processed using SPSS 22.0 statistical software.

[0161] 5. Experimental Results

[0162] 5.1 Effects on behavioral activities and body weight

[0163] The results showed that during the modeling period, the control group mice exhibited no abnormal behavior, while the modeling group mice showed irritability, disordered fur, and huddling behavior. Furthermore, the mean weight of the modeling group mice after modeling was slightly lower than the mean weight before modeling, and significantly lower than the control group (statistically significant, P<0.01), consistent with the abnormal behavior and slow weight gain observed in mice after CUMS modeling. See Table 1.

[0164] Table 1. Effects on animal body weight during the modeling period ( )

[0165]

[0166] Note: Compared with the control group, ##P<0.01.

[0167] 5.2 Effects on SPT sucrose preference index and number of suspension tail rests

[0168] The results of saccharide preference analysis showed that, compared with the control group, the saccharide preference index of mice in the CUMS group and the CUMS+PCPA group was significantly reduced (P<0.01), indicating that the depression-insomnia model met expectations. After drug intervention, compared with the CUMS+PCPA group, the saccharide preference index of mice in the traditional Chinese medicine composition, traditional Chinese medicine control group 1, and traditional Chinese medicine control group 2 showed a significant rebound, with statistically significant differences (P<0.05 or P<0.01), indicating that the above three groups of drugs had a certain therapeutic effect on the depression model. The saccharide preference index of the medium-dose group of the traditional Chinese medicine composition was higher than that of traditional Chinese medicine control group 1 and traditional Chinese medicine control group 2 (P<0.05), suggesting that the therapeutic effect of the traditional Chinese medicine composition in improving saccharide preference was better than that of the two traditional Chinese medicine control groups.

[0169] The tail suspension test results showed that, compared with the control group, the number of times mice remained still was significantly increased in the PCPA group, CUMS group, and CUMS+PCPA group (P<0.05 or P<0.01). Compared with CUMS+PCPA, the number of times mice remained still was significantly decreased in the positive control group, the medium-dose group of the traditional Chinese medicine composition, and the high-dose group (P<0.05 or P<0.01). Compared with the traditional Chinese medicine control group 1 and the traditional Chinese medicine control group 2, the medium-dose group of the traditional Chinese medicine composition showed a significant decrease in the number of times mice remained still (P<0.05), indicating that the therapeutic effect of the traditional Chinese medicine composition in improving the number of times mice remained still was better than that of the two traditional Chinese medicine control groups (see Table 2).

[0170] Table 2. Effects on sugar water preference index and number of rest periods ( (n=10)

[0171]

[0172] Note: Compared with the control group, * P<0.05, **P<0.01; compared with the PCPA+CUMS group, # P<0.05, ## P<0.01; compared with the medium-dose group, △ P<0.05.

[0173] 5.3 Effects on FST immobility cumulative time and PST sleep duration

[0174] The results showed that, compared with the control group, the cumulative immobility time of mice in the CUMS and CUMS+PCPA groups was prolonged, which was statistically significant (P<0.05). Compared with CUMS+PCPA, the cumulative immobility time of mice in the positive control group, the medium-dose group of the traditional Chinese medicine composition, the high-dose group, and the two traditional Chinese medicine control groups was significantly reduced, which was statistically significant (P<0.05 or P<0.01); the cumulative immobility time of mice in the medium-dose group of the traditional Chinese medicine composition was significantly reduced compared with the two traditional Chinese medicine control groups (P<0.05).

[0175] PST results showed that, compared with the control group, the sleep duration of mice in the PCPA group, CUMS group, and CUMS+PCPA group was significantly shortened (P<0.01). Compared with CUMS+PCPA, the sleep duration of mice in the positive control group, the medium-dose group of the traditional Chinese medicine composition, and the high-dose group was significantly prolonged (P<0.01). The sleep duration of mice in the medium-dose group of the traditional Chinese medicine composition was significantly prolonged compared with the traditional Chinese medicine control groups 1 and 2 (P<0.05), indicating that the traditional Chinese medicine composition was more effective than the two traditional Chinese medicine control groups in improving mouse sleep (see Table 3).

[0176] Table 3 Effects on cumulative immobility time and sleep duration ( (n=10)

[0177]

[0178] Note: Compared with the control group, ** P<0.01; compared with the PCPA+CUMS group, # P<0.05, ## P<0.01; compared with the medium-dose group, △ P<0.05.

[0179] 5.4 Effects on changes in motion behavior during open field tests

[0180] The results showed that, compared with the control group, the total distance, total average speed, center distance, center average speed and center entry number were reduced in the CUMS group, and the total distance, total average speed and center distance were reduced in the CUMS+PCPA group, which were statistically significant (P<0.05 or P<0.01). Compared with CUMS+PCPA, the total distances of the positive control drug group, the low, medium, and high dose groups of the traditional Chinese medicine composition group, and the traditional Chinese medicine control group 1 and 2 were all significantly increased (P<0.05 or P<0.01); the total average speeds of the positive control drug group, the medium and high dose groups of the traditional Chinese medicine composition group, and the traditional Chinese medicine control group 2 were all significantly increased (P<0.05 or P<0.01); the center distances of the medium and high dose groups of the traditional Chinese medicine composition group and the traditional Chinese medicine control group 1 were all increased (P<0.05 or P<0.01); and the number of center entry times of the medium and high dose groups of the traditional Chinese medicine composition group, the traditional Chinese medicine control group 1, and the traditional Chinese medicine control group 2 were all increased (P<0.05 or P<0.01). Compared with the traditional Chinese medicine control group 1 and the traditional Chinese medicine control group 2, the medium dose group of the traditional Chinese medicine composition group showed increased total distances, total average speeds, center distances, and center average speeds (see Table 4).

[0181] Table 4. Effects on changes in motion behavior during open field tests ( (n=10)

[0182]

[0183] Note: Compared with the control group, * P<0.05, ** P<0.01; compared with the PCPA+CUMS group, # P<0.05, ## P<0.01.

[0184] 5.5 Effects on related neurotransmitters in brain tissue

[0185] The results showed that, compared with the control group, the 5-HT content in the brain tissue of the PCPA group, CUMS group, and CUMS+PCPA group was significantly decreased, the GABA content in the PCPA group and CUMS+PCPA group was significantly decreased, and the GLU content in the CUMS group and CUMS+PCPA group was significantly increased, which were statistically significant (P<0.05 or P<0.01). Compared with CUMS+PCPA, the 5-HT and GABA content in the brain tissue of mice in each treatment group was increased, but no statistical difference was observed. The GLU content in the brain tissue of mice in the positive control group, the low-dose and high-dose groups of the traditional Chinese medicine composition, the traditional Chinese medicine control group 1, and the traditional Chinese medicine control group 2 was significantly decreased, which were statistically significant (P<0.05 or P<0.01). The medium-dose group of the traditional Chinese medicine composition had higher 5-HT and GABA content in the brain tissue of mice compared with the traditional Chinese medicine control group 1 and the traditional Chinese medicine control group 2, as shown in Table 5.

[0186] Table 5 Effects on related neurotransmitters in brain tissue ( (n=10)

[0187]

[0188] Note: Compared with the control group, * P<0.05, ** P<0.01; compared with the PCPA+CUMS group, # P<0.05, ## P<0.01.

[0189] 5.6 Pathological examination of brain tissue

[0190] Pathological results show that ( Figure 1-2Table 6): ① Control group (6 animals): Neuronal structure was intact, neatly arranged, nucleoli were clear, and Nissl bodies were basically normal. ② PCPA group, CUMS group, and CUMS + PCPA group all showed: Most neurons were disordered and loosely arranged. Some cells had irregular morphology, pyknosis, and deep staining; some Nissl bodies were broken and reduced; some showed vacuolar degeneration in the hippocampus; lesion severity grade (+, 18 / 18). ③ Positive drug group (6 animals): No abnormalities were observed in the animal brain tissue (-, 2 / 6); A small number of neurons were disordered and loosely arranged; some cells had irregular morphology, pyknosis, and deep staining; some Nissl bodies were broken and reduced; some showed vacuolar degeneration in the hippocampus (+, 4 / 6). ④ Low-dose group of traditional Chinese medicine combination (6 animals): Most neurons were disordered and loosely arranged; some cells had irregular morphology, pyknosis, and deep staining. Some Nissl bodies were broken and reduced; some showed vacuolar degeneration in the hippocampus; lesion severity grade (+, 6 / 6). ⑤ Medium-dose group of traditional Chinese medicine composition (6 animals): No abnormalities were observed in the brain tissue of the animals (-, 3 / 6); a small number of neurons showed disordered and loose arrangement; some cells had irregular morphology, pyknosis, and deep staining; some Nissl bodies were broken and reduced; some showed vacuolar degeneration in the hippocampus (+, 3 / 6). ⑥ High-dose group of traditional Chinese medicine composition (6 animals): No abnormalities were observed in the brain tissue of the animals (-, 5 / 6); a small number of neurons showed disordered and loose arrangement; some cells had irregular morphology, pyknosis, and deep staining; some Nissl bodies were broken and reduced; some showed vacuolar degeneration in the hippocampus (+, 1 / 6). ⑦ Control group 1 of traditional Chinese medicine (6 animals): No abnormalities were observed in the brain tissue of the animals (-, 3 / 6); a small number of neurons showed disordered and loose arrangement; some cells had irregular morphology, pyknosis, and deep staining; some Nissl bodies were broken and reduced; some showed vacuolar degeneration in the hippocampus (+, 3 / 6). ⑧ Control group 2 (6 animals): No abnormalities were found in the brain tissue of the animals (-, 2 / 6); a small number of neurons were arranged in a disordered and loose manner; some cells had irregular morphology, pyknosis, and deep staining; some Nissl bodies were broken and reduced; some showed vacuolar changes in the hippocampus (+, 4 / 6).

[0191] Table 6 Analysis of Pathological Examination Results in Brain Tissue

[0192]

[0193] 5.7 Immunohistochemical detection

[0194] Compared with the control group, the expression of MTNR1A and MTNR1B proteins in the PCPA group, CUMS group, and CUMS+PCPA group were significantly downregulated (P<0.05 or P<0.01). The positive control group, the medium and high dose groups of the traditional Chinese medicine composition could reverse the CUMS+PCPA-induced inhibition of MTNR1A receptor expression, and the expression level was significantly higher than that in the CUMS+PCPA group (P<0.05 or P<0.01). The positive control group, the low, medium, and high dose groups of the traditional Chinese medicine composition, and traditional Chinese medicine control group 1 could reverse the CUMS+PCPA-induced inhibition of MTNR1B receptor expression. Compared with traditional Chinese medicine control group 1 and traditional Chinese medicine control group 2, the medium dose group of the traditional Chinese medicine composition showed increased expression levels of both MTNR1A and MTNR1B, with MTNR1A reaching a significant level (P<0.05). Results are as follows. Figure 3 , Figure 4 As shown in Table 7.

[0195] Table 7. Average optical density values ​​of MTNR1A and MTNR1B protein expression in mouse brain tissue ( (n=4)

[0196]

[0197] Note: Compared with the control group, * P<0.05, ** P<0.01; compared with the PCPA+CUMS group, # P<0.05, ## P<0.01; compared with the medium-dose group, △ P<0.05.

[0198] 5.8 Conclusion

[0199] Experimental studies have revealed that the medium- and high-dose groups of the traditional Chinese medicine composition exhibit regulatory capabilities against depression and related sleep disorders. This composition not only effectively improves core symptoms of depression, such as anhedonia, behavioral hopelessness, and reduced exploratory behavior, but also significantly prolongs sleep time, achieving simultaneous relief of depression and insomnia at the behavioral level. Research confirms that it can restore neurotransmitter balance by regulating the function of the 5-HT, GABA, and glutamate systems, and reverse the inhibition of MTNR1A and MTNR1B receptor expression in key brain regions such as the hippocampus, thereby achieving multi-level regulation from circadian rhythms to neural plasticity at the pathophysiological level. Unexpectedly, under the same dosage conditions, the combination of *Codonopsis pilosula*, *Pueraria lobata*, *Epimedium brevicornu*, and jujube was more effective in treating depression accompanied by insomnia than the combination of *Codonopsis pilosula*, *Pueraria lobata*, and *Epimedium brevicornu* and the jujube alone, and the efficacy was positively correlated with dosage. The combination of *Codonopsis pilosula*, *Pueraria lobata*, and *Epimedium brevicornu* showed some effect in treating depression, but did not show a significant effect in treating depression accompanied by insomnia. Most importantly, this composition showed good effects on a depression model accompanied by decreased melatonin levels, achieving dual regulation of hormone levels and sleep structure. This provides a modern pharmacological basis for the traditional Chinese medicine concept of "holistic regulation" revealed by this herbal composition, opening up a new path for the treatment of depression accompanied by insomnia that focuses on both symptom relief and pathological reversal.

[0200] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0201] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A traditional Chinese medicine composition for preventing and / or treating depression with insomnia and hormone level reduction, characterized in that, it comprises the following components in parts by weight: 15-24 parts of radix pseudostellariae, 10-16 parts of radix puerariae, 5-8 parts of herba epimedii, and 8-12 parts of traditional Chinese medicine A; and the traditional Chinese medicine A comprises one or more of the following: fructus jujubae, semen ziziphi spinosae, radix polygalae, poria cocos, cistanche and radix ginseng. 2.The traditional Chinese medicine composition of claim 1, wherein the traditional Chinese medicine A comprises one or more of the following: fructus jujubae, semen ziziphi spinosae, radix polygalae, poria cocos, cistanche and radix ginseng. 3.The traditional Chinese medicine composition of claim 1, wherein it comprises the following components in parts by weight: 16-20 parts of radix pseudostellariae, 12-16 parts of radix puerariae, 6-8 parts of herba epimedii, and 10-12 parts of traditional Chinese medicine A.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, 4.The traditional Chinese medicine composition of claim 1, wherein it comprises the following components in percentage by weight: 31-55% of radix pseudostellariae, 20-39% of radix puerariae, 9-21% of herba epimedii, and 15-25% of traditional Chinese medicine A.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, 5.A preparation method of the traditional Chinese medicine composition of claim 1, comprising the steps of: separately preparing extract A1 and extract A2, and mixing the extract A1 and the extract A2.

4. The preparation method of the traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, 6.The preparation method of claim 5, wherein the step of preparing the extract A1 comprises: mixing the radix pseudostellariae and the traditional Chinese medicine A with water, performing first extraction, collecting water extract, performing first concentration on the water extract to prepare water extract concentrate, precipitating the water extract concentrate with ethanol, collecting precipitate to prepare total polysaccharide of radix pseudostellariae and traditional Chinese medicine A. 7.The preparation method of claim 5, wherein the step of preparing the extract A2 comprises: mixing the radix puerariae and the herba epimedii with aqueous ethanol solution, performing second extraction, collecting alcohol extract, performing second concentration on the alcohol extract to prepare alcohol extract concentrate, and collecting supernatant from the alcohol extract concentrate to separate and prepare total flavonoids of radix puerariae and herba epimedii. 8.The preparation method of claim 5, wherein in the step of preparing the extract A1, the ratio of the total mass of the radix pseudostellariae and the traditional Chinese medicine A to the mass of water is 1: (8-12).

5. The preparation method according to claim 4, characterized in that, 9.The preparation method of claim 5, wherein in the step of preparing the extract A1, the temperature of the first extraction is 85-95℃, and the time of the first extraction is 3-9h.

6. The preparation method according to claim 4, characterized in that, 10.The preparation method of claim 5, wherein in the step of preparing the extract A2, the ratio of the total mass of the radix puerariae and the herba epimedii to the mass of the aqueous ethanol solution is 1: (10-15).

7. The preparation method according to claim 4, characterized in that, 11.The preparation method of claim 5, wherein in the step of preparing the extract A2, the temperature of the second extraction is 70-90℃, and the time of the second extraction is 1-3h.

8. The method of any one of claims 4 to 7, wherein the method further comprises the step of: 12.The preparation method of claim 5, wherein one or more of the following conditions are met:

9. The method of any one of claims 4 to 7, wherein the method further comprises the step of: (1) in the step of preparing the extract A1, the temperature of the first concentration is 65-85℃; (2) in the step of precipitating the water extract concentrate with ethanol solution in the step of preparing the extract A1, the volume percentage of ethanol in the total system of the mixture is 75-85%; (3) in the step of preparing the extract A2, the volume percentage of ethanol in the aqueous ethanol solution is 75-85%; (4) in the step of preparing the extract A2, the temperature of the second concentration is 60-80℃; (5) in the step of preparing the extract A2, the alcohol extract concentrate is allowed to stand at a temperature less than or equal to 10℃ for more than 40h, and optionally, the alcohol extract concentrate is allowed to stand at a temperature of 4-10℃ for 40-50h; and (6) in the step of preparing the extract A2, the total flavonoids of radix puerariae and herba epimedii in the supernatant are obtained by using macroporous adsorption resin separation. ​ 10. The use of the traditional Chinese medicine composition of any one of claims 1-3 or the traditional Chinese medicine composition prepared by the preparation method of any one of claims 4-9 in the preparation of a product for preventing and / or treating depression with insomnia and reduced hormone levels.

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