CN109550035B

CN109550035B - New application of traditional Chinese medicine composition in preparation of antidepressant

Info

Publication number: CN109550035B
Application number: CN201811648411.6A
Authority: CN
Inventors: 高嵩; 徐建; 唐晓桐; 田淋淋; 吴萍; 赵源慧; 徐云
Original assignee: Jilin Xiuzheng Pharmaceutical New Medicine Development Co ltd
Current assignee: Tongyao Pharmaceutical Group Corp
Priority date: 2018-12-30
Filing date: 2018-12-30
Publication date: 2021-08-13
Anticipated expiration: 2038-12-30
Also published as: CN109550035A

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to application of a traditional Chinese medicine composition in preparation of antidepressant medicines, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of immature bitter orange, 4-8 parts of bighead atractylodes rhizome, 4-8 parts of rhizoma pinellinae praeparata, 5-15 parts of coptis chinensis, 3-7 parts of codonopsis pilosula, 2-6 parts of liquorice, 2-6 parts of poria cocos, 4-12 parts of mangnolia officinalis, 2-6 parts of malt and 2-6 parts of dried ginger. Experiments show that the traditional Chinese medicine composition can improve the cell survival rate of a PC12 nerve cell damage model caused by corticosterone, recover the mitochondrial membrane potential, inhibit the expression of apoptosis genes and/or promote the expression of apoptosis inhibiting genes; the composition has the advantages that the immobility time of tail suspension and forced swimming of the mouse are obviously reduced, the content of 5-hydroxytryptamine in serum is increased, the depression-like behavior of the mouse is obviously improved, and the results show that the composition has obvious curative effect on depression.

Description

New application of traditional Chinese medicine composition in preparation of antidepressant

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to a new application of a traditional Chinese medicine composition in preparation of antidepressant medicines.

Background

Depression is a clinical syndrome characterized mainly by emotional disorders such as depressed mood, speech loss, behavioral retardation, autonomy and autonomy. In recent years, with the change of work and living environment, the stress on people is gradually increased, and how to prevent and improve depression becomes a topic of attention.

At present, the western medicine mainly adopts medicaments such as an antidepressant, a tranquilizer, a hypnotic, an antipsychotic and the like for treating the depression, and has the advantages of large toxic and side effects, strong dependence and high price.

The prior art also discloses various traditional Chinese medicine compositions with antidepressant effect, for example, patent CN104173933 discloses a traditional Chinese medicine composition for treating depression, which is composed of 29 medicinal materials of Ziziphora bungeana, tribulus terrestris, poria with hostwood, radix paeoniae alba, safflower, bighead atractylodes rhizome, salvia miltiorrhiza, pinellia ternate, fiveleaf akebia fruit, flos rosae rugosae, schisandra chinensis, peach kernel, epimedium herb, curcuma zedoary, rheum officinale, cortex lycii radicis, fructus psoraleae, caulis spatholobi, mangnolia officinalis, caulis polygoni multiflori, immature bitter orange, medlar, platycodon grandiflorum, desmodium, pericarpium citri reticulatae viride, Japanese ardisia herb, morinda root, platycladi seed and liquorice. Patent CN104306933B discloses a Chinese medicinal composition for treating depression, which is prepared from 16 Chinese medicinal materials including fructus Lycii, radix Angelicae sinensis, caulis Lonicerae, arillus longan, ramulus Cinnamomi, rhizoma Zingiberis, rhizoma Polygonati, herba Taraxaci, radix Scutellariae, rhizoma anemarrhenae, radix Polygalae, radix Glycyrrhizae, fructus Schisandrae Bicoloris, radix rehmanniae, flos Caryophylli, and rhizoma Polygonati Odorati. The traditional Chinese medicine composition has a certain curative effect on depression, but the prescription has more medicinal ingredients and more complex components.

Disclosure of Invention

In view of the above, the invention aims to provide a new application of a traditional Chinese medicine composition in preparation of an antidepressant, and the traditional Chinese medicine composition has fewer medicinal ingredients and a good antidepressant effect.

The invention provides application of a traditional Chinese medicine composition in preparation of an antidepressant, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of immature bitter orange, 4-8 parts of bighead atractylodes rhizome, 4-8 parts of rhizoma pinellinae praeparata, 5-15 parts of coptis chinensis, 3-7 parts of codonopsis pilosula, 2-6 parts of liquorice, 2-6 parts of poria cocos, 4-12 parts of mangnolia officinalis, 2-6 parts of malt and 2-6 parts of dried ginger.

The prior art discloses immature bitter orange pill for relieving distension and fullness, namely Shixiao pill, which is a digestion-promoting agent and comprises the following components: 100g of immature bitter orange (fried), 60g of largehead atractylodes rhizome (fried), 60g of rhizoma pinellinae praeparata, 100g of coptis root, 50g of codonopsis pilosula, 40g of liquorice (prepared), 40g of poria cocos, 80g of mangnolia officinalis (prepared), 40g of malt (fried) and 40g of dried ginger. The formula is from secret book of lan ventricular (Lab, secret book of Lidong Yuanzhou), has the effects of relieving oppression and fullness, and invigorating spleen and stomach, and is clinically used for treating dyspepsia, chronic gastritis, chronic bronchitis, gastrointestinal neurosis and the like.

The invention takes immature bitter orange, largehead atractylodes rhizome, rhizoma pinellinae praeparata, coptis root, pilose asiabell root, liquorice, tuckahoe, officinal magnolia bark, malt and dried ginger as active ingredients of a traditional Chinese medicine composition, and the traditional Chinese medicine composition is used for preparing the anti-melancholia medicine. The experimental result shows that the traditional Chinese medicine composition can improve the cell survival rate of a PC12 nerve cell damage model caused by corticosterone, recover the mitochondrial membrane potential, inhibit the expression of apoptosis genes and/or promote the expression of apoptosis inhibiting genes; the results show that the composition has obvious curative effect on depression and has more obvious curative effect compared with the prior traditional Chinese medicine composition with complex components.

In a specific embodiment, the recovery effect of the traditional Chinese medicine composition on corticosterone-induced PC12 nerve cell injury model apoptosis is investigated by an annexin V-FITC/PI apoptosis double staining kit (BD). The result shows that the traditional Chinese medicine composition can obviously inhibit PC12 cell apoptosis induced by corticosterone, so the invention provides the application of the traditional Chinese medicine composition in preparing the nerve cell apoptosis inhibitor, and the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of immature bitter orange, 4-8 parts of bighead atractylodes rhizome, 4-8 parts of rhizoma pinellinae praeparata, 5-15 parts of coptis chinensis, 3-7 parts of codonopsis pilosula, 2-6 parts of liquorice, 2-6 parts of poria cocos, 4-12 parts of mangnolia officinalis, 2-6 parts of malt and 2-6 parts of dried ginger.

In some embodiments, the neural cell is a PC-12 cell.

In some embodiments, the neural cell apoptosis is corticosterone-induced neural cell apoptosis.

In a specific embodiment, the effect of the traditional Chinese medicine composition on the expression of autophagy and apoptosis-related proteins Atg5, LC3II/I, Atg7, Bax, Bcl2, PARP, p-mTOR and mTOR in a corticosterone-induced PC12 nerve cell injury model is detected by a western blotting method, and the result shows that the traditional Chinese medicine composition promotes the protein expression of Bcl2/Bax, inhibits the expression of PARP (fig. 6) and inhibits corticosterone-induced neuronal mitochondrial apoptosis. At the same time, protein expression of Atg5, LC3II/I and Atg7 was up-regulated (FIG. 8), promoting autophagy. Secondly, the experimental result shows that after corticosterone modeling, the expression of the p-mTOR/mTOR protein is obviously increased, the autophagy is obviously reduced, and after the composition is dried, the expression of the p-mTOR/mTOR protein is obviously reduced (figure 7), and the autophagy is obviously increased. Therefore, the invention also provides application of the traditional Chinese medicine composition in preparing a medicine for inhibiting and/or promoting expression of apoptosis-related protein and/or expression of autophagy-related protein, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of immature bitter orange, 4-8 parts of bighead atractylodes rhizome, 4-8 parts of rhizoma pinellinae praeparata, 5-15 parts of coptis chinensis, 3-7 parts of codonopsis pilosula, 2-6 parts of liquorice, 2-6 parts of poria cocos, 4-12 parts of mangnolia officinalis, 2-6 parts of malt and 2-6 parts of dried ginger.

Wherein the apoptosis-related protein comprises Bax protein, Bcl2 protein and parp protein, and the autophagy-related protein comprises LC3II protein, LC3I protein, Atg7 protein, Atg5 protein, mTOR protein and p-mTOR protein.

In some specific embodiments, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts of immature bitter orange, 6 parts of bighead atractylodes rhizome, 6 parts of rhizoma pinellinae praeparata, 10 parts of coptis chinensis, 5 parts of codonopsis pilosula, 4 parts of liquorice, 4 parts of poria cocos, 8 parts of mangnolia officinalis, 4 parts of malt and 4 parts of dried ginger.

In some embodiments, the method of preparing the traditional Chinese medicine composition comprises: extracting fructus Aurantii Immaturus, cortex Magnolia officinalis, and Atractylodis rhizoma by steam distillation, and collecting volatile oil, water extractive solution and residue respectively; making the volatile oil into clathrate of beta-cyclodextrin-volatile oil, mixing the residue with rhizoma Pinelliae, Coptidis rhizoma, radix Codonopsis, Glycyrrhrizae radix, Poria, fructus Hordei Germinatus and Zingiberis rhizoma, decocting in water for 2 times, filtering, and mixing filtrates; and mixing the volatile oil, the water extract and the filtrate to obtain the traditional Chinese medicine composition.

In some embodiments, the number of times of decocting with water is 2, each for 1.5 hours; the amount of the water is 8-10 times of the total mass of the medicine residues, the pinellia ternate, the coptis chinensis, the codonopsis pilosula, the liquorice, the poria cocos and the malt and dried ginger.

In some embodiments, the steam distillation extraction further comprises a step of soaking in water, wherein the mass of the water is 10 times of the total weight of the immature bitter orange, the magnolia bark and the white atractylodes rhizome, and the soaking time is 1 hour.

Those skilled in the art can add various pharmaceutically acceptable common adjuvants into the effective amount of the Chinese medicinal composition, and make into common preparations such as tablet, pill, granule, capsule, dripping pill, syrup, etc. by conventional methods.

The invention provides a new application of a traditional Chinese medicine composition in preparation of antidepressant drugs, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of immature bitter orange, 4-8 parts of bighead atractylodes rhizome, 4-8 parts of rhizoma pinellinae praeparata, 5-15 parts of coptis chinensis, 3-7 parts of codonopsis pilosula, 2-6 parts of liquorice, 2-6 parts of poria cocos, 4-12 parts of mangnolia officinalis, 2-6 parts of malt and 2-6 parts of dried ginger. Experiments show that the traditional Chinese medicine composition can obviously improve the cell survival rate of a PC12 nerve cell damage model caused by corticosterone, recover the mitochondrial membrane potential, inhibit the expression of apoptosis genes and/or promote the expression of apoptosis inhibiting genes; the mouse tail suspension immobility time is obvious, the forced swimming immobility time is obviously reduced, the content of 5-hydroxytryptamine in serum is obviously increased, the mouse depression-like behavior is effectively improved, and the results show that the composition has obvious curative effect on depression.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a statistical chart showing the survival rate of cells induced by corticosterone molding using the Chinese medicinal composition of example 1;

FIG. 2 is a diagram showing an immunofluorescence analysis of the composition of example 1 for mitochondrial membrane potential damage caused by corticosterone molding;

FIG. 3 is a fluorescent quantitative analysis chart of mitochondrial membrane potential damage caused by corticosterone molding with the Chinese medicinal composition of example 1;

FIG. 4 is a flow cytometry plot showing the effect of the Chinese medicinal composition of example 1 on apoptosis induced by corticosterone modeling;

FIG. 5 is a bar graph showing the effect of the Chinese medicinal composition of example 1 on apoptosis induced by corticosterone molding;

FIG. 6 shows a Western blot of Chinese medicinal composition on corticosterone-induced PC12 nerve cell injury model Bcl2/Bax, PARP protein expression;

FIG. 7 is a Western blot of p-mTOR/mTOR protein expression in a corticosterone-induced PC12 neural cell injury model with a traditional Chinese medicinal composition;

FIG. 8 is a Western blot of the expression of Atg5, LC3II/I and Atg7 proteins in a corticosterone-induced PC12 nerve cell injury model by the traditional Chinese medicine composition.

Detailed Description

The invention discloses a new application of a traditional Chinese medicine composition in preparation of an antidepressant, and a person skilled in the art can realize the application by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

The invention is further illustrated by the following examples:

example 1

Consists of the following components: 100g of immature bitter orange, 80g of mangnolia officinalis, 100g of coptis chinensis, 60g of pinellia ternate, 40g of dried ginger, 40g of malt, 60g of bighead atractylodes rhizome, 40g of poria cocos, 50g of codonopsis pilosula and 40g of liquorice.

The preparation method comprises the following steps: soaking fructus Aurantii Immaturus, cortex Magnolia officinalis, and Atractylodis rhizoma in 10 times of water for 1 hr, extracting by steam distillation for 6 hr, collecting volatile oil, water extractive solution and residue, respectively, and dissolving with equal amount of ethanol; weighing beta-cyclodextrin with 8 times of volatile oil amount to prepare a saturated aqueous solution at 45-55 ℃, adding the volatile oil dissolved in ethanol while stirring, continuously stirring for 3 hours at the temperature of 45-55 ℃, refrigerating overnight, filtering, and airing to obtain a volatile oil beta-cyclodextrin inclusion compound for later use. Collecting the distilled water solution in another container for later use; decocting the residues and rhizoma Coptidis with 10 times and 8 times of water for 1.5 hr twice, filtering, mixing the filtrate with the above water solution,

example 2 Effect on cell survival in corticosterone-induced PC12 neural cell injury model

Purpose of the experiment

The recovery effect of the traditional Chinese medicine composition on the nerve cell injury model at different concentrations is observed by performing immature bitter orange disintegrating granule intervention on the PC12 nerve cell injury model induced by corticosterone.

2 experimental materials and experimental equipment:

2.1 Experimental reagents

PC12 cells (ATCC), imported fetal bovine serum (CLARK), imported horse serum (Life Technology), 96-well cell culture plate (NEST), MTT powder (Sigma), corticosterone (Sigma).

2.2 Experimental drugs

Example 1 Chinese medicinal composition. Dissolved in DMSO respectively to prepare 200mg/mL stock solutions.

2.3 main experimental equipments, see table 1.

TABLE 1 Experimental Equipment

3 Experimental methods

3.1 principle of the experiment

MTT is a yellow compound, a dye that accepts hydrogen ions, and acts on the respiratory chain in mitochondria of living cells, and the tetrazolium ring is cleaved by succinate dehydrogenase and cytochrome C to produce blue formazan crystals, the amount of which is only proportional to the number of living cells (i.e., the absence of succinate dehydrogenase in dead cells does not reduce MTT). The formazan crystals formed by the reduction were dissolved in MTT solution containing 50% N, N-dimethylformamide and 20% sodium dodecylbenzenesulfonate (pH 4.7), and the OD value at 490nm was measured by a microplate reader to reflect the number of living cells.

3.2 Experimental procedures:

3.2.1 seeding of cells: PC12 cells were made into single cell suspensions in culture medium containing 7.5% fetal bovine serum + 2.5% horse serum, and inoculated into 96-well plates at 5000 cells per well, 100ul per well volume.

3.2.2 culturing cells: the traditional Chinese medicine composition of the example 1 is added after 24h of culture under the same general culture conditions, and the concentration is 25-200 mug/mL respectively.

3.2.3 color generation: after 48h incubation, 10ul of MTT solution (5mg/ml in PBS) was added to each well. Incubation was continued for 4 hours, the culture was terminated, and the culture supernatant in the wells was carefully aspirated, 150ul DMSO was added to each well, and the mixture was shaken for 10 minutes to allow the crystals to be fully thawed.

3.2.4 color comparison: the 490nm wavelength is selected, the light absorption value of each pore is measured on an enzyme linked immunosorbent instrument, and the result is recorded.

The results of the experiment are shown in FIG. 1.

MTT results show that the traditional Chinese medicine composition provided by the invention can obviously improve the cell survival rate of a PC12 nerve cell damage model caused by corticosterone, and has dose dependence.

Example 3 Effect on corticosterone-induced PC12 neuronal cell injury model mitochondrial Membrane potential

Purpose of the experiment

The traditional Chinese medicine composition is interfered in a corticosterone-induced PC12 nerve cell damage model, and the recovery effect of the traditional Chinese medicine composition on the mitochondrial membrane potential of the nerve cell damage model is observed.

2 experimental materials and experimental equipment:

2.1 Experimental reagents

Mitochondrial membrane potential probe kit (C2006, cloudy day).

2.2 Experimental drugs

Example 1A Chinese medicinal composition, dissolved in DMSO, is prepared as a 200mg/mL stock solution.

2.3 the main experimental equipment is the Cytation 5 cell phase system (Bio Tek).

3 Experimental methods

3.1 principle of the experiment

The breakdown of mitochondrial transmembrane potential is often accompanied in the course of apoptosis, which is widely recognized as one of the earliest events in the apoptotic process. There are many methods for detecting mitochondrial membrane potential, and JC-1 flow assay is a relatively common method. JC-1 has both monomer and multimer states, where green fluorescence is detectable in the presence of monomer at low concentrations, red light is detectable in the presence of multimer at high concentrations of the FL-1 channel (and FITC co-channel) in flow-based assays, and FL-2 channel (and PE co-channel) in flow-based assays. A reversible transition between monomer and multimer is formed as a result of the change in JC-1 concentration.

In normal cells, when the membrane potential is normal, JC-1 enters mitochondria through the polarity of mitochondrial membranes and forms polymers emitting red fluorescence due to the increase of the concentration, and apoptotic cells depolarize the mitochondrial transmembrane potential, and JC-1 is released from mitochondria, the concentration is reduced and the monomer emitting green fluorescence is reversed. Thus, the change in mitochondrial membrane potential can be detected qualitatively (shift in cell population) and quantitatively (fluorescence intensity of cell population) by detecting green and red fluorescence.

3.2 Experimental procedures:

3.2.1 preparing JC-1 dyeing working solution: an appropriate amount of JC-1 (200X) was taken and JC-1 was diluted at a rate of adding 8mL of ultrapure water to 50. mu.L of JC-1 (200X). Shaking vigorously to dissolve and mix JC-1 evenly. Then adding 2ml JC-1 staining buffer solution (5 x) and mixing uniformly to obtain the JC-1 staining working solution. PC12 cells were plated in 6-well plates and 24 hours later 25, 50, 100, 200ug/ml of the Chinese medicinal composition of example 1 was added, followed by culturing for 48 hours, and the cells were collected and examined as follows.

3.2.2 resuspend the cells in 0.5mL of cell culture medium, which may contain serum and phenol red. Adding 0.5mlJC-1 dyeing working solution, reversing for several times and mixing evenly. The cells were incubated at 37 ℃ for 20 minutes in a cell incubator.

3.2.3 during the incubation, an appropriate amount of JC-1 staining buffer (1X) was prepared by adding 4mL of distilled water to 1mL of JC-1 staining buffer (5X) and placed in an ice bath.

After incubation at 3.2.437 ℃ was completed, the cells were pelleted by centrifugation at 600g and 4 ℃ for 5 minutes. Discard the supernatant and take care not to aspirate the cells as much as possible. Wash 2 times with JC-1 staining buffer (1X) and analyze with fluorescence microscopy.

The results of the experiment are shown in fig. 2 and 3.

The results show that after corticosterone molding, the mitochondrial membrane potential is obviously reduced, and the cells are obviously apoptotic. After the treatment of the traditional Chinese medicine composition in the embodiment 1, the mitochondrial membrane potential is obviously recovered, the apoptosis is reduced, and the result shows that the traditional Chinese medicine composition has the anti-apoptosis effect.

Example 4 Effect on corticosterone-induced apoptosis in PC12 neural cell injury model

Purpose of the experiment

The recovery effect of the composition on the apoptosis of the nerve cell injury model is observed by carrying out drug intervention on a corticosterone-induced PC12 nerve cell injury model.

2 experimental materials and experimental equipment:

2.1 Experimental reagents

AnnexinV-FITC/PI cell apoptosis double staining kit (BD).

2.2 Experimental drugs: example 1 Chinese medicinal composition.

2.3 main experimental equipments: flow cytometer (BD)

3 Experimental methods

3.1 principle of the experiment

In normal cells, phosphatidylserine is distributed only inside the lipid bilayer of the cell membrane, and the cell undergoes the earliest apoptosis, and the membrane Phosphatidylserine (PS) turns from the inside of the lipid membrane to the outside, which is earlier than the apoptosis phenomena such as cell shrinkage, chromatin condensation, fragmentation of DNA, and increased permeability of the cell membrane. Annexin v is a phospholipid-binding protein with high affinity for phosphatidylserine, and thus can bind to the cell membrane of early apoptotic cells through phosphatidylserine exposed outside the cell. Therefore, annexin V is taken as one of sensitive indicators for detecting early apoptosis of cells. Propidium Iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane, but in cells in the middle and late stages of apoptosis and dead cells, PI can penetrate the cell membrane to red the nucleus due to increased permeability of the cell membrane. Therefore, by matching Annexin V with PI, cells at different apoptosis stages can be distinguished.

3.2 Experimental procedures:

3.2.10.25% pancreatin digestion, collecting cells, washing with PBS for 1 time, centrifuging at 1000rpm for 5 minutes, and collecting cells;

3.2.2 Add 300 u L of 1 x Binding Buffer suspension cells;

3.2.3Annexin V-FITC labeling: adding 5 mu L Annexin V-FITC, mixing uniformly, keeping out of the sun, and incubating for 15 minutes at room temperature;

3.2.4PI labelling: 5 μ L of PI was added for staining 5 minutes before loading.

3.2.5 was supplemented with 200. mu.L of 1 XBinding Buffer for detection by flow cytometry analysis.

4 results of the experiment, see table 2 and fig. 4 to 5.

TABLE 2 detection of apoptosis Rate

As can be seen from the results of table 2 and fig. 4 to 5, compared with the blank group (Ctrl), the corticosterone-induced PC12 neural cell injury model group (Cort) significantly increases the early apoptosis and the late apoptosis of PC12 cells, and the traditional Chinese medicine composition of the present invention at different concentrations can significantly reduce the corticosterone-induced PC12 apoptosis rate and inhibit the corticosterone-induced PC12 apoptosis.

Experiment 5 protein immunoblotting method for detecting influence of traditional Chinese medicine composition on autophagy and apoptosis-related protein expression in corticosterone-induced PC12 nerve cell injury model

Purpose of the experiment

By means of a protein immunoblotting technology, autophagy and apoptosis related proteins Atg5, LC3II/I, Atg7, Bax and Bcl in a corticosterone-induced PC12 nerve cell injury model detected by immature bitter orange disintegrating tablet B₂Effects of PARP, p-mTOR and mTOR expression.

2 experimental materials and experimental equipment:

2.1 Experimental reagents

Antibodies Atg5, LC3II/I, Atg7, Bax, Bcl₂PARP, p-mTOR, Actin (CST), rabbit and mouse secondary antibodies (cloudy day), BCA protein assay kit (cloudy day).

2.2 Experimental drugs: EXAMPLE 1 Chinese medicinal composition

2.3 main experimental equipments: flow cytometry (BD).

3 Experimental methods

3.1 principle of the experiment

Western immunoblotting (Western Blot) is a method in which a protein is transferred to a membrane and then detected using an antibody. For known expressed proteins, the corresponding antibody can be used as a primary antibody for detection. The protein sample separated by PAGE is transferred to a solid phase carrier, and the solid phase carrier adsorbs the protein in a non-covalent bond form and can keep the type and the biological activity of the polypeptide separated by electrophoresis unchanged. Taking protein or polypeptide on a solid phase carrier as an antigen, carrying out immunoreaction with a corresponding antibody, then carrying out reaction with a second antibody labeled by enzyme or isotope, and carrying out substrate chromogenic or autoradiography to detect the protein component expressed by the specific target gene separated by electrophoresis.

3.2 Experimental procedures:

3.2.1 protein sample preparation: collecting cells, lysing RIPA cell lysate, fully lysing, centrifuging at 12000rpm/4 ℃ for 10min, taking supernatant, determining protein concentration by using a BCA protein concentration determination kit, denaturing the protein at 100 ℃ for 5min, and storing at-80 ℃.

3.2.2SDS-PAG electrophoresis: a30. mu.g solution of protein was loaded in volume, 80V running on the concentrate gel and 100V running on the isolate gel.

3.2.3 transfer of film: sealing with 5% skimmed milk powder for 1h after rotating the membrane at 100V for 80 min.

3.2.4 antibody incubation

The Western-anti dilution is used to dilute the antibodies according to the appropriate ratio. Shaking table overnight at 4 ℃. Western washing was added, washed 3 times, and diluted horseradish peroxidase (HRP) -labeled secondary antibody was added. Washing 3 times at 37 ℃ for 1h, and developing by ECL.

3.2.5 Western immunoblot image analysis

The grey values of each sample specific target band and the corresponding Actin band were measured separately using the BioRad pdquestv8.0.1 gel image processing system and the corresponding relative grey values were calculated. The relative gray scale values reflect the expression level of the protein of interest. The calculation method comprises the following steps: the relative gray-scale value is the gray-scale value of the target band/the gray-scale value of the same sample Actin, and the results are shown in fig. 6-8.

4 statistical methods

All data are expressed as (Mean ± Sd) and statistical analysis was performed using GraphPad Prism 5 software. The intra-group mean comparison adopts paired t test, the inter-group comparison adopts One-factor analysis of variance (One-ANOVA), and the two-by-two comparison among the groups is carried out by adopting t test after the variances are uniform. P <0.05 was statistically significant.

5 results of the experiment

Autophagy is a process by which a cell engulfs and encapsulates self-cytoplasmic proteins or organelles into vesicles, fuses with lysosomes to form autophagosomes, degrading the encapsulated contents, thereby fulfilling the metabolic needs of the cell itself and the renewal of certain organelles. The research proves that in the process of nerve cell injury caused by depression, along with the reduction of autophagy in different degrees, the increase of autophagy can obviously reduce the damage of nerve cells and improve the symptoms of depression. In depression, the depression can inhibit activation of Beclin-1 by promoting PI3K/AKT/mTOR signaling pathway, inhibit Atg5 from recruiting autophagy precursor, LC3 and lysosome to form autophagosome, and inhibit autophagy.

The results show that the composition of the invention can promote the protein expression of Bcl2/Bax, inhibit the expression of PARP (figure 6) and inhibit the mitochondrial apoptosis of nerve cells induced by corticosterone. At the same time, protein expression of Atg5, LC3II/I and Atg7 was up-regulated (FIG. 8), promoting autophagy. Secondly, the experimental result shows that after corticosterone modeling, the expression of p-mTOR/mTOR protein is obviously increased, and autophagy is obviously reduced, after the composition disclosed by the invention is dried, the expression of p-mTOR/mTOR protein is obviously reduced (figure 7), and autophagy is obviously increased, which indicates that the composition disclosed by the invention can inhibit an mTOR signal pathway and further inhibit autophagy reduction caused by corticosterone, so that depression is treated.

Example 6 therapeutic efficacy test on Depression mice

1. Treatment of depression in mice

1.1 Experimental animals

Animals 100 healthy C57BL/6J male mice, SPF grade, body mass 20 + -2 g. Purchased from yi si experimental animal technology, llc of vinca, animal licenses: SCXK (Ji) -2014-. The adaptive breeding is carried out for 1 week at the temperature of 21-25 ℃, the humidity of 30-70 percent and the illumination for 12 hours, and the drinking and the ingestion are normal during the period.

1.2 Main reagents and instruments

Mirtazapine tablets: harbin trigeminy pharmaceutical industries, ltd, lot number: 180204. ease pill: jiuzhitang gmbh, batch number: 180301. the positive Chinese medicine is prepared according to the embodiment 1 of the patent CN 104173933; the mouse serum 5-HT ELISA kit is purchased from Nanjing institute of bioengineering. Refrigerated centrifuge (Eppendorf, Germany, type 5424R); tail-suspended experimental observation boxes (model: TST, Bona Biotechnology Co., Ltd., Xuzhou); forced swimming glass jar (Beijing Zhi rat Duobao Biotechnology, Inc., model: DB 009).

1.3 animal modeling, grouping and interventional therapy

1.3.1 mouse depression model establishment:

after the body mass, Tail Suspension Test (TST) and Forced Swimming Test (FST) of 100C 57BL/6J mice are measured and initially screened, 90 mice with similar indexes are selected. 10 mice are randomly selected from the normal group, the remaining 105 mice are randomly divided into 7 groups, each group comprises 15 mice, the mice adopt modeling methods of chronic unpredictable mild stress model (CUMS) and isolated breeding (single-mouse single-cage breeding), and the mice are depressed by 28 days of stress. The stress comprises 24h fasting and water deprivation, cold water swimming (12 ℃, 5min), inclined squirrel cage (45-degree inclination, 8h), squirrel cage shaking (1 time/s, 5min), tail clamping for 60s, wet cage for 8h hot water swimming (37 ℃, 5min), night illumination for 12h, and tail clamping for 90 s. The 9 stimulations were given randomly 1 per day, with no repeated stimulation every other day, 3 times per stimulation on average, and no stimulation on day 18. Molding standard: after 28d stimulation, compared with the blank group and the mice before modeling, the percent of immobility time of the tail suspension test experiment (TST) and the Forced Swimming Test (FST) of the mice is remarkably prolonged, and the body mass is remarkably reduced or has no remarkable difference.

1.4 methods of treatment: the groups of 6 mice were: the traditional Chinese medicine composition comprises a positive western medicine group (mirtazapine tablet), a positive traditional Chinese medicine group (CN104173933 patent traditional Chinese medicine composition) and high, medium and low dose groups of the traditional Chinese medicine composition in the embodiment 1, wherein the administration of each group is 10ml/kg through intragastric administration for 28 days; blank, model group: purified water, 10ml/kg, was administered by gavage for 28 consecutive days.

1.4 Observation index and detection method

1.4.1 tail overhang test experiment: after the tail of the mouse distal end was fixed at about 1.5cm, the head was suspended downward, about 35cm from the ground. The total length of the recording time is 360s, and the determination index is the percentage of the time of immobility when the mouse is suspended, so that the helplessness degree of the mouse is reflected.

1.4.2 forced swimming experiments: the mice are placed in a transparent glass water tank with the diameter of 25cm and the height of 30cm, water with the height of 15cm is injected, the water temperature is 21-25 ℃, the total recording time is 360s, the recorded data is the time percentage of the mice floating or slight limb movement within 120-360 s, and the time percentage is used for reflecting the helplessness degree of the mice.

1.4.3 body weight: before modeling, after modeling and after treatment, the body weight of each group of mice is recorded to reflect the physiological conditions of the mice after modeling and after treatment.

1.4.4 serum 5-HT assay: cutting tail of mouse, taking 50 μ L of blood, standing at room temperature for 2h to fully coagulate the blood, centrifuging at 3000r/min at room temperature for 15min, separating supernatant, freezing and storing at-20, and operating according to the ELISA kit instruction.

1.5 results

1.5.1 the body weight of the mice in each group was varied, and the results are shown in Table 3.

Table 3 comparison of body mass of each group of mice before molding, after molding and after 28d administration (n-10,

g)

compared with the blank group after the molding,^※P<0.05; compared with the model group after 28d administration,^#P<0.05，^##P<0.01。

the results show that the body weight difference of the mice in each group before the model building is not statistically significant (P is more than 0.05). After the mice in the normal group are normally bred for 28 days, the statistical significance is achieved (P is less than 0.05) compared with a blank group before modeling; the other 6 groups of mice had a reduced body weight average compared to the blank group, with statistical significance (P < 0.05). After modeling, the weights of other 6 groups of mice were compared with those before modeling of each group, and the mice had no statistical significance (P > 0.05). After 28 days of drug treatment, the positive western medicine group, the positive Chinese medicine group, the high dose group and the medium dose group have statistical significance (P is less than 0.05, P is less than 0.01) compared with the modeling, and have no statistical difference (P is more than 0.05) compared with the blank group.

1.5.2 change in the dead time of tail suspension, the results are shown in Table 4.

Table 4 comparison of percent time spent tail suspension before, after and after 28d dosing for each group of mice (n-10,

％)

group of	Dosage (g/(kg/d)	Before molding	After molding	After 28 days of administration
					Blank group	----	28.56±4.41	27.94±5.07	29.25±5.72
Model set	----	29.62±5.34	69.83±6.49^※※※	56.25±7.31^※※※
					Positive western medicine group	11.7	27.72±6.18	65.82±5.91^※※※	33.74±3.52^##
Positive Chinese medicine	2.34	28.59±4.83	63.92±5.83^※※※	35.14±4.09^#
					High dose group	11.7	29.01±6.16	64.38±5.55^※※※	29.12±4.85^###△
Middle dose group	7.02	28.77±5.23	63.17±6.16^※※※	32.24±6.13^##
					Low dose group	2.34	26.28±5.49	64.73±5.82^※※※	33.79±5.14^##

Compared with the blank group after the molding,^※※※P<0.001; compared with the model group after 28d administration,^#P<0.05，^##P<0.01; compared with the positive Chinese medicinal group, delta P<0.05。

The results show that the percent of immobility time of the tail suspension of each group of mice is similar before modeling, and no statistical difference exists (P is more than 0.05). After modeling, the mice in other groups have higher percent of immobility time of suspending tail compared with the blank group, and have statistical significance (P is less than 0.001). After 28 days of administration, the mice in the positive western medicine group, the positive Chinese medicine group and the high, medium and low dose groups of example 1 have reduced tail suspension immobility time and have statistical significance compared with the model group (P <0.05, P <0.01 and P < 0.001). Wherein, the effect of the group in the embodiment 1 is obviously better than that of the positive traditional Chinese medicine group, and the statistical difference is realized (P < 0.05).

1.5.2 Change in immobility time for forced swimming

Table 5 comparison of percent immobility time in forced swimming for each group of mice before, after and after molding (n-10,

％)

compared with the blank group after the molding,^※※※P<0.001; compared with the model group after 28d administration,^#P<0.05; compared with the positive Chinese medicinal group^△P<0.05。

The results show that the percentage of immobility time in forced swimming of the mice in each group before molding is not statistically significant (P > 0.05). After model building, compared with a blank group, the time percentage of the immobile time of forced swimming of other groups of mice is increased before model building, and the statistical significance is achieved (P is less than 0.001). After 28 days of administration, the percentage of immobility time for forced swimming of mice in the positive western medicine group, the positive Chinese medicine group and the high, medium and low dose groups in the example 1 group is reduced compared with the model group, and the statistical significance is achieved (P is less than 0.05, P is less than 0.01, and P is less than 0.001). Wherein, the effect of the low dose group in the embodiment 1 is obviously better than that of the positive traditional Chinese medicine group in the same dose.

1.5.35-HT content

Table 6 comparison of 5-HT levels in groups of mice after 28d administration (n-10,

ng/mL)

in comparison with the blank set, the results,^※※※P<0.001; in comparison with the set of models,^#P<0.05，^##P<0.01; comparison with the Positive Western medicine group<0.05。

The results showed that the model group had a statistically significant decrease in 5-HT concentration (P <0.001) after 28 days of administration compared to the blank group. Compared with the model group, the positive western medicine group and the high, medium and low dose groups of example 1 have the 5-HT concentration which is increased and have statistical significance (P is less than 0.05 and P is less than 0.01). Wherein, under the same dosage, the effect of the low-dosage group in the embodiment 1 is obviously better than that of the positive traditional Chinese medicine group.

1.6 conclusion

The results show that the traditional Chinese medicine composition can obviously reduce the immobility time of tail suspension and forced swimming of mice, increase the content of 5-hydroxytryptamine in serum, and obviously improve the depression-like behavior of the mice, which indicates that the composition has obvious curative effect on depression.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in preparing antidepressant medicines is characterized in that the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of immature bitter orange, 4-8 parts of bighead atractylodes rhizome, 4-8 parts of rhizoma pinellinae praeparata, 5-15 parts of coptis chinensis, 3-7 parts of codonopsis pilosula, 2-6 parts of liquorice, 2-6 parts of poria cocos, 4-12 parts of mangnolia officinalis, 2-6 parts of malt and 2-6 parts of dried ginger.

2. The use of claim 1, wherein the Chinese medicinal composition is prepared from the following raw materials in parts by weight: 10 parts of immature bitter orange, 6 parts of bighead atractylodes rhizome, 6 parts of rhizoma pinellinae praeparata, 10 parts of coptis chinensis, 5 parts of codonopsis pilosula, 4 parts of liquorice, 4 parts of poria cocos, 8 parts of mangnolia officinalis, 4 parts of malt and 4 parts of dried ginger.

3. The use of claim 1 or 2, wherein the preparation method of the traditional Chinese medicine composition comprises the following steps: extracting fructus Aurantii Immaturus, cortex Magnolia officinalis, and Atractylodis rhizoma by steam distillation, and collecting volatile oil, water extractive solution and residue respectively; making the volatile oil into clathrate of beta-cyclodextrin-volatile oil, mixing the residue with rhizoma Pinelliae, Coptidis rhizoma, radix Codonopsis, Glycyrrhrizae radix, Poria, fructus Hordei Germinatus and Zingiberis rhizoma, decocting in water for 2 times, filtering, and mixing filtrates; and mixing the volatile oil, the water extract and the filtrate to obtain the traditional Chinese medicine composition.

4. The use as claimed in claim 3, further comprising the step of soaking in water for 1 hr, wherein the water is 10 times the weight of fructus Aurantii Immaturus, cortex Magnolia officinalis and Atractylodis rhizoma; the times of decocting with water are 2 times, and each time lasts for 1.5 hours; the amount of the water is 8-10 times of the total mass of the medicine residues, the pinellia ternate, the coptis chinensis, the codonopsis pilosula, the liquorice, the poria cocos and the malt and dried ginger.

5. The use of claim 3, wherein the medicament is in the form of tablets, pills, granules, capsules, pills or syrup.