An antidepressant Chinese medicine composition and its preparation method and application

Abstract

The present invention belongs to the field of traditional Chinese medicine, and in particular to a kind of antidepressant Chinese medicine composition and its preparation method and application. The Chinese medicine composition of the present invention comprises, by weight, 55-65 parts of aconite, 55-65 parts of dried ginger, 85-95 parts of liquorice, 85-95 parts of cinnamon twig, 85-95 parts of white peony root, 290-310 parts of oyster, 85-95 parts of cornus, 40-55 parts of black plum, 145-155 parts of poria, 145-155 parts of spiny jujube seeds, 85-95 parts of jujube, 145-155 parts of yam, 55-65 parts of Morinda officinalis, 85-95 parts of ophiopogon, 25-35 parts of Amomum villosum, 25-35 parts of agarwood, 25-35 parts of cinnamon bark, 115-125 parts of polygonatum, 10-20 parts of tortoise shell glue, 10-20 parts of donkey-hide glue, and 10-20 parts of antler glue. Animal experiments show that the Chinese medicine composition of the present invention can effectively improve the behavioral manifestations of rats and mice with depression, reduce Nissl body damage in brain tissue, regulate the expression of p-TrkB / TrkB, p-AKT / AKT and p-CREB / CREB proteins in the hippocampus of brain tissue, and prevent and treat depression by improving the plasticity of hippocampal neurons.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and specifically relates to an antidepressant traditional Chinese medicine composition and a preparation method and application thereof. Background Art

[0002] Depression has become a common and frequently occurring disease in modern society, and its incidence is still increasing. Symptoms are characterized by low emotions, loss of interest, slow thinking, and reduced speech and movement. Patients with severe depression have thoughts of world-weariness or suicide. Such diseases can cause great pain to patients and their families. Modern medical research believes that the etiology of depression is complex, and its onset may involve multiple mechanisms such as neurotransmitter disorders, brain-derived neurotrophic factor deficiency, hypothalamic-pituitary-adrenal axis disorders, inflammatory reactions, abnormal energy metabolism, and neuronal regeneration disorders. At present, the main drugs used in clinical treatment of depression include 5-HT reuptake inhibitors (SSRIs), NE reuptake inhibitors (NRIs), 5-HT / NE dual reuptake inhibitors (SNRIs), tricyclic antidepressants (TCAs), and monoamine oxidase inhibitors (MAOIs). Although these drugs have certain clinical efficacy, they have defects such as slow onset, narrow spectrum of action, large side effects, and easy relapse after discontinuation of medication.

[0003] In recent years, with the development of modern research on traditional Chinese medicine, traditional Chinese medicine has shown strong advantages in the treatment of depression. Traditional Chinese medicine has the characteristics of "multiple components, multiple targets, and multiple pathways", and is safe and has few side effects. Therefore, the compliance of using traditional Chinese medicine to treat depression in clinical practice has been greatly improved. There are already many traditional Chinese medicine compositions for the treatment of depression in the prior art, such as:

[0004] Patent CN102233071B discloses a Chinese medicine composition for treating depression and a preparation method thereof, wherein the Chinese medicine composition is made of the following raw materials in parts by weight: 1-6 parts of ginseng, 1-5 parts of polygala, 1-5 parts of calamus, 1-6 parts of tuckahoe, 1-6 parts of morinda officinalis, 1-5 parts of bupleurum, 1-5 parts of immature bitter orange, 1-6 parts of red peony root and 1-5 parts of liquorice. The Chinese medicine composition has the effects of invigorating qi, nourishing the heart and calming the mind, regulating qi and soothing the liver, and promoting blood circulation and relieving depression.

[0005] Patent CN115429864B discloses a Chinese medicine composition for treating depression, comprising the following Chinese medicine raw materials in weight proportions: 0.5-6 parts of bupleurum, 0.5-6 parts of Hypericum perforatum, 1-5 parts of white peony root, 0.8-5 parts of curcuma, 0.8-5 parts of citron, 1-6 parts of Pseudostellariae Radix, 1-5 parts of Atractylodes macrocephala, 0.5-4 parts of Poria, 0.8-5 parts of Ziziphus jujuba seeds, 1-5 parts of valerian, and 1-6 parts of Albizzia julibrissin. The above medicinal flavors are used together to soothe the liver and regulate qi, strengthen the spleen and nourish the heart, calm the mind and relieve depression, and are used for mild and moderate depression caused by liver depression, spleen deficiency, and mental malnutrition.

[0006] However, most of the existing Chinese medicine compositions treat depression based on the liver, and generally have the disadvantage of insufficient Yang Qi, weak warming and agitation, poor Qi flow, and imbalance of Yin and Yang, which leads to unsatisfactory treatment effects. Therefore, in-depth research on the formula is still needed to make the Chinese medicine composition more effective and reliable, so as to achieve the effect of treating both the symptoms and the root cause. Summary of the invention

[0007] According to a first aspect of the present invention, an antidepressant traditional Chinese medicine composition is provided, wherein the raw materials thereof comprise, by weight, 55-65 parts of aconite root, 55-65 parts of dried ginger, 85-95 parts of liquorice, 85-95 parts of cinnamon twig, 85-95 parts of white peony root, 290-310 parts of oyster, 85-95 parts of cornus fruit, 40-55 parts of ebony, 145-155 parts of poria, 145-155 parts of spiny jujube seed, 85-95 parts of jujube, 145-155 parts of yam, 55-65 parts of Morinda officinalis, 85-95 parts of ophiopogon, 25-35 parts of amomum, 25-35 parts of agarwood, 25-35 parts of cinnamon bark, 115-125 parts of polygonatum, 10-20 parts of tortoise shell glue, 10-20 parts of donkey-hide glue, and 10-20 parts of antler glue.

[0008] The present invention targets the pathogenesis of depression syndrome "Yang deficiency, Qi stagnation, imbalance of Yin and Yang", takes "warming Yang to relieve depression, and harmonizing Ying and Wei" as the treatment principle, starts from the perspective of warming Yang, harmonizing Ying and Wei to relieve depression, and nourishing blood and Yin to balance Yin and Yang, and pays attention to the interdependence and mutual unity between Qi and blood and internal organs. The present invention is based on warming Yang, harmonizing Ying and Wei as the core, supplemented by nourishing blood and Yin, balancing Yin and Yang, so as to effectively warm Yang and replenish Qi, harmonize Yang surface and Yin interior, and effectively relieve the symptoms of depression.

[0009] The present invention uses cinnamon twig and white peony root as the main medicine to harmonize the Ying and Wei and to communicate yin and yang. Cinnamon twig is pungent and warm, warming from the yang and supporting the Wei, while white peony root is sour and cold, sour and cold going to the yin and benefiting the Ying; aconite, dried ginger, barberry, cinnamon bark, and agarwood are used as the ministerial medicines to warm and nourish the spleen and kidney, warm the yang and suppress the yang, and guide the fire back to the origin to help cinnamon twig and Wei, while oyster, cornus fruit, black plum, poria, spiny jujube seed, and ophiopogon are used as the ministerial medicines to sour and sweeten the yin, nourish the blood and calm the mind to help white peony root benefit the Ying; jujube, yam, amomum, polygonatum, tortoise shell glue, donkey-hide glue, and deer horn glue are used as adjuvants, and yam, jujube, and amomum are combined to Turtle shell glue, donkey-hide glue and deer antler glue can raise the generative Qi of the spleen and stomach and harmonize the Ying and Wei. Tortoise shell glue, donkey-hide glue and deer antler glue can nourish the essence and blood, and benefit the Yang and transform the Yin. At the same time, Amomum villosum, Polygonatum sibiricum and Ophiopogon japonicus can be used together to nourish the heart and spleen, replenish the Yin essence and blood, and benefit the Yang and transform the Yin, thereby harmonizing the Ying and Wei, calming the mind and settling the mind, and enhancing the effect of the whole prescription in harmonizing the Ying and Wei, benefiting the Yang and transforming the Yin, so as to achieve the purpose of replenishing deficiency, correcting deviation and balancing Yin and Yang. Licorice is used as a guiding drug to harmonize all the drugs, harmonize the Yang surface and Yin interior, and make the Qi, Wei, blood and Ying run in parallel without conflict, which is the harmony of hardness and softness. The whole formula harmonizes the Ying and Wei, guides the Yang to come out of the Yin during the day and enter the Yin at night, guides the Qi to rise and fall in an orderly manner, strengthens the middle earth, and connects the heart and kidney; when the Ying Qi is full, it nourishes the blood vessels, nourishes the heart Qi, and the heart veins are full, the mind is clear, and the soul is stable; when the Wei Qi is well regulated, it can convey the spirit of the internal organs and communicate the inside and outside; the whole formula warms the Yang and relieves depression on the basis of nourishing the blood and Yin, and rises and falls in an orderly manner. It can effectively warm the Yang and replenish the Qi, promote the flow of Qi and relieve depression, harmonize the Yang surface and Yin, and fit the pathogenesis of depression, so that the spirit and soul are nourished, and the depression is relieved, and the coordination of the internal organs can be restored, and the pathological phenomenon of the partial victory and decline of Yin and Yang can be corrected to restore it to a normal state. This prescription is especially suitable for people with yang deficiency depression, accompanied by inattention, low mood, poor sleep and fatigue.

[0010] The raw material sources of the present invention are as follows:

[0011] Aconite: The lateral tuberous root (sub-root) of the Ranunculaceae plant Aconitum (cultivated product).

[0012] Dried ginger: the dried rhizome of Zingiber oj-jicinale Rosc., a plant of the ginger family.

[0013] Licorice: The roots and rhizomes of Glycyrrhiza uralensis Fisch., a plant of the genus Glycyrrhiza in the legume family.

[0014] Cinnamon twig: twig of the cinnamon plant of the Lauraceae family.

[0015] White peony root: the root of the Paeonia lactiflora plant of the Ranunculaceae family.

[0016] Oyster: The shell of the oyster family, such as the Omi oyster, the long oyster or the Dalian Bay oyster.

[0017] Cornus officinalis: the dried mature pulp of Cornus officinalis Sieb.et Zucc., a plant of the Cornaceae family.

[0018] Black plum: the dried, nearly ripe fruit of Prunus mume (Sieb.) Sieb. et Zuce., a plant of the Rosaceae family.

[0019] Poria: The sclerotium of Poria cocos Schw. Wolf, a plant of the Polyporaceae family, contains parts of pine roots.

[0020] Ziziphus jujuba kernel: the dried mature seeds of Ziziphus jujuba Mill.var.spinosa (Bunge)Hu exH.F.Chou, a plant of the Rhamnaceae family.

[0021] Jujube: the dried mature fruit of Ziziphus jujuba Mill.var.inermis (Bunge) Rehd., a plant of the Rhamnaceae family.

[0022] Chinese yam: the rhizome of Dioscorea opposita Thunb., a plant of the Dioscoreaceae family. Huai yam refers to the Chinese yam produced in Guangdong, Jiangsu, Anhui and other places.

[0023] Morinda officinalis: The dried root of Morinda officinalis How, a plant of the Rubiaceae family.

[0024] Ophiopogon japonicus: the dried tuberous root of Ophiopogon japonicus (Ophiopogon japonicus), a plant of the Liliaceae family.

[0025] Amomum villosum: the ripe fruit of the herbaceous plants Amomum villosum Louv., A. longiligulare T.L. Wu, or A. xanthioides Wall. of the Zingiberaceae family.

[0026] Agarwood: The resin-containing wood of the Thymelaeaceae trees Aquilaria agallocha Roxb. and Aquilaria sinensis (Lour.) Gilg.

[0027] Cinnamon: the bark of the cinnamon tree of the Lauraceae family.

[0028] Polygonatum: The rhizome of Polygonatum sibiricum, a plant of the Liliaceae family.

[0029] Turtle shell glue: solid glue made by boiling and concentrating tortoise shells.

[0030] Donkey-hide gelatin: a solid gelatin made by boiling and concentrating the skin of the donkey Equus asinus L. and other donkeys of the Equine family.

[0031] Deer antler glue: a glue block made by boiling the antlers of the sika deer or red deer, which are animals of the Cervidae family.

[0032] In some embodiments, the raw material composition, measured by mass, includes: 60 parts of aconite, 60 parts of dried ginger, 90 parts of licorice, 90 parts of cinnamon twig, 90 parts of white peony root, 300 parts of oyster, 90 parts of cornus, 45 parts of ebony, 150 parts of poria, 150 parts of spiny jujube seeds, 90 parts of jujube, 150 parts of yam, 60 parts of Morinda officinalis, 90 parts of Ophiopogon japonicus, 30 parts of Amomum villosum, 30 parts of agarwood, 30 parts of cinnamon, 120 parts of polygonatum, 15 parts of tortoise shell glue, 15 parts of donkey-hide gelatin, and 15 parts of deer antler glue.

[0033] In some embodiments, the aconite root is cooked aconite root, which is a processed product of aconite root.

[0034] In some embodiments, the licorice is roasted licorice, which is a honey-roasted licorice product.

[0035] In some embodiments, the Fructus Corni is processed Fructus Corni, which is a processed product of Fructus Corni.

[0036] In some embodiments, pharmaceutically acceptable excipients are also included.

[0037] In some embodiments, the auxiliary material is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant, and a lubricant.

[0038] In some embodiments, the dosage form of the Chinese medicine composition is ointment, decoction, granules, powder, tablet, pill or capsule.

[0039] According to a second aspect of the present invention, a method for preparing the above-mentioned Chinese medicine composition is provided. When the dosage form of the Chinese medicine composition is an ointment, the preparation method comprises the following steps:

[0040] (1) adding the raw medicinal materials except agarwood, tortoise shell glue, donkey-hide gelatin, and deer antler glue to 10 times the amount of water, soaking for 1 hour, and then decocting twice, each decoction time is continued for 1.5 hours after boiling, combining the decoctions obtained from the two decoctions, filtering through an 80-mesh sieve, and obtaining a decoction liquid;

[0041] (2) heating the decoction obtained in step (1) at 70-90° C. and concentrating it to a first clear paste having a relative density of 1.30-1.35 (at room temperature);

[0042] (3) Mix tortoise shell glue, donkey hide glue and deer antler glue, add water three times the amount of donkey hide glue, and then heat to 60-80° C. to melt, and then add to the first clear paste obtained in step (2) after melting, stir evenly, to obtain a second clear paste;

[0043] (4) Passing agarwood through a 120-mesh sieve to obtain agarwood powder, heating the second clear paste obtained in step (3) to 60° C., adding the agarwood powder, stirring and mixing evenly to obtain a mixed medicine;

[0044] (5) adding medium honey to the mixed drug obtained in step (4) at a mass ratio of 1:1, then heating to 60° C. and stirring evenly to obtain a finished paste with a relative density of 1.43-1.46 (30° C.);

[0045] (6) Cooling the finished paste obtained in step (5) to room temperature, canning and sterilizing the finished paste.

[0046] According to a third aspect of the present invention, there is provided a use of the above-mentioned Chinese medicine composition in the preparation of a drug for treating depression. Specifically, the depression is yang deficiency type depression.

[0047] The beneficial effects of the present invention include:

[0048] (1) Animal experiments show that the Chinese medicine composition of the present invention can effectively improve the behavioral performance of depression model rats and mice, reduce the damage of Nissl bodies in brain tissue, regulate the expression of multiple proteins such as p-TrkB / TrkB, p-AKT / AKT and p-CREB / CREB in the hippocampus of brain tissue, and prevent and treat depression by improving the plasticity of hippocampal neurons. This suggests that the Chinese medicine composition of the present invention can effectively treat depression.

[0049] (2) Clinical Application The Chinese medicine composition of the present invention can rapidly improve the persistent depression, decreased speech, mental and motor retardation and other symptoms of depression patients, and can relieve the symptoms of fatigue, slow reaction, memory loss, insomnia and other symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Base peak ion chromatogram under positive ion mode for characterization of the components of the traditional Chinese medicine ointment sample prepared in Example 1. Note: 3, 4, 5, 8, 10, 11, 12, 13, 14 represent liquiritin, paeoniflorin, magiformin, ursolic acid, betulinic acid, nastrin, ophiopogon saponin B, cis-cinnamaldehyde and anhydrous glucose, respectively.

[0051] Figure 2 This is the base peak ion chromatogram under negative ion mode for characterizing the components of the traditional Chinese medicine ointment sample prepared in Example 1. Note: 1, 2, 6, 7, 9, 15 represent cinnamaldehyde, 6-gingerol, morroniside, citric acid, spinosin and agarwood tetraol, respectively.

[0052] Figure 3 This is the secondary mass spectrum of gingerol, an active ingredient in the traditional Chinese medicine ointment prepared in Example 1.

[0053] Figure 4This is the secondary mass spectrum of spinosin, an active ingredient in the traditional Chinese medicine ointment prepared in Example 1.

[0054] Figure 5 This is the secondary mass spectrum of the active ingredient agarwood tetraol in the traditional Chinese medicine ointment prepared in Example 1.

[0055] Figure 6 This is the secondary mass spectrum of the active ingredient paeoniflorin in the traditional Chinese medicine ointment prepared in Example 1.

[0056] Figure 7 These are the results of the effect of the traditional Chinese medicine ointment on the body weight of the corticosterone-induced depression mouse model in animal experiment 1. Note: control represents the normal control group, model represents the model group, low represents the low-dose traditional Chinese medicine ointment group, medium represents the medium-dose traditional Chinese medicine ointment group, high represents the high-dose traditional Chinese medicine ointment group, and positive represents the positive drug group.

[0057] Figure 8 This is the effect of the Chinese herbal ointment in animal experiment 1 on the hippocampal neurons of mice with corticosterone-induced depression model (20×). Note: A: normal control group; B: model group; C: low-dose Chinese herbal ointment group; D: medium-dose Chinese herbal ointment group; E: high-dose Chinese herbal ointment group; F: positive drug group.

[0058] Fig. 9 This is a bar graph of Trkb, CREB, BDNF and p-Akt protein expression in the mouse hippocampus tissue of animal experiment 1. Note: In the figure, A: normal control group; B: model group; C: low-dose Chinese medicine ointment group; D: medium-dose Chinese medicine ointment group; E: high-dose Chinese medicine ointment group; F: positive drug group.

[0059] Fig.10 The results of comparison of the effects of each group in animal experiment 2 on SPT (A), NSF (B) and FST (C) of CUMS depression model rats. Note: *p<0.05 indicates significant difference.

[0060] Fig.11 The morphological changes of neuronal cells in the hippocampus of each group of rats in animal experiment 2 (Nissl staining, 20×). Note: A: control group, B: model group, C: 8.4g / kg SAO group, D: 14g / kg SAO group, E: fluoxetine group, F: 14g / kg SAO+ANA-12 group, G: ANA-12 group.

[0061] Fig.12 These are the bar graphs of BDNF, p-TrkB, p-AKT and p-CREB protein expression in the hippocampal tissues of rats in each group of animal experiment 2. Note: A: control group, B: model group, C: 8.4g / kg SAO group, D: 14g / kg SAO group, E: fluoxetine group, F: 14g / kg SAO+ANA-12 group, G: ANA-12 group.

[0062] Fig.13 Comparison results of BDNF, p-TrkB, p-AKT and p-CREB protein expression in the hippocampus tissue of each group of rats in animal experiment 2. Note: *p<0.05 indicates significant difference. DETAILED DESCRIPTION

[0063] The present invention is further described in detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto. The materials involved in the following examples can all be obtained from commercial channels.

[0064] Example 1

[0065] The antidepressant Chinese medicine ointment of the present embodiment is composed of the following raw materials in parts by mass: 60g of cooked aconite, 60g of dried ginger, 90g of roasted licorice, 90g of cinnamon twig, 90g of white peony root, 300g of oyster, 90g of processed cornus fruit, 45g of ebony, 150g of poria, 150g of spiny jujube seed, 90g of jujube, 150g of Chinese yam, 60g of Morinda officinalis, 90g of Ophiopogon japonicus, 30g of Amomum villosum, 30g of agarwood powder, 30g of cinnamon, 120g of polygonatum, 15g of tortoise shell glue, 15g of donkey-hide glue, and 15g of deer antler glue.

[0066] The preparation method thereof comprises the following steps:

[0067] (1) Put cooked aconite root, dried ginger, roasted licorice root, white peony root, oyster, processed cornus fruit, black plum, poria, spiny jujube seed, jujube, Chinese yam, Morinda officinalis, Ophiopogon japonicus, and polygonatum into a large gauze medicine bag, put it into a medicine pot, add water to cover the medicine surface and soak for 20 minutes, bring to a boil over high heat, and then simmer over low heat for 30 minutes;

[0068] (2) Place cinnamon twig, amomum villosum and cinnamon bark into a small gauze medicine bag, place it into the above medicine pot, and continue to boil for 20 minutes, then filter out the medicine liquid and set aside;

[0069] (3) adding the same amount of water to the above medicine tank, boiling the medicine residue in the same way, filtering out the medicine liquid, and combining it with the medicine liquid filtered out for the first time to obtain a combined medicine liquid for standby use;

[0070] (4) Place tortoise shell glue, donkey-hide gelatin and deer antler glue in a container, add about 45 mL of warm water at about 80°C, heat to melt, mix evenly with the above combined liquid medicine, divide into three equal parts, take one portion in the morning, noon and evening respectively. When taking, divide agarwood powder into three equal parts, add one portion of agarwood powder to each portion and take with water. A course of treatment is 3 months.

[0071] The components of the prepared traditional Chinese medicine ointment were characterized, and the characterization method and results are as follows:

[0072] (1) Chromatographic conditions: The chromatographic column was an ACE Excel 3Super C18 column (2.1×100 mm×1.8 μm), the mobile phase was 0.1% formic acid water (A)-acetonitrile (B), gradient elution, and the elution program was: 0-1 min 10% B, 1-2.5 min 10%-25% B, 2.5-8.5 min 25%-95% B, 8.5-12 min 95%-95% B, 12-12.01 min 95%-100% B. The flow rate was 0.3 mL / min; the column temperature was 30°C; and the injection volume was 2 μL.

[0073] (2) Mass spectrometry conditions: ESI ion source, data acquisition (positive and negative ion M mode), data full scan acquisition mode is IDA-production. Primary mass scan range is m / z 50-1000, mass scan range is m / z 50-1000 in IDA mode; capillary voltage: 5.5 kV, cone voltage: 80 V, ion source temperature: 600 °C, desolvation gas temperature: 600 °C, nebulizer gas (N 2 ) Flow rate: 55L·h -1 , desolvation gas (N 2 ) Flow rate: 55L·h -1 ,Data acquisition and processing were performed using Analyst TF 1.6 software.

[0074] (3) Under the above LC-MS conditions, the Chinese medicine ointment sample (concentration 5.18 mg / mL) prepared in Example 1 was injected and analyzed. After reverse deduction of the chemical formula, a total of 15 chromatographic peaks were identified, which were mainly flavonoids and terpenoids. The positive and negative ion chromatograms of the sample are shown in Figure 2. Figure 1-2 As shown; the secondary ion current diagram of some active ingredients is shown Figure 3-6 The mass spectrum information related to the base peak ion chromatogram is shown in Table 1.

[0075] Table 1 Mass spectrometry information of chemical components in traditional Chinese medicine ointment samples

[0076]

[0077] Example 2

[0078] The antidepressant Chinese medicine ointment of this embodiment has the following raw materials in parts by mass:

[0079] 60g of cooked aconite, 60g of dried ginger, 90g of roasted licorice, 90g of cinnamon twig, 90g of white peony root, 300g of oyster, 90g of processed cornus fruit, 45g of black plum, 150g of poria, 150g of spiny jujube kernel, 90g of jujube, 150g of Chinese yam, 60g of Morinda officinalis, 90g of Ophiopogon japonicus, 30g of Amomum villosum, 30g of agarwood, 30g of cinnamon, 120g of Polygonatum sibiricum, 15g of tortoise shell glue, 15g of donkey-hide glue, and 15g of deer antler glue.

[0080] The preparation method thereof comprises the following steps:

[0081] (1) Take cooked aconite root, dried ginger, roasted licorice root, cinnamon twig, white peony root, oyster, processed cornus fruit, black plum, poria, spiny jujube seed, jujube, Chinese yam, Morinda officinalis, Ophiopogon japonicus, Amomum villosum, Aquilaria sinensis, cinnamon bark, and polygonatum, add 10 times the amount of water to soak for 1 hour, decoct for 1.5 hours, collect the decoction, decoct the residue with 10 times the amount of water for 1.5 hours, collect the decoction, combine the two decoctions to obtain a combined decoction;

[0082] (2) Melt tortoise shell glue, donkey-hide glue and deer antler glue with warm water and add them to the combined water decoction obtained in step (1), filter and concentrate the obtained medicinal solution under reduced pressure to obtain 212.8 g of extract.

[0083] Example 3

[0084] The antidepressant Chinese medicine ointment of this embodiment has the following raw materials in parts by mass:

[0085] 60g of cooked aconite, 60g of dried ginger, 90g of roasted licorice, 90g of cinnamon twig, 90g of white peony root, 300g of oyster, 90g of processed cornus fruit, 45g of black plum, 150g of poria, 150g of spiny jujube kernel, 90g of jujube, 150g of Chinese yam, 60g of Morinda officinalis, 90g of Ophiopogon japonicus, 30g of Amomum villosum, 30g of agarwood, 30g of cinnamon, 120g of Polygonatum sibiricum, 15g of tortoise shell glue, 15g of donkey-hide glue, and 15g of deer antler glue.

[0086] The preparation method thereof comprises the following steps:

[0087] (1) adding the raw medicinal materials except agarwood, tortoise shell glue, donkey-hide gelatin, and deer antler glue to 10 times the amount of water, soaking for 1 hour, and then decocting twice, each decoction time is continued for 1.5 hours after boiling, combining the decoctions obtained from the two decoctions, filtering through an 80-mesh sieve, and obtaining a decoction liquid;

[0088] (2) heating the decoction obtained in step (1) at 80° C. and concentrating it to a first clear paste having a relative density of 1.30 (at room temperature);

[0089] (3) Mix tortoise shell glue, donkey hide glue and deer antler glue, add about 45 mL of water, and then heat to 70° C. to melt, and then add to the first clear paste obtained in step (2) after melting, and stir evenly to obtain a second clear paste;

[0090] (4) Passing agarwood through a 120-mesh sieve to obtain agarwood powder, heating the second clear paste obtained in step (3) to 60° C., adding the agarwood powder, stirring and mixing evenly to obtain a mixed medicine;

[0091] (5) adding medium honey to the mixed drug obtained in step (4) at a mass ratio of 1:1, then heating to 60° C. and stirring evenly to obtain a finished paste with a relative density of 1.44 (room temperature);

[0092] (6) Cool the finished paste obtained in step (5) and then can it for sterilization. The decoction is sealed and stored in 100 mL / bottle, and taken in the morning on an empty stomach, 20 mL at a time, and a course of treatment is 3 months.

[0093] Example 4

[0094] The antidepressant Chinese medicine ointment of this embodiment has the following raw materials in parts by mass:

[0095] Cooked aconite 55g, dried ginger 55g, roasted licorice root 85g, cinnamon twig 85g, white peony root 85g, oyster 290g, processed cornus fruit 85g, black plum 40g, poria 145g, spiny jujube seed 145g, jujube 85g, Chinese yam 145g, Morinda officinalis 55g, Ophiopogon japonicus 85g, Amomum villosum 25g, agarwood 25g, cinnamon 25g, Polygonatum 115g, tortoise shell glue 10g, donkey-hide glue 10g, deer antler glue 10g.

[0096] The preparation method is the same as that of Example 3.

[0097] Example 5

[0098] The antidepressant Chinese medicine ointment of this embodiment has the following raw materials in parts by mass:

[0099] Cooked aconite 65g, dried ginger 65g, roasted licorice root 95g, cinnamon twig 95g, white peony root 95g, oyster 310g, processed cornus fruit 95g, black plum 55g, poria 155g, spiny jujube seed 155g, jujube 95g, Chinese yam 155g, Morinda officinalis 65g, Ophiopogon japonicus 95g, Amomum villosum 35g, agarwood 35g, cinnamon bark 35g, Polygonatum 125g, tortoise shell glue 20g, donkey-hide glue 20g, deer antler glue 20g.

[0100] The preparation method is the same as that of Example 3.

[0101] In order to explore the therapeutic effect of the antidepressant Chinese medicine composition of the present invention on depression, the antidepressant Chinese medicine ointment prepared in Examples 2 and 3 was used to conduct the following animal experiments.

[0102] Animal Experiment 1

[0103] 1. Experimental Materials

[0104] 1.1 Experimental animals: 60 SPF-grade ICR mice, 3-4 weeks old, weighing 18-22 g, provided by Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., Animal Production License No. SCXK(Zhejiang)2019-0001, Animal Use License No.: SYXK(Sichuan)2018-100.

[0105] 1.2 Experimental instruments: chemiluminescence imaging system (ChemiScope6100, Shanghai Qinxiang Scientific Instrument Co., Ltd.); spectrometer (BioTekμQuant, USA); transfer tank and components (Bio-Rad Laboratories Mini Trans-Blot Cell); high-speed refrigerated centrifuge (SCILOGEX D3024R, USA); high-speed cryogenic tissue grinder (servicebio KZ-III-F); rotary slicer (Leica-2016, Leica, Germany); digital slice scanner (Pannoramic 250, 3DHISTECH).

[0106] 1.3 Experimental drugs:

[0107] The antidepressant Chinese medicinal ointment prepared in Example 2.

[0108] Imipramine hydrochloride, batch number AF22031806, specification 20 mg / bottle, provided by Chengdu Aifa Biotechnology Co., Ltd.

[0109] 1.4 Reagents: Corticosterone (Batch No. AFBH0905), provided by Chengdu Aifa Biotechnology Co., Ltd.; Toluidine blue dye (Batch No. CR2102004), provided by Wuhan Sewell Biotechnology Co., Ltd.; BDNF, Trkb, CREB, p-Akt, Akt antibodies, provided by abcam-Abcam (Shanghai) Trading Co., Ltd., UK; GAPDH (Batch No. Mab5465), provided by Hangzhou Lianke Biotechnology Co., Ltd.; BCA protein concentration determination kit (Batch No. AR0146), provided by Wuhan Boster Bioengineering Co., Ltd.

[0110] 2. Experimental Methods

[0111] 2.1 Animal grouping: 60 SPF-level ICR mice, male, weighing 18-22 g, were adaptively fed for 5-7 days and then divided into 6 groups using a random number table, with 10 mice in each group; they were divided into a normal control group, a model group, a low-dose Chinese medicine ointment group, a medium-dose Chinese medicine ointment group, a high-dose Chinese medicine ointment group, and a positive drug group.

[0112] 2.2 Establishment of animal model and drug administration: Except for the normal control group, which did not receive other treatment and maintained normal drinking water and food intake, the mice in other groups were subcutaneously injected with 0.01 g / kg of corticosterone to establish the model, once a day for a total of 28 days. Model establishment and drug administration were carried out simultaneously.

[0113] The normal control group and the model group were gavaged with an equal amount of normal saline, and the positive drug group and the Chinese medicine ointment groups of various doses were gavaged with drugs according to the body weight standard. The specific dosages were:

[0114] Low-dose Chinese medicine ointment group: the antidepressant Chinese medicine ointment prepared in Example 2, with a dosage of 4.10 g / kg;

[0115] Medium-dose Chinese medicine ointment group: the antidepressant Chinese medicine ointment prepared in Example 2, with a dosage of 12.3 g / kg;

[0116] High-dose Chinese medicine ointment group: the antidepressant Chinese medicine ointment prepared in Example 2, with a dosage of 20.5 g / kg;

[0117] Positive drug group: imipramine, the dosage was 0.02 g / kg.

[0118] Dosing volume 10mL·kg -1 , once a day, recording feed consumption, body weight changes, mouse status, etc., and continuous gavage administration for 28 days.

[0119] 2.3 Body weight detection: During the adaptation period, modeling and 0-4 weeks after administration, the body weight changes of each group of mice were recorded under the same breeding environment conditions.

[0120] 2.4 Sucrose preference test (SPT): Before the experiment, mice were subjected to adaptive training for 3 days, that is, two bottles were placed in the cage, one bottle contained 1wt% sucrose water, and the other bottle contained regular drinking water. The positions of the two bottles were swapped every day (from left to right, from right to left). After the adaptive training, the mice were fasted and deprived of water for 24 hours. Prepare 1 bottle of regular drinking water and 1 bottle of 1wt% sucrose water in the mouse cage. The experiment was terminated after 1 hour, and the mice's intake of drinking water and sucrose water was recorded. Result determination: Sucrose preference value (%) = [sucrose water intake / (sucrose water intake + pure water intake)] × 100%.

[0121] 2.5 Forced swimming test (FST): The mice were pre-tested 1 day before the test. The mice were placed in a transparent cylinder (25 cm high, 15 cm in diameter, water temperature 25 ± 2 ° C, water depth 10 cm) for a predicted duration of 12 minutes. They were then taken out, dried and placed in a cage. On the day of the test, the mice were placed in the same environment again for a test duration of 6 minutes. Result determination: The last 4 minutes of the test for each mouse were recorded, and the time the mouse remained in the "immobile state" in the water was recorded.

[0122] 2.6 Novelty Suppression of Food Intake (NSF): The mice were fasted for 24 hours before the test. During the test, ordinary mouse food pellets were placed on a white paper platform in the center of the cage. Each mouse was placed in a corner of the box and allowed to explore for up to 5 minutes. If the mouse had not eaten at the end of the timer, it was calculated as 5 minutes, and the time when the mouse first ate the food pellets was recorded as the latency. The latency period was not counted when the mouse smelled or carried the food. The latency period was only recorded when the mouse started to gnaw on the food.

[0123] 2.7 Nissl staining analysis of brain tissue: After the behavioral test, the mice were anesthetized with 4% chloral hydrate, and the brain tissues were quickly removed on ice and fixed in 4% paraformaldehyde for 15 min. Paraffin sections were made after dehydration, embedding, and slicing, and then placed in Nissl staining solution. The reaction was terminated when the Nissl bodies became clear, and the changes of Nissl bodies were observed and photographed under a microscope.

[0124] 2.8 Western blot method to detect brain tissue protein expression: The brain tissue of the hippocampus region of mice was separated and placed in a 2 mL grinding tube, and then 3 mm steel beads and RIPA lysis solution (by mass ratio, sample: lysis solution = 1:10) were added to each tube, and placed in a high-speed low-temperature tissue grinder (temperature -20°C, grind 4 times, each time for 60 s); taken out and placed in a 4°C refrigerator for 30 minutes for lysis, taken out after 30 minutes and placed in a centrifuge (4°C, 12000 rpm for 10 minutes); after centrifugation, the supernatant was taken and the protein concentration was determined using a BCA protein concentration assay kit. 30 μg of protein sample was taken, electrophoresed at a stable voltage of 100 V, and the protein was transferred to PVDF membrane, and the buffer (Note: the buffer is 5% skim milk diluted with TBST Buffer, provided by Wuhan Google Biotechnology Co., Ltd.) was blocked on a shaker at room temperature for 2 hours, and primary antibodies BDNF (1:1000), Trkb (1:1000), p-Akt (1:1000), Akt (1:2000), CREB (1:1000) were added. The membrane was washed with Tris-HCl buffer solution for 10 min × 3 times, and horseradish peroxidase-labeled secondary antibody (1:10000) (Note: the secondary antibody is HRP-labeled goat anti-rabbit) was added, reacted at 37°C for 1 hour, and the membrane was washed for 10 min × 3 times. After exposure, it was developed and fixed, and the band absorbance was measured using ChemiScope6100, and GAPDH was selected as the internal reference.

[0125] 2.9 Statistical analysis: Statistical analysis was performed using Graph Pad Prism 9.0.0 (86) and presented as mean ± standard deviation. The differences among the groups were analyzed by one-way analysis of variance (ANOVA) using SPSS statistical software. The differences among the groups were considered significant when P < 0.05.

[0126] 3. Experimental results

[0127] 3.1 Effects on body weight of mice with corticosterone-induced depression model

[0128] The body weight of mice in each group was measured as follows Figure 7 As shown, from Figure 7 It can be seen that after administration, the body weight of mice in the model group was significantly reduced on the 28th day compared with the normal control group (P < 0.05); compared with the model group, the body weight of mice in the positive drug group and each dose of Chinese medicine ointment group was significantly increased (P < 0.05). From the above results, it can be seen that the Chinese medicine composition of the present invention can increase the body weight of depression model mice and improve the growth retardation of depression mice.

[0129] 3.2 Effects on sucrose preference in mice with corticosterone-induced depression

[0130] The results of sucrose preference test of each group of mice are shown in Table 2:

[0131] Table 2 Results of sucrose preference test for mice in each group

[0132] Group Sucrose preference value / (%) Normal control group 69.4±1.4 Model Group <![CDATA[60.6±2.2 # ]]> Low-dose Chinese medicine ointment group <![CDATA[82.5±0.5 * ]]> Medium dose Chinese medicine ointment group <![CDATA[69.0±0.9 * ]]> High-dose Chinese medicine ointment group <![CDATA[76.6±1.1 * ]]> Positive drug group 63.5±2.7

[0133] Note: In the table, # indicates P < 0.05 compared with the normal control group; * Indicates P < 0.05 compared with the model group.

[0134] As can be seen from Table 2, the sucrose preference value of the model group mice was significantly lower than that of the normal control group, and the difference was significant (P<0.05), indicating that the depression mouse model was successfully established. The sucrose preference values ​​of the mice in the low, medium and high doses of Chinese medicine ointment groups were significantly higher than those in the model group, and the difference was significant (P<0.05 or P<0.01), and the effects of the low, medium and high doses of Chinese medicine ointment groups were better than those of the positive drug group, among which the low dose of Chinese medicine ointment group had the best effect.

[0135] 3.3 Effects of forced swimming on corticosterone-induced depression model mice

[0136] The results of the swimming immobility time of mice in each group are shown in Table 3:

[0137] Table 3 Results of the swimming immobility time of mice in each group

[0138] Group Swimming immobility time / (s) Normal control group 118.2±6.3 Model Group <![CDATA[172.4±14.0 # ]]> Low-dose Chinese medicine ointment group 115.4±3.3* Medium dose Chinese medicine ointment group 110.6±2.9* High-dose Chinese medicine ointment group 92.0±1.6* Positive drug group 113.4±5.1*

[0139] Note: In the table, # indicates P < 0.05 compared with the normal control group; * Indicates P < 0.05 compared with the model group.

[0140] As can be seen from Table 3, compared with the normal control group mice, the immobility time of the model group mice in the water was significantly increased, and the difference was significant (P<0.05), indicating that the depression mouse model was successfully established. The immobility time of the positive drug group and the low, medium and high doses of Chinese medicine ointment groups in the water was significantly lower than that of the model group, and the difference was significant (P<0.05). Among them, the effects of the medium and high doses of Chinese medicine ointment groups were better than those of the positive drug group, and the high dose of Chinese medicine ointment group had the best effect.

[0141] 3.4 Effects of novelty suppression of food intake in the corticosterone-induced depression mouse model

[0142] The results of the feeding latency test of each group of mice are shown in Table 4:

[0143] Table 4 Results of feeding latency test of mice in each group

[0144] Group Feeding latency / (s) Normal control group 151.8±7 Model Group <![CDATA[267.8±33 # ]]> Low-dose Chinese medicine ointment group 158.8±25* Medium dose Chinese medicine ointment group 212.4±54* High-dose Chinese medicine ointment group 150.6±24* Positive drug group 176.8±48*

[0145] Note: In the table, # indicates P < 0.05 compared with the normal control group; * Indicates P < 0.05 compared with the model group.

[0146] As can be seen from Table 4, compared with the normal control group, the feeding latency of the model group mice on the 28th day was significantly increased (P < 0.05); compared with the model group, the feeding latency of the mice in the low, medium and high doses of Chinese medicine ointment groups and the positive drug group was significantly reduced (P < 0.05). From the above results, it can be seen that the Chinese medicine composition of the present invention can reduce the feeding latency of depression mice and improve the feeding state of depression mice.

[0147] 3.5 Nissl staining results of brain tissue

[0148] The results of the determination of hippocampal neurons in each group of mice are shown in Figure 8 As shown. Figure 8 It can be seen that compared with the normal control group, the neurons in the model group were abnormal in morphology, with lighter staining, some neurons were atrophied and deformed, the matrix was looser, and the Nissl bodies were significantly reduced. Compared with the model group, the number of neurons in the low, medium, and high doses of Chinese medicine ointment groups and the positive drug group was large and dense, and the morphology was full, indicating that the Chinese medicine composition of the present invention showed a protective effect on the neuroplasticity of depression model mice.

[0149] 3.6 Western blot protein expression analysis results

[0150] The expression of Trkb, CREB, BDNF and p-Akt proteins in the hippocampus tissue of each group of mice is shown in the figure. Fig. 9 The results of the determination of the expression of related proteins in mouse hippocampus tissue are shown in Table 5.

[0151] Table 5 Results of determination of related protein expression in mouse hippocampus tissue

[0152] Group BDNF Trkb CREB p-Akt / Akt Normal control group 0.95±0.093 0.68±0.006 0.54±0.035 1.99±0.41 Model Group <![CDATA[0.34±0.090 # ]]> <![CDATA[0.21±0.035 # ]]> <![CDATA[0.08±0.015 # ]]> <![CDATA[0.33±0.08 # ]]> Low-dose Chinese medicine ointment group 0.64±0.158* 0.44±0.036* 0.25±0.049* 0.89±0.24 Medium dose Chinese medicine ointment group 0.49±0.082* 0.53±0.035* 0.32±0.057* 1.46±0.50* High-dose Chinese medicine ointment group 0.62±0.103* 0.6±0.030* 0.46±0.076** 1.97±0.45** Positive drug group 0.73±0.032* 0.69±0.021* 0.55±0.035** 2.47±0.33**

[0153] Note: In the table, # indicates P < 0.05 compared with the normal control group; * Compared with the model group, P < 0.05, ** Indicates P < 0.01 compared with the model group.

[0154] from Fig. 9 As can be seen from Table 5, compared with the normal control group, the expression levels of BDNF, Trkb, CREB and p-Akt / Akt in the model group were significantly decreased (P < 0.05); compared with the model group, the expression levels of BDNF, Trkb, CREB and p-Akt in the low, medium and high doses of Chinese medicine ointment groups were significantly increased (P < 0.05).

[0155] In summary, the Chinese medicine composition of the present invention may exert its anti-depressant effect by affecting the BDNF / Trkb signaling pathway and downstream related proteins in depression model mice through the active ingredients contained therein, such as morroniside, loganin, paeoniflorin, gingerol (i.e., 6-gingerol), and spinosum. Its mechanism may be related to upregulating the expression of Trkb, p-Akt, BDNF and CREB proteins and improving the plasticity of hippocampal neurons.

[0156] Animal Experiment II

[0157] 1. Experimental Materials

[0158] 1.1 Experimental animals: 84 SD rats, half male and half female, weighing 220±20 g, provided by Beijing Huafukang Biotechnology Co., Ltd., Animal Production License No.: SCXK(Beijing)2019-0008. Animal Use License No.: SYXK(Sichuan)2018-100, Ethics Review Approval No.: SYLL(2023)-028.

[0159] 1.2 Experimental instruments and materials: The main experimental instruments and materials are shown in Table 6.

[0160] Table 6 Experimental instruments and materials

[0161]

[0162]

[0163]

[0164]

[0165] 1.3 Experimental drugs:

[0166] The antidepressant Chinese medicinal ointment prepared in Example 3.

[0167] Fluoxetine, code number mrbk2101, specification 100 mg / bottle, provided by Chengdu Aifa Biotechnology Co., Ltd.

[0168] ANA-12 (TrkB antagonist), number AFBJ1075, was provided by Chengdu Aifa Biotechnology Co., Ltd.

[0169] 2. Experimental Methods

[0170] 2.1 Animal grouping: 84 SD rats, half male and half female, weighing 220±20 g, were adaptively fed for 5-7 days and then divided into 7 groups using a random number table, with 12 rats in each group; the 7 groups were normal control group (referred to as control group), model group (referred to as CUMS group), medium-dose Chinese medicine ointment group (referred to as 8.4 g / kg SAO group), high-dose Chinese medicine ointment group (referred to as 14 g / kg SAO group), fluoxetine positive drug control group (referred to as fluoxetine group), high-dose Chinese medicine ointment + ANA-12 group (referred to as 14 g / kg SAO + ANA-12 group) and ANA-12 group.

[0171] 2.2 Establishment of CUMS rat depression model and drug administration:

[0172] The rats were simulated with various chronic stresses that humans face in daily life: (1) 12 hours of light-dark reversal; (2) 12 hours of fasting; (3) 12 hours of water deprivation; (4) 12 hours of cage tilt at 45 degrees; (5) 5 minutes of swimming in 40°C hot water; (6) 24 hours of wet bedding; (7) 24 hours of mothball stimulation. Except for the normal control group, the remaining rats were subjected to the above chronic unpredictable stimulation for 35 consecutive days. Each stress stimulus was discontinuous and irregular. Modeling and drug administration were performed simultaneously.

[0173] The control group and the model group were gavaged with an equal amount of normal saline, and the other groups were gavaged with drugs according to body weight. The specific dosages were:

[0174] 8.4 g / kg SAO group: the antidepressant Chinese medicine ointment prepared in Example 3, the dosage was 8.4 g / kg / d;

[0175] 14 g / kg SAO group: the antidepressant Chinese medicine ointment prepared in Example 3, with a dosage of 14 g / kg / d;

[0176] Fluoxetine group: Fluoxetine, dosage 0.02 g / kg / d;

[0177] 14g / kg SAO+ANA-12 group: The antidepressant Chinese medicine ointment prepared in Example 3 and ANA-12 were administered simultaneously, wherein the dosage of the Chinese medicine ointment was 14g / kg / d, and the dosage of ANA-12 was 0.0005g / kg;

[0178] ANA-12 group: ANA-12, dosage 0.0005 g / kg.

[0179] Dosing volume 10mL·kg -1 , once a day, by oral gavage for 35 consecutive days.

[0180] 2.3 Sucrose preference test (SPT): Before the experiment, rats were subjected to adaptive training for 3 days, that is, two bottles were placed in the cage, one bottle contained 1wt% sucrose water, and the other bottle contained regular drinking water. The positions of the two bottles were swapped every day (from left to right, from right to left). After the adaptive training, the rats were fasted and deprived of water for 24 hours. Prepare 1 bottle of regular drinking water and 1 bottle of 1wt% sucrose water in the mouse cage. The experiment was terminated after 1 hour, and the mice's intake of drinking water and sucrose water was recorded. Result determination: Sucrose preference value (%) = [sucrose water intake / (sucrose water intake + pure water intake)] × 100%.

[0181] 2.4 Forced swimming test (FST): A pre-experiment was conducted on rats 1 day before the test: the rats were placed in a transparent cylinder (25 cm high, 25 cm in diameter, water temperature 25 ± 2 °C, water depth 10 cm) for a predicted duration of 12 min, then taken out, dried and placed in a cage. On the day of the test, the rats were placed in the same environment again for a test duration of 6 min. Result determination: The last 4 min of the test for each rat was recorded, and the time the rat remained in the "immobile state" in the water was recorded.

[0182] 2.5 Novelty Suppressed Feeding Test (NSF): NSF is designed to evaluate the anxiolytic and / or antidepressant effects of chronic antidepressant drug treatment. The tester consists of a 50×50×20cm plastic box. During the test, after 48 hours of fasting and water deprivation, a weighed ordinary rat food pellet was placed on a white paper platform in the center of the plastic box. Each rat was placed in a corner of the box, and after the rat adapted for 1 minute, it was allowed to explore for up to 5 minutes. The test ended when the rat chewed a portion of the food. The amount of food consumed in the cage was taken as the amount of food consumed within 5 minutes. If there was no food at the end of the timing, it was calculated as 5 minutes, and the time when the rat first ate the food pellet was recorded as the latency.

[0183] 2.6 Analysis of brain tissue Nissl staining: After fasting and water deprivation for 24 hours after the behavioral experiment, three rats were killed, and the brain tissue was quickly peeled off on ice and fixed in 4% paraformaldehyde for 15 minutes. After dehydration, embedding, and slicing, paraffin sections were made, Nissl staining was performed, and the conditions of hippocampal neurons in each group were observed and photographed. The remaining rats were removed from the brain on ice and the hippocampus was quickly isolated, quickly frozen with liquid nitrogen, and stored at -80°C for later use.

[0184] 2.7 Western blot method to detect brain tissue protein expression: 30μg protein sample was placed at a stable voltage of 100V to start electrophoresis and transfer the protein to the PVDF membrane. The sealing buffer (Note: the buffer is 5% skim milk diluted with TBSTBuffer, provided by Wuhan Google Biotechnology Co., Ltd.) was placed on a room temperature shaking table for two hours. BDNF (1:1000), Trkb (1:1000) and CREB (1:1000) antibodies were added. Rinse with Tris-hcl buffered saline for 10min×3 times, add horseradish peroxidase-labeled secondary antibody (1:10000) (Note: the secondary antibody is HRP-labeled goat anti-rabbit), react at 37℃ for 1 hour, rinse for 10 minutes, expose 3 times, and then develop and fix. The absorbance of the strip was measured using Tianneng GIS chassis control software V2.0, and β-actin was selected as the internal reference.

[0185] 2.8 Statistical analysis: Statistical analysis was performed using the statistical software Graph Pad Prism 9.0.0 (86). All group data were analyzed using one-way analysis of variance (ANOVA) and expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.

[0186] 3. Experimental results

[0187] 3.1 Behavioral test results: 35 days after the successful establishment of the CUMS rat model, SPT, FST and NSF behavioral tests were performed. The test results are as follows: Fig.10 shown.

[0188] from Fig.10 It can be seen that compared with the control group, the model group can significantly reduce the rats' preference for sugar water, while intragastric administration of 0.02g / kg fluoxetine and 8.4g / kg and 14g / kg Chinese medicine ointment can significantly reverse the decline in the rats' preference for sugar water ( Fig.10 A). In the rat NSF and FST, compared with the control group, the immobility time of the rats in the CUMS model group was significantly prolonged (P<0.05), while the immobility time of the rats in the fluoxetine group and the Chinese medicine ointment groups of various doses was significantly shortened (P<0.05) ( Fig.10 B and Fig.10 C).

[0189] 3.2 Nissl staining results of brain tissue: The results of Nissl staining of brain tissue are as follows Fig.11 As shown. Fig.11 It can be seen that compared with the normal control group, the neuronal cells in the model group were sparsely arranged, the number was small, some of the nuclei were condensed, and the Nissl bodies decreased or disappeared. After the administration of fluoxetine and medium and high doses of Chinese medicine ointment, the arrangement of neuronal cells became regular, the number of neuronal cells and Nissl bodies increased, and the cell morphology was basically restored.

[0190] 3.3 Western blot protein expression analysis results:

[0191] Western blot protein expression analysis results Fig.12 , 13 As shown, Fig.12 The expression of BDNF, p-Trkb, p-AKT and p-CREB proteins in the hippocampus tissue of rats in each group is shown in the figure. Fig.13 Comparison of BDNF, p-TrkB, p-AKT and p-CREB protein expression in hippocampal tissue of rats in each group.

[0192] from Fig.12 , 13 It can be seen that compared with the control group, the expression of BDNF protein and the downstream p-TrkB / TrkB, p-AKT / AKT, and p-CREB / CREB ratios in the hippocampus of the model group rats were significantly reduced after 35 days of chronic stress (P<0.05). Compared with the model group, each drug-treated group significantly increased the expression of BDNF in the hippocampus of rats (P<0.05). At the same time, continuous administration of Chinese medicine ointment significantly inhibited the decrease of the BDNF downstream p-TrkB / TrkB, p-AKT / AKT, and p-CREB / CREB ratios in the hippocampus of rats (P<0.05), and the effect of Chinese medicine ointment in the hippocampus was blocked by the intervention of antagonist ANA-12 (P<0.05), suggesting that the BDNF-TrkB signaling pathway may be involved in its antidepressant mechanism. Since BDNF is an important neuromodulator of synaptic transmission and long-term potentiation between the hippocampus and other brain regions, it plays an important role in maintaining hippocampal neuron function, promoting neuron regeneration, and inhibiting neuron apoptosis. TrkB receptor has a high affinity with BDNF. After specific binding with BDNF, it can simultaneously activate multiple signaling pathways such as downstream MAPK, PI3K / Akt, and cAMP, and participate in and mediate the occurrence and development of depression. At the same time, CREB has been proven to be related to the plasticity of the nervous system. CREB is further activated, which can promote the release of neurotransmitters in the synaptic space and interfere with the proliferation and differentiation of nerve cells. Therefore, it shows that the anti-depressant mechanism of Chinese medicine ointment may be closely related to the influence on neural plasticity.

[0193] In summary, the Chinese medicine composition of the present invention can significantly increase the sugar water preference of rats in SPT, shorten the immobility time of rats in FST and NSF, and reduce the pathological damage of depression caused by CUMS to the brain tissue of rats, indicating that it has a good preventive and therapeutic effect on depression model rats, and its antidepressant mechanism may be closely related to the impact on neural plasticity.

[0194] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An antidepressant Chinese medicine composition, characterized in that: The raw material composition is as follows by mass: 55-65 parts of aconite, 55-65 parts of dried ginger, 85-95 parts of liquorice, 85-95 parts of cinnamon twig, 85-95 parts of white peony root, 290-310 parts of oyster, 85-95 parts of cornus fruit, 40-55 parts of black plum, 145-155 parts of poria, 145-155 parts of spiny jujube seeds, 85-95 parts of jujube, 145-155 parts of yam, 55-65 parts of Morinda officinalis, 85-95 parts of ophiopogon, 25-35 parts of amomum, 25-35 parts of agarwood, 25-35 parts of cinnamon, 115-125 parts of polygonatum, 10-20 parts of tortoise shell glue, 10-20 parts of donkey-hide gelatin, and 10-20 parts of antler glue.

2. The Chinese medicine composition according to claim 1, characterized in that: The raw material composition is as follows by mass: 60 parts of aconite, 60 parts of dried ginger, 90 parts of liquorice, 90 parts of cinnamon twig, 90 parts of white peony root, 300 parts of oyster, 90 parts of cornus, 45 parts of ebony, 150 parts of poria, 150 parts of spiny jujube seeds, 90 parts of jujube, 150 parts of yam, 60 parts of Morinda officinalis, 90 parts of Ophiopogon japonicus, 30 parts of Amomum villosum, 30 parts of agarwood, 30 parts of cinnamon, 120 parts of polygonatum, 15 parts of tortoise shell glue, 15 parts of donkey-hide gelatin, and 15 parts of antler glue.

3. The Chinese medicine composition according to claim 1 or 2, characterized in that: The aconite root is cooked aconite root, the licorice root is roasted licorice root, and the cornus fruit is processed cornus fruit.

4. The Chinese medicine composition according to claim 1 or 2, characterized in that: Also included are pharmaceutically acceptable excipients.

5. The Chinese medicine composition according to claim 4, characterized in that: The auxiliary material is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant, and a lubricant.

6. The Chinese medicine composition according to claim 1 or 2, characterized in that: The dosage form of the Chinese medicine composition is ointment, decoction, granules, powder, tablet, pill or capsule.

7. The method for preparing the Chinese medicine composition according to any one of claims 1 to 6, characterized in that: When the dosage form of the Chinese medicine composition is an ointment, the preparation method thereof comprises the following steps: (1) Add the raw medicinal materials except agarwood, tortoise shell glue, donkey-hide gelatin and deer antler glue to 10 times the amount of water, soak for 1 hour, and then decoct twice, each decoction time is continued for 1.5 hours after boiling, and the decoctions obtained from the two decoctions are combined and filtered through an 80-mesh sieve to obtain a decoction liquid; (2) heating the decoction obtained in step (1) at 70-90° C. and concentrating it into a first clear paste having a relative density of 1.30-1.35; (3) Mix tortoise shell glue, donkey-hide gelatin and deer antler glue, add water three times the amount of donkey-hide gelatin, and heat to 60-80°C to melt. After melting, add the mixture to the first clear paste obtained in step (2), stir evenly, and obtain a second clear paste. (4) Passing agarwood through a 120-mesh sieve to obtain agarwood powder, heating the second clear paste obtained in step (3) to 60° C., adding the agarwood powder, and stirring evenly to obtain a mixed medicine; (5) adding medium honey to the mixed drug obtained in step (4) at a mass ratio of 1:1, then heating to 60° C. and stirring evenly to obtain a finished paste with a relative density of 1.43-1.46; (6) The finished paste obtained in step (5) is cooled to room temperature and then canned and sterilized.

8. Use of the Chinese medicine composition according to any one of claims 1 to 6 in the preparation of a medicine for treating depression.

9. The use according to claim 8, characterized in that: The depression is yang deficiency type depression.

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