CN116726082A

CN116726082A - Traditional Chinese medicine composition for treating stress-induced depression anxiety and learning and memory disorder as well as preparation method and application thereof

Info

Publication number: CN116726082A
Application number: CN202310749816.3A
Authority: CN
Inventors: 陈颖; 朱晓新; 杨庆; 翁小刚; 王娅杰; 李玉洁; 蔡维艳; 李琦; 杨紫玉; 董李晋川
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2022-07-01
Filing date: 2023-06-25
Publication date: 2023-09-12

Abstract

The invention discloses a traditional Chinese medicine composition for treating stress-induced depression anxiety and learning and memory disorder, and a preparation method and application thereof.

Description

Traditional Chinese medicine composition for treating stress-induced depression anxiety and learning and memory disorder as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine compound composition for treating stress-induced depression anxiety and learning and memory disorder, and a compatibility preparation method and application thereof.

Background

Stress is an adaptive physiological and pathological feedback process of the body to cope with external bad stimulus. The continuous bad stimulus causes excessive feedback of the organism to cause chronic stress, destroys the regulation capacity of the organism, causes mental and psychiatric pressure, causes neuroendocrine disturbance, and further causes diseases such as mood disorder (such as depression anxiety) and learning and memory disorder.

Patent document CN 102552436A discloses a medicament for treating anxiety and depression disorders, which is prepared from the following raw materials: radix Paeoniae alba, atractylodis rhizoma, bupleuri radix, coptidis rhizoma, prunellae Spica, and cortex Cinnamomi. The medicine is prepared from pure traditional Chinese medicines, and has obvious treatment effect on anxiety and depression based on sufficient traditional Chinese medicine and Western medicine theory.

Patent document CN108543037B discloses a traditional Chinese medicine composition, a preparation method and application thereof, wherein the traditional Chinese medicine composition consists of coptis chinensis, phellodendron bark, gardenia, red sage root, turmeric and grassleaf sweelflag rhizome, can improve learning and memory dysfunction of a 5×fad transgenic mouse, and provides a reliable theoretical basis for preparing medicines for treating or assisting in treating Alzheimer disease.

In the prior art, regarding the treatment of diseases such as mood disorder (such as depression anxiety) and learning and memory disorder, in the aspect of traditional Chinese medicine treatment, the traditional Chinese medicine has excessive medicine taste, difficult material obtaining and high price; has no obvious drug effect, poor treatment effect and the like. At present, no report that a traditional Chinese medicine composition is formed by compounding coptis chinensis, white paeony root and evodia rutaecarpa extract for treating depression anxiety and learning and memory disorders caused by stress is seen in the prior art, and the invention is particularly provided in view of the problem.

Disclosure of Invention

The invention overcomes the defects existing in the prior art and provides a traditional Chinese medicine composition for preventing and/or treating stress disorder diseases, a preparation method and application thereof.

The first aspect of the invention provides a traditional Chinese medicine composition for preventing and/or treating stress-induced depression anxiety and learning and memory disorders, wherein the traditional Chinese medicine composition comprises coptis chinensis, white paeony root and evodia rutaecarpa.

Further, the traditional Chinese medicine composition raw material medicine consists of coptis chinensis, white paeony root and evodia rutaecarpa.

The traditional Chinese medicine composition is prepared by mixing medicinal powder of raw materials or extracts of the raw materials.

In one embodiment of the invention, the coptis chinensis is coptis chinensis extract.

In one embodiment of the invention, the coptis chinensis is a concentrate of coptis chinensis extract.

In one embodiment of the present invention, the coptis chinensis is a powder obtained by drying a concentrate of coptis chinensis extract.

Further, the extraction solvent of the coptis extract is selected from the group consisting of: water, alcohol, ketone, ether, and ester.

Further, the alcohol is selected from: the ketone is an acetone solution, the ether is an diethyl ether solution or a petroleum ether solution, and the ester is an ethyl acetate solution or an ethyl formate solution.

Preferably, the extraction solvent of the coptis chinensis extract is alcohol or water.

In one embodiment of the present invention, the extraction solvent of the coptis chinensis extract is an ethanol solution.

In one embodiment of the present invention, the extraction solvent of the coptis chinensis extract is water.

Further, the white peony root may be raw white peony root and/or fried white peony root, preferably the white peony root is fried white peony root.

In one embodiment of the present invention, the white peony root is an extract of white peony root (e.g. raw white peony root, fried white peony root).

In one embodiment of the present invention, the white peony root is a concentrate of white peony root (e.g. raw white peony root, fried white peony root) extract.

In one embodiment of the present invention, the white peony root is a powder obtained by drying a concentrate of white peony root (e.g. raw white peony root, fried white peony root) extract.

Further, the extraction solvent of the white peony root extract is selected from the group consisting of: water, alcohol, ketone, ether, and ester.

Preferably, the extraction solvent of the white peony root extract is alcohol or water.

In one embodiment of the present invention, the extraction solvent of the white peony root extract is water.

In one embodiment of the present invention, the extraction solvent of the white peony root extract is ethanol solution.

Further, the evodia rutaecarpa may be processed evodia rutaecarpa and/or evodia rutaecarpa, preferably the evodia rutaecarpa is processed evodia rutaecarpa.

In one embodiment of the present invention, the evodia rutaecarpa is an extract of evodia rutaecarpa (e.g., evodia rutaecarpa).

In one embodiment of the present invention, the evodia rutaecarpa is a concentrate of an extract of evodia rutaecarpa (e.g., evodia rutaecarpa).

In one embodiment of the present invention, the evodia rutaecarpa is a powder obtained by drying a concentrate of an evodia rutaecarpa (e.g., evodia rutaecarpa preparata) extract.

Further, the extraction solvent of the evodia rutaecarpa extract is selected from the group consisting of: water, alcohol, ketone, ether, and ester.

Preferably, the extraction solvent of the evodia rutaecarpa extract is alcohol or water.

In one embodiment of the invention, the extraction solvent of the evodia rutaecarpa extract is ethanol solution.

In one embodiment of the invention, the extraction solvent of the evodia rutaecarpa extract is water.

Further, the drying is selected from: freeze drying, normal pressure drying, reduced pressure drying, boiling drying, spray drying, infrared drying, and microwave drying.

Further, in the traditional Chinese medicine composition, the raw materials of coptis chinensis, evodia rutaecarpa and white paeony root are prepared according to the mass parts (calculated by crude drug): 1-30:1-10:1-30 (such as 1-25:1-5:1-25, 1-20:1-5:1-20, 1-15:1-5:1-15, 1-14:1-5:1-14, 1-13:1-5:1-13, 1-12:1-5:1-12, 1-11:1-5:1-11, 1-10:1-5:1-10, 1-9:1-5:1-9, 1-30:1-5:1-5, 1-25:1-5:1-5, 1-20:1-5:1-5, 1-15:1-5:1-5, 1-10:1-5:1-5, 1-5:1-3:1-3), preferably, the mass parts (by weight of the raw materials of coptis, the fructus evodiae and the white peony root are: 1-25:1:1-25, more preferably, the mass part ratio (based on crude drug) of the raw materials of coptis chinensis, fructus evodiae and radix paeoniae alba is as follows: 1-20:1:1-20, more preferably, the mass part ratio (based on crude drug) of the raw materials of coptis chinensis, fructus evodiae and radix paeoniae alba is as follows: 1-15:1:1-15.

In one embodiment of the invention, the raw materials of coptis chinensis, evodia rutaecarpa and white paeony root are prepared from the following components in parts by weight (calculated by crude drug): 12:1:12.

In one embodiment of the invention, the raw materials of coptis chinensis, evodia rutaecarpa and white paeony root are prepared from the following components in parts by weight (calculated by crude drug): 5:1:1.

Further, the traditional Chinese medicine composition also comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are selected from the following materials: preservatives, colorants, pH adjusters, buffers, antioxidants, metal ion chelating agents, bacteriostats, isotonicity adjusters, carriers, diluents, binders, lubricants, sweeteners, absorbents, disintegrants, wetting agents, coating materials.

Further, the dosage form of the traditional Chinese medicine composition is selected from the following components: decoction, powder, oral liquid, tablet, capsule, pill, injection, inhalant, suppository, emulsion, nanoparticle, gel, powder, suspension, cream, jelly, spray, etc.

In a second aspect, the present invention provides a method for preparing the Chinese medicinal composition according to the first aspect, which comprises the following steps:

(1) Weighing the raw materials according to the weight ratio;

(2) Making into medicinal powder or extracting to obtain crude drug extract;

(3) Mixing the medicinal powder or extract of the medicinal materials, or adding one or more pharmaceutically acceptable adjuvants.

The extraction method of the step (2) is selected from the following steps: preferably, the extraction method in the step (2) is a solvent reflux method, and the solvent reflux method comprises a reflux method and/or a continuous reflux method.

In some embodiments of the invention, the extraction process is a reflux process.

In some embodiments of the invention, the extraction process is a continuous reflux process.

In some embodiments of the invention, the crude drug coptis chinensis is extracted by a solvent reflux method, in particular to the coptis chinensis decoction pieces are extracted by the solvent reflux method.

In some embodiments of the present invention, the raw material medicine, i.e., the evodia rutaecarpa (e.g., the evodia rutaecarpa) is extracted by a solvent reflux method, specifically, the decoction pieces of the evodia rutaecarpa (e.g., the decoction pieces of the evodia rutaecarpa) are extracted by a solvent reflux method.

In some embodiments of the present invention, the radix Paeoniae alba (such as raw radix Paeoniae alba and parched radix Paeoniae alba) is extracted by solvent reflux method, specifically, radix Paeoniae alba decoction pieces (such as raw radix Paeoniae alba decoction pieces and parched radix Paeoniae alba decoction pieces) are extracted by solvent reflux method.

Further, the extraction time of the reflux method is as follows: 1 to 5 hours (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours), preferably 1 to 3 hours, more preferably 2 hours.

Further, the extraction temperature of the reflux method is as follows: 50 to 100 ℃ (e.g., 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃), preferably 70 to 90 ℃, more preferably 80 ℃.

Further, the step (2) further comprises a step of soaking the crude drug with an extraction solvent before the extraction step.

Further, the soaking time is 1-20 hours (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18.5, 19, 19.5, 20) hours.

In one embodiment of the invention, the soaking time is 1 hour.

Further, the step (2) further comprises the steps of concentrating and drying after the step of extracting.

Further, the concentrating may be: normal pressure concentration, reduced pressure concentration, film concentration and multiple effect concentration.

In some embodiments of the invention, the concentrating is atmospheric concentrating.

In some embodiments of the invention, the concentrating is reduced pressure concentrating.

Further, the drying may be freeze drying, normal pressure drying, reduced pressure drying, boiling drying, spray drying, infrared drying, or microwave drying.

In some embodiments of the invention, the drying is reduced pressure drying.

In some embodiments of the invention, the drying is freeze drying.

In some embodiments of the present invention, after the crude drug coptis chinensis is extracted by a solvent reflux method, the method further comprises the steps of concentrating under reduced pressure and drying under reduced pressure.

In some embodiments of the present invention, the method further comprises the steps of concentrating under reduced pressure and drying under reduced pressure after the radix paeoniae alba serving as the raw material is extracted by a solvent reflux method.

In some embodiments of the present invention, after the raw material evodia rutaecarpa is extracted by a solvent reflux method, the method further comprises the steps of concentrating under reduced pressure and drying under reduced pressure.

Further, the extraction solvent of step (2) is selected from the group consisting of: one of water, alcohol, ketone, ether and ester, preferably the alcohol is selected from: one of an ethanol solution, an isopropanol solution and a tertiary butanol solution, wherein the ketone is an acetone solution, the ether is an diethyl ether solution or a petroleum ether solution, and the ester is an ethyl acetate solution or an ethyl formate solution;

preferably, the extraction solvent in the step (2) is alcohol or water.

In one embodiment of the present invention, for the crude drug coptis chinensis, the extraction solvent in the step (2) is an ethanol solution.

In one embodiment of the present invention, the extraction solvent in the step (2) is an ethanol solution for the raw material drug evodia rutaecarpa.

In one embodiment of the present invention, for the raw material white peony root, the extraction solvent in the step (2) is water.

Further, the concentration of the ethanol solution is 40% -95% (specifically, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%).

In one embodiment of the present invention, the concentration of the ethanol solution is 50% for the drug substance coptis chinensis.

In one embodiment of the present invention, the concentration of the ethanol solution is 85% for the drug substance evodia rutaecarpa.

Further, in the step (2), the number of times of extraction of the crude drug (such as coptis, evodia rutaecarpa and white paeony root) is 1 or more, for example, 2, 3, 4, 5, 6, 7, 9 and 10 times, preferably, the number of times of extraction is 2 times.

Further, in the step (2), for single extraction or multiple extractions, the mass-volume ratio of the raw materials (such as coptis chinensis, evodia rutaecarpa and white paeony root) to the extraction solvent is 1:5 to 20, preferably 1:5 to 15, more preferably 1:5 to 10.

In one embodiment of the invention, the mass-to-volume ratio of the raw materials (such as coptis chinensis, fructus evodiae and radix paeoniae alba) to the extraction solvent is 1:10.

In one embodiment of the invention, for the crude drug coptis chinensis, the extraction times are 2 times, the extraction solvent is ethanol solution, and the concentration of the extraction solvent is 50%.

In one embodiment of the present invention, the number of times of extraction is 2 for the crude drug evodia rutaecarpa, the extraction solvent is ethanol solution, and the concentration of the extraction solvent is 85%.

In one embodiment of the present invention, the number of times of extraction is 2 for the raw material white peony root, and the extraction solvent is water.

In a third aspect, the present invention provides the use of a Chinese medicinal composition according to the first aspect for the preparation of a medicament for the prevention and/or treatment of stress-induced depression anxiety and learning and memory disorders.

Preferably, the stress is selected from: (1), somatic stress and psychological stress; (2), acute stress and chronic stress; and, (3) one of physiological stress and pathological stress.

In one embodiment of the invention, the stress is acute stress and/or chronic stress.

Further, the prevention and/or treatment of stress-induced depressive anxiety and learning memory disorders includes prevention and/or treatment of acute stress-induced: depression anxiety, reduced consciousness, difficulty in directing, inability to cope with external stimuli, fear, anger, despair, overactive or retired.

Further, the prevention and/or treatment of stress-induced depressive anxiety and learning memory disorders includes prevention and/or treatment of chronic stress-induced: mental fatigue and work capacity, learning and memory disorder, distraction, lack of creativity and self-confidence, poor communication effect, anxiety, tension, confusion, agitation, increase in mind, emotional sensitivity, reaction allergy, depression, withdrawal, depression, dysphoria or dissatisfaction.

The pharmaceutical composition is prepared from pure traditional Chinese medicines, not only is diagnosis and treatment aiming at the description of symptoms for treating stress-induced anxiety depression and learning memory disorder, but also focuses on the pathogenesis background of the whole disease and pathogenesis of the whole disease, has the unique advantages of multiple ways and multiple targets, goes deep into the root, thereby regulating the emotion, viscera and function stable state of organisms, has obvious curative effect, no toxic or side effect, is easy to accept by patients, has few raw material components, is abundant and easy to obtain, has low price, is simple in preparation process, and is suitable for popularization and use.

Drawings

Fig. 1 is a flow chart of experimental flow of the mood influence of a traditional Chinese medicine compound composition on stress-induced depression anxiety rats.

Fig. 2 shows sucrose bias for each group of rats (significant differences in # P <0.05, significant differences in # P <0.001, and significant differences in # P <0.01 for each group of rats administered as compared to the model rats).

Fig. 3 shows the results of open field detection of rats in each group (significant differences in # P <0.05, significant differences in # P <0.01, and significant differences in P <0.05, for rats in each group compared to normal rats).

Fig. 4 shows the results of forced swimming test for rats of each group (model rats showed very significant differences of # P <0.01 compared to normal rats; rats of each group administered showed significant differences of =p <0.05 compared to model rats; P <0.01 showed very significant differences).

Fig. 5 is a flow chart of an experiment of the influence of the compound Chinese medicinal composition on learning and memory ability of a tail suspension stress mouse model.

Fig. 6 shows the effect of the present compound traditional Chinese medicine composition on the seeking latency of the positioning navigation experiment (significant difference between P <0.05 and P <0.001 in the model group compared with the normal group, and significant difference between # # # P <0.001 in each administration group compared with the model group).

Fig. 7 shows the effect of the compound composition on seeking latency in a space exploration experiment (significant differences in P <0.05, P <0.01, and significant differences in # P <0.05, and significant differences in # P <0.001, for each of the mice in the dosing group, compared to the mice in the control group).

Fig. 8 shows the effect of the present herbal compound composition on the false latency in the diving tower experiment (the difference between #p <0.05 is significant in each group of dosed mice compared to model mice).

Fig. 9 is a total ion chromatogram of a mouse brain cortex sample.

Fig. 10 is a graph of the multivariate statistical analysis of the cortical metabolites of mice of each group (panel a is a PCA score plot, r2x=0.55, q2 (cum) =0.272; panel b is an OPLS-DA score plot, r2x=0.363, r2y=0.696, q2 (cum) =0.508).

Detailed Description

In order that the technical content of the present invention may be more clearly understood, the following detailed description of the embodiments is given only for better understanding of the content of the present invention and is not intended to limit the scope of the present invention.

Example 1 the present herbal Compound composition improves stress-induced depression anxiety rats' mood

1. Experimental materials

1.1 laboratory animals

SPF SD pregnant mice were obtained from SCXK 2016-0011, a laboratory animal technology Co., ltd., beijing, and approved by the animal ethical committee of the Chinese medical college of China. Animals were kept in Chinese traditional medicine institute of Chinese medical science SYXK (Beijing) 2020-0042. After pregnant mice were produced, every 10 rats were kept with the female mice, 5 rats were kept per cage after 28 days of age had passed off breast milk, and were free to drink water.

1.2 major laboratory instruments and apparatus

TABLE 1

1.3 Main Experimental reagents and solutions

TABLE 2

1.4 preparation of Main Experimental reagents and solutions

(1) Preparation of the Chinese herbal compound composition

The coptis chinensis decoction pieces are purchased from Beijing Wei Ren traditional Chinese medicine decoction piece factory, and are subjected to solvent reflux extraction in a pharmaceutical room of a Chinese and Japanese hospital, and are dried in vacuum to obtain extract solid powder, wherein the yield is 19.3%, specifically 3000g of coptis chinensis decoction pieces is taken, and 50% ethanol water with the volume of 10 times is used for extraction twice; the extraction solution was recovered in a rotary evaporator with ethanol and then dried in vacuo to give a brown residue (579 g). The prepared evodia rutaecarpa decoction pieces are purchased from Beijing Wei Ren traditional Chinese medicine decoction piece factories, are subjected to solvent reflux extraction in a pharmaceutical room of a Chinese and Japanese hospital, and are dried in vacuum to obtain extract solid powder with the yield of 17.7%, specifically, the evodia rutaecarpa decoction pieces (600 g) are taken and extracted twice by using 85% ethanol water with the volume of 10 times; the extraction solution was recovered in a rotary evaporator with ethanol and then dried in vacuo to give a brown residue (106 g). The fried white peony root decoction pieces are purchased from Beijing Wei Ren traditional Chinese medicine decoction piece factory, and are subjected to solvent reflux extraction in a pharmaceutical room of a Chinese and Japanese hospital, and are subjected to vacuum drying to obtain extract solid powder, wherein the yield is 9.37%, specifically, the white peony root decoction pieces (3000 g) are crushed, 10 times of volume of water is used for reflux extraction for two times, and the water extract is subjected to vacuum drying to obtain light yellow powder (279 g). According to the crude drug amount, coptis chinensis: preparing fructus evodiae: the extracts of the fried white peony root (12:1:12, 5:1:1) are respectively weighed and fully mixed to obtain the traditional Chinese medicine compound composition 1 (12:1:12) and the traditional Chinese medicine compound composition 2 (5:1:1). The compound Chinese medicine composition 1 is dissolved into 30.1mg/mL by pure water and is uniformly shaken before administration. The compound traditional Chinese medicine composition 2 is dissolved into 12.3mg/mL by pure water and is uniformly shaken before administration.

(2) Sertraline

Weighing sertraline 20mg, and grinding into powder. Dissolving into 2mg/mL with pure water, and shaking before administration.

(3) 1% sucrose water

50g of sucrose was weighed, dissolved to 1% by adding pure water and shaken well before the experiment.

2. Experimental method

2.1 animal grouping, establishment of IBS model and administration

Fig. 1 is a flow chart of an experiment of the mood influence of a stress-induced depression anxiety rats by the traditional Chinese medicine compound composition. Female milk mice were sorted on day 7 of birth, with 15 total three-nest female milk mice being the normal group. The remaining female milk mice begin childhood stress from day 7 to day 20 of birth. The molded milk mice start at nine points daily for two consecutive weeksThe colon was stimulated with a PTCA balloon, the liquid paraffin lubricated PTCA balloon was inserted 2cm into the anus of the suckling mouse, and air was injected until the balloon was fully inflated, for 1min. After colon stimulation, single-cage single-master-slave separation is performed on the forelimbs of the tethered suckling mice for three hours. After the mother-son separation was completed, the PTCA balloon was again used to stimulate the rat colon and the rats were returned to the mother cage. Female milk mice were weaned from breast milk 28 days after birth and divided into cages. The model rats were trained and tested for sugar preference from 28 days to 35 days of birth, and were divided into 5 groups (normal group, model group, 1 group of the present Chinese herbal medicine composition, 2 groups of the present Chinese herbal medicine composition, and house Qu Linzu) according to sugar preference results in combination with body weight, each group being 12. Model and dosing group rats were tethered 9 hours daily at night from 48 to 70 days of birth and started gavage dosing 9 a day early. The medicine groups are respectively according to 10mL Kg ^-1 The normal group and the model group of the rats subjected to the stomach infusion administration are given with equal amounts of pure water, and the positive group is given with equal amounts of sertraline solution. And carrying out sugar water preference detection, open field detection and forced swimming detection on the rats from 56 days to 70 days.

2.2 behavioural detection

(1) Sugar water preference detection

The rats were trained in sugar water for 48 hours at week 5 of birth, fasted during training, given 1% sucrose water for the first 24 hours, given 1% sucrose water and purified water simultaneously for the second 24 hours, and exchanged sucrose water and purified water orientations three times for the second 24 hours. The sugar water preference detection is carried out on rats at the 5 th and 9 th weeks of birth after sugar water training. After the rats are kept in a single cage and forbidden for 14 hours, one bottle of 1% sucrose water and one bottle of purified water are administered to the rats. The drinking amount (g) of two bottles of water in 2h is calculated by weighing, and the sucrose preference degree (%) =the drinking amount of the sucrose water/(the drinking amount of the sucrose water+the drinking amount of purified water) ×100% is calculated by a formula.

(2) Open field experiment

Open field testing was performed at week 10 of birth. After placing the rat wall in the border area of the square test box (100X 50 cm), the test was started for 5min. The behavioural detection instrument records and counts the activity of the animal in the test time. And calculating the total journey, the movement time, the central area movement journey ratio and the central area movement time ratio index.

(3) Forced swimming

Forced swim tests were performed at week 10 of birth in rats. The day before the test, rats were pre-swim for 15min in water at 23 ℃. In the formal experiment, rats were placed in a transparent cylinder (diameter 20 cm. Times.20 cm) with a water depth of 25cm and a water temperature of 23℃and the test was started for 5 minutes. The behavioural detection instrument records the stationary time of the statistical rats in the test time.

3. Results

3.1 sugar water preference test results

As in fig. 2 (where n=12;) As shown, model rats showed a significant decrease in the saccharophila compared to normal rats after childhood stress and prior to non-dosing. After constraint stress and administration, the model rats had significantly lower saccharophilia than normal rats; the sugar degree of rats in each administration group is remarkably increased compared with that in the model group.

3.2 open field test

Open field tests are often used to test spontaneous activity and exploration behaviors of mice, with central area exploration times, central area exploration time, and central area exploration distance to evaluate the mice' ability to explore autonomously in a new environment.

As in fig. 3 (where n=12;) As shown, the model, dosing group rats had no significant difference in time of locomotion from normal rats; compared with a normal rat, the model rat has obviously increased total movement distance, central area time ratio and central area movement distance ratio; the total movement distance of the rats in the group 1 (12:1:12) and the group 2 (5:1:1) of the traditional Chinese medicine compound composition is not obviously different from that of the rats in the model; the total movement distance of sertraline group rats is obviously increased compared with that of model rats; the time ratio of the central area of the group of Chinese herbal compound composition 1 (12:1:12) and the movement path ratio of the central area of the sertraline group of rats are not significantly different from those of the model rats; the Chinese herbal medicine compound combinationThe central zone time ratio and central zone movement path ratio were significantly reduced in group 2 (5:1:1) rats compared to model rats.

3.3 forced swimming

As in fig. 4 (where n=12;) As shown, the immobility time of model rats was significantly increased compared to normal rats; compared with a model rat, the immobility time of the group 2 (5:1:1) and sertraline rats of the traditional Chinese medicine compound composition is obviously reduced; the immobility time of the group of rats of the traditional Chinese medicine compound composition 1 (12:1:12) is not significantly different from that of the model rats.

EXAMPLE 2 improving action of the Chinese herbal Compound composition on learning and memory ability of mice model with stress of Tail suspension

1. Experimental materials

1.1 laboratory animals

Male ICR mice, SPF grade, 23-25g, 114 total, supplied by Peking Violet laboratory animals Co., ltd., animal license number: SCXK (Beijing) 2021-0006, all experimental procedures met the requirements of the ethical committee of animal research. The animals eat and feed water freely, the raising environment of experimental animals and the conditions of room temperature of 25+/-2 ℃ and humidity of 55+/-10% are kept in the process of all experiments, the circadian rhythm of 12h illumination and 12h darkness is maintained, and the animals adapt to the environment until the weight of the mice reaches 30g before the experiments, and the model is built.

1.2 major laboratory instruments and apparatus

The water maze test device is provided by Chinese traditional medicine institute of Chinese traditional medicine department. The device for simulating real-time monitoring of the weightless tail suspension is provided by the medical plant research institute of the national academy of medical science.

1.3 Main Experimental reagents and solutions

Donepezil hydrochloride tablet (Anapplication, 10 mg/tablet x 7) was purchased from the medical company, inc., of sanitation (China), production lot number: 2102079.

1.4 preparation of Main Experimental reagents and solutions

(1) Preparation of the Chinese herbal compound composition

The traditional Chinese medicine compound is prepared by adopting coptis chinensis, evodia rutaecarpa and white paeony root in a ratio of 12:1:12, setting the equivalent dosage of the medicine as the low dosage of the traditional Chinese medicine compound composition according to the calculated medicine crude drug amount, preparing the low dosage of the traditional Chinese medicine compound composition (coptis chinensis extract 0.0139 mg/ml < -1 >, evodia rutaecarpa extract 0.00106 mg/ml < -1 >, white paeony root extract 0.00675 mg/ml < -1 > and 0.0217 mg/ml < -1 > in total) by pure water according to the extract yield and the compatibility ratio, and the high dosage of the traditional Chinese medicine compound composition (the coptis chinensis extract 0.0279 mg/ml < -1 >, the evodia rutaecarpa extract 0.00212 mg/ml < -1 > and the white paeony root extract 0.0135 mg/ml < -1 > and the total 0.0435 mg/ml < -1 >).

(2) Positive medicine preparation.

Donepezil hydrochloride tablet (Anapplication, 10 mg/tablet. Times.7) was taken as a positive drug, and 2mg was ground into powder with a mortar. Dissolving with pure water to obtain 0.08mg.ml-1 solution, and shaking before administration.

2. Experimental method

Animals were acclimatized to the environment prior to the experiment until administration of each group was initiated after 30 g. The behavioural test was started 4 weeks after modeling, including a 9 day water maze test (6 days during the directional navigation phase and 3 days during the space exploration phase), and dosing continued until the behavioural test ended for a total of 40 days.

2.1 grouping and administration of animals

114 mice were randomly divided into 5 groups, including normal group (22), model group (24), donepezil group (24), low dose group of the present compound Chinese medicine composition (22) and high dose group of the present compound Chinese medicine composition (22). The mice of each dosage group of the traditional Chinese medicine compound are respectively subjected to gastric administration according to the concentration of 20mL kg < -1 >, the control group and the model group are respectively subjected to equivalent pure water administration, and the donepezil solution is adopted as a positive medicament by the donepezil Ji Zu administration of equivalent donepezil hydrochloride solution.

2.2 Tail suspension Molding

Before the experiment, the commercial wet injury pain-relieving plaster is cut into 7cm multiplied by 1cm long strips, the middle part of the plaster is folded in half, two ends of the plaster are attached to the 1cm position of the root of the tail of the mouse, the folded part of the plaster is left to form a circle, the mouse is convenient to hang, and 3-4 circles of medical plaster (0.95 cm multiplied by 7 cm) are wound outside the plaster, so that the plaster is tightly attached to the tail of the mouse. So that the medical adhesive plaster is in an annular structure, and the medical adhesive plaster is spirally wrapped outside the annular structure to avoid slipping. The metal chain with a hook at one end is hooked on the annular plaster, the other end is connected with the annular hanging ring of the micro switch of the tail hanging instrument, the length of the chain is adjusted according to the requirement, and the chain is fixed by a metal clip. The length of the hook rope is adjusted to suspend the tail of the mouse, the forelimbs are grounded, the hindlimbs are lifted off the ground, and the body forms an angle of 30 degrees with the ground. Each mouse was kept individually, and model was built continuously for 24 hours throughout the day, giving sufficient diet and water. The molding was performed for 4 weeks and the animals remained tail-suspended during molding until the end of the behavioural test. And during the behavioural detection, the mice are taken out, immediately put back into the tail cage after the detection is finished, and the modeling is continued. The molding period keeps the tail hanging ring clean and tidy and the tail hanging chamber is quiet.

2.3 behavioural detection

(1) Water maze test

The activity of the mice is detected by a real-time monitoring system of the water maze of the mice. Experiments were performed with reference to the Morris classical protocol and with appropriate modifications. The device is formed by a circular pool filled with water, adding food pigment to darken the pool, dividing the pool into four quadrants equally, placing a cylindrical platform in one quadrant to make the platform lower than the water surface by 1.5cm, and positioning the moving track of the mouse in real time by a software system to collect data. Mice were subjected to the water maze test 4 weeks after molding. Positioning and sailing training is carried out for 5 days, 3 times per day, one swimming quadrant at a time, adaptation is carried out for 10s before and after each training, the detection time is 90s, and the platform searching latency of each group of mice is detected. After the space exploration stage is positioned and sailed, the safety platform is moved to an adjacent quadrant, the seeking latency is used as an evaluation index, and the memory capacity (namely the working memory capacity) of the animal on the platform at the new moving position is detected. At this time, the experimental mode is the same as the water maze positioning navigation stage, but the quadrants where the safety platform is located are different every day, and the rest experimental operations are the same as the positioning navigation stage.

(2) Bench jump experiment

The diving platform experiment is a passive conditional reflex behavior detection method for testing learning and memory capacity of animals to space identification. The mice are placed into a test box with the faces facing outwards, animals are adapted to a diving platform box body for 3min, then 60V voltage is given to a power grid (a stimulation area), and the mice in each group are electrified for 5min, so that the mice in each group acquire stimulated memory. And (3) carrying out a consolidation stage test after 24 hours, detecting the memory capacity of the animal on the diving platform safety area, placing the animal on an insulating diving platform at the beginning of the test, and electrifying a stimulation area. The test time was 5min with no adaptation period. The time from the placement of the animal on the safety platform to the first jump off platform was recorded and noted as the error latency.

2.4 metabonomics study

2.4.1LC/MS conditions

Samples were introduced using a pre-chilled autosampler (Open autosampler UltiMate OAS-3300TXRS,Thermo Fisher Scientifc) and eluted through a Waters Acquity UPLC BEH C18 column (2.1 mm x 100mm,1.7 μm particles) using a transit II with an UltiMate pump (Thermo Fisher Scientifc). The mobile phase consisted of solvents a (0.1% acetonitrile formate) and B (0.1% aqueous formic acid), and was eluted gradient for 30min. The flow rate is 0.3mL/min, and the sample injection amount is 2 mu L. The column temperature was maintained at 60℃and the sample was maintained at 4℃throughout the analysis. Q Exactive Orbitrap the mass spectrometer (Thermo Fisher Scientifc) operates in a positive mode with a heated electrospray ionization source (HESI). The instrument was controlled using the traceFinder 4.1 clinical study and the Aria MX (Thermo Fisher scientific). In MS full scan mode, the mass range is set to 99 to 1515Da with a resolution of 75,000.

2.4.2LC-MS/MS data preprocessing

Raw data were preprocessed using MZmine (2.53) software. Chromatograms were constructed using an ADAP module with at least 7 scans per peak, a group intensity threshold of 10,000, a maximum intensity of 200,000, and an m/z bias of 0.001 or 5ppm.

2.4.3 dimension reduction and Pattern recognition

Multivariate statistical analysis was performed using SIMCA-P software (version 14.1, umetrics, sweden), including unsupervised Principal Component Analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA). First, principal component analysis is used to observe potential clustering trends and possible outliers and to calculate R2X. Then, to maximize the isolation of sample classes and to find potential biomarkers, OPLS-DA was performed. VIP values reflect the impact of each variable on classification. The study selects a discrimination variable according to the VIP value greater than 1 in the OPLS-DA model. Finally, a variance test (ANOVA) analysis (SPSS 20.0) was used to determine if biomarker candidates that differ between the three groups were statistically significant.

2.4.4 biomarker identification

In this study, the workflow for metabolite structure identification was first submitted to an online database such as Metlin (https:// Metlin. Scrips. Edu/index. Php), HMDB (http:// www.hmdb.ca /), massBank (http:// www.massbank.jp /), metFrag (http:// msbi. Ipb-mulle. De/MetFrag /), and the like for database retrieval using the peaks extracted by MZmine. In addition, the MS/MS data excludes neutral loss peaks, isotope peaks and product ions.

3. Results

3.1 Water maze test

(1) Positioning navigation stage

In the positioning navigation experiment, the difference between the seeking latency of the normal group and the model group increases gradually along with the increase of the experimental period, and the seeking latency of the model group increases remarkably on the fourth and fifth days (P<0.05)、(P<0.001 A) is provided; compared with the model group, the 5 th day of the seeking latency of the donepezil group and the traditional Chinese medicine compound composition is reduced, and the similar time (P) of the donepezil to the normal group is obviously reduced<0.001 The low-dose group seeking latency of the traditional Chinese medicine compound composition is lower than that of the high-dose group of the traditional Chinese medicine compound composition, and the result is shown in figure 6 (wherein n=22-24;)。

(2) Space exploration stage

On the sixth day of swimming, the platform is removed for space exploration experiments. The mouse seeking latency in the space exploration experiment may reflect the working memory capacity of the mouse. And if the working memory capacity is good, the mice arrive at the periphery of the target earlier, and the incubation period is explored.

The results showed that the model group had a significantly higher 3-day seeking latency than the normal group (P<0.05)、(P<0.01)、(P<0.01 A) is provided; donepezil compared to model groupThe seeking latency of the group and the administration group of the Chinese medicinal compound composition is reduced; wherein the first and second donepezil groups have significantly reduced seeking latency (P<0.001)、(P<0.05 The low-dose group seeking latency of the traditional Chinese medicine compound composition is obviously reduced (P)<0.05 More closely to the normal control group in the second and third day experiments, the results are shown in fig. 7 (where n=22 to 24;)。

3.2 bench jump experiments

Fig. 8 (wherein n=9 to 12;) The effect of the traditional Chinese medicine compound composition on the error latency in the bench jump experiment is shown. The results show that the jump error latency of the model group is reduced compared with the normal group; the donepezil group and the high dose administration group of the Chinese herbal compound composition significantly prolong the false latency (P) of the model mice<0.05)。

The results show that the space memory capacity and the working memory capacity of the tail suspension model are obviously reduced in the water maze positioning navigation and exploration experiment stage, and the compound administration has a certain effect of improving the space memory disorder and the working memory disorder of the tail suspension model mice, and the low-dose effect of the compound administration is better than that of the high-dose. Meanwhile, the tail crane can lead the mice to have hypomnesis in the group safety area in the stage of the bench jump experiment consolidation, but the high-dose administration of the traditional Chinese medicine compound composition can also obviously improve the passive stimulation learning and memory capacity of the tail crane mice for space identification.

3.3 non-targeted metabonomics

3.3.1 Metabolic analysis

The UHPLC-QTOF-MS Total Ion Current (TIC) chromatogram of a cortical sample is shown in FIG. 9. It can be seen that small molecule metabolites can be well separated within 30min of elution time due to the high performance of the UHPLC system and small particle size (1.7 m) of the Waters Acquity UPLC BEH C column packing.

3.2. Multivariate statistical analysis

The PCA scoring graph (figure 10) shows that the separation of the control group and the model group is more obvious than the separation of the control group and the traditional Chinese medicine compound composition group, which shows that the traditional Chinese medicine compound composition can relieve metabolic disorder caused by stress of tail suspension. The OPLS-DA scoring graph (figure 10) shows that the separation among the three groups is more obvious, and the traditional Chinese medicine compound composition can restore metabolic disorder caused by tail suspension stress to the normal direction. Potential biomarkers identified by non-targeted metabolomics are shown in table 3. It can be seen that the compound Chinese medicinal composition plays a role in improving memory mainly by regulating biosynthesis of neurotransmitters and metabolism of amino acids, fatty acids and the like.

TABLE 3 Table 3

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Claims

1. The traditional Chinese medicine composition for preventing and/or treating stress-induced depression anxiety and learning and memory disorder is characterized by comprising the following raw materials in parts by weight: 1-30:1-10:1-30.

2. The traditional Chinese medicine composition of claim 1, wherein the raw materials comprise coptis chinensis, fructus evodiae and radix paeoniae alba in parts by weight: 1-25:1:1-25, preferably, the raw materials comprise coptis chinensis, fructus evodiae and radix paeoniae alba in parts by weight: 1-20:1:1-20, more preferably, the raw material medicines of coptis chinensis, fructus evodiae and radix paeoniae alba comprise the following components in parts by weight: 1-15:1:1-15.

3. The traditional Chinese medicine composition according to claim 1 or 2, wherein the traditional Chinese medicine composition is prepared by mixing extracts of crude drugs, and the traditional Chinese medicine composition further comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are selected from the group consisting of: preservative, colorant, pH regulator, buffer, antioxidant, metal ion chelating agent, bacteriostat, isotonic regulator, carrier, diluent, adhesive, lubricant, sweetener, absorbent, disintegrating agent, wetting agent and coating material, wherein the dosage form of the traditional Chinese medicine composition is selected from the group consisting of: decoction, powder, oral liquid, tablet, capsule, pill, injection, inhalant, suppository, emulsion, nanoparticle, gel, powder, suspension, cream, jelly, and spray.

4. The Chinese medicinal composition of claim 1, wherein the coptis is coptis chinensis extract;

preferably, the coptis chinensis is a concentrate of coptis chinensis extract;

preferably, the coptis is medicinal powder obtained by drying a concentrate of coptis extract;

preferably, the extraction solvent of the coptis extract is selected from: water, alcohol, ketone, ether, and ester.

5. A Chinese medicinal composition according to claim 1, wherein said white peony root is raw white peony root and/or fried white peony root, preferably said white peony root is fried white peony root;

preferably, the white peony root is white peony root extract;

preferably, the white peony root is a concentrate of white peony root extract;

preferably, the white peony root is a medicinal powder obtained by drying a concentrate of the white peony root extract;

preferably, the extraction solvent of the white peony root extract is selected from: water, alcohol, ketone, ether, and ester.

6. The traditional Chinese medicine composition of claim 1, wherein the fructus evodiae is processed fructus evodiae and/or fructus evodiae, preferably the fructus evodiae is processed fructus evodiae;

preferably, the evodia rutaecarpa is an evodia rutaecarpa extract;

preferably, the evodia rutaecarpa is a concentrate of evodia rutaecarpa extract;

preferably, the evodia rutaecarpa is medicinal powder obtained by drying the concentrate of the evodia rutaecarpa extract;

preferably, the extraction solvent of the evodia rutaecarpa extract is selected from the group consisting of: water, alcohol, ketone, ether, and ester.

7. The method for preparing the Chinese medicinal composition according to claim 1, which comprises the following steps:

(1) Weighing the raw materials according to the weight ratio;

(2) Extracting to obtain crude drug extract;

(3) The preparation method comprises the steps of mixing extracts of the raw materials, or adding one or more pharmaceutically acceptable auxiliary materials into the extracts of the raw materials, wherein the extraction method in the step (2) is selected from the following steps: solvent extraction, percolation, decoction, reflux, ultrasonic extraction, supercritical fluid extraction, and steam distillation,

preferably, the extraction method in the step (2) is a solvent reflux method, and the solvent reflux method comprises a reflux method and/or a continuous reflux method;

preferably, the extraction time of the reflux method is as follows: 1-5h;

preferably, the extraction temperature of the reflux method is 50-100 ℃;

preferably, the extraction solvent of step (2) is selected from: one of water, alcohol, ketone, ether, and ester;

preferably, the alcohol is selected from: one of an ethanol solution, an isopropanol solution and a tertiary butanol solution, wherein the ketone is an acetone solution, the ether is an diethyl ether solution or a petroleum ether solution, and the ester is an ethyl acetate solution or an ethyl formate solution;

preferably, the extraction solvent in the step (2) is alcohol or water;

preferably, the concentration of the ethanol solution is 40% -95%;

preferably, the mass volume ratio of the bulk drug to the extraction solvent is 1:5-20

Preferably, for the crude drugs of coptis chinensis and evodia rutaecarpa, the extraction solvent in the step (2) is ethanol solution;

preferably, for the raw material medicine white peony root, the extraction solvent in the step (2) is water.

8. The preparation method of claim 7, wherein the raw materials of coptis chinensis, evodia rutaecarpa and white paeony root are extracted by adopting a solvent reflux method, the step (2) further comprises the steps of concentration and drying after the extraction step,

preferably, the steps of concentrating under reduced pressure and drying under reduced pressure are further included after the raw materials of coptis chinensis, white paeony root and evodia rutaecarpa are extracted by adopting a solvent reflux method.

9. Use of a Chinese medicinal composition according to claim 1 for the preparation of a medicament for the prevention and/or treatment of stress-induced depression anxiety and learning memory disorders, preferably wherein the stress is selected from the group consisting of: (1), somatic stress and psychological stress; (2), acute stress and chronic stress; and, (3) one of physiological stress and pathological stress.

10. The use according to claim 9, wherein said preventing and/or treating stress-induced depressive anxiety and learning memory disorders comprises preventing and/or treating acute stress-induced: worry, depression anxiety, reduced consciousness, difficulty in orientation, inability to cope with external stimuli, fear, anger, despair, overactivity or withdrawal;

preferably, the prevention and/or treatment of stress-induced depressive anxiety and learning memory disorders comprises prevention and/or treatment of chronic stress-induced: mental fatigue and work capacity, learning and memory disorder, distraction, lack of creativity and self-confidence, poor communication effect, anxiety, tension, confusion, agitation, increase in mind, emotional sensitivity, reaction allergy, depression, withdrawal, depression, dysphoria or dissatisfaction.