CN118001323A - Traditional Chinese medicine composition for preventing and treating anxiety disorder and preparation method and application thereof - Google Patents
Traditional Chinese medicine composition for preventing and treating anxiety disorder and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a traditional Chinese medicine composition for preventing and treating anxiety disorder, and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 1-20 parts of cassia twig, 5-30 parts of red paeony root, 5-25 parts of fructus aurantii, 1-20 parts of platycodon grandiflorum, 1-20 parts of costustoot and 1-10 parts of liquorice. The traditional Chinese medicine composition has the effects of soothing liver, relieving depression, regulating qi and blood, and can effectively regulate neurotransmitters, repair nerve injury and improve qi and blood circulation.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composition for preventing and treating anxiety disorder, and a preparation method and application thereof.
Background
Anxiety disorder (anxiety), also known as anxiety neurosis, is one of the most common of the major classes of disorders, neurosis, and is characterized by an anxiety emotional experience. Chronic anxiety, i.e. generalized anxiety (generalized anxiety) and acute anxiety, also known as panic attacks (PANIC ATTACK), can be divided into two forms. The main manifestations are: there is no stress concern of clear and objective subjects, and restlessness is often accompanied by sleep disorder and autonomic nerve dysfunction symptoms such as dizziness, palpitation, dyspnea, dry mouth, hand tremble, sweating, frequent urination, urgent urination, tremor, and restlessness. The anxiety is not caused by a real threat or the tension is very disproportionate to reality.
The main etiology of anxiety is in several ways, firstly, the risk of anxiety is higher in people with family history than in general people. Secondly, it was found that gamma-aminobutyric acid is one of the bases of the pathogenesis, and it is also possibly involved in the hyperfunction of the norepinephrine system, abnormality of the 5-tryptamine system, and the like. Furthermore, the intra-cerebral emotional control loop is composed of forehead leaves, amygdala, hippocampus, hypothalamus, etc., and abnormal structure, function or association of these regions may cause emotional control disorders, which form the pathological structural basis of anxiety disorders. Psychological factors also play an important role in the development of anxiety disorders, such as generalized anxiety disorder due to unresolved conscious conflicts, information processing duration, etc.; social anxiety disorders are due to excessive patient attention and evaluation by others who are in mind, and may also be associated with some negative experience before adulthood; specific phobia may be associated with fear objects and traumatic experiences.
Traditional Chinese medicine has unique advantages in improving symptoms of anxiety patients, and is a current research hotspot. Traditional Chinese medicine considers that liver depression and qi stagnation and spleen deficiency malnutrition are main reasons for anxiety disorder. Liver depression, anxiety, emotional depression and anxiety lead to liver failure and diarrhea, qi stagnation, unfavorable channels and collaterals, fullness and distention and pain in chest and hypochondrium or lower abdomen, emotional depression and frequent sighing. Long-term anxiety, poor mood, stasis and impairment of spleen, insufficient qi and blood circulation and transformation of spleen deficiency, no nourishment of liver blood, and insufficient liver blood is not easy to be smooth. Therefore, the principle of soothing liver, relieving depression, strengthening spleen and promoting qi circulation is developed in clinic. However, in clinical practice, it is found that the therapeutic effect of the traditional Chinese medicine composition designed by purely using the therapeutic method of soothing liver-qi stagnation and strengthening spleen to promote qi circulation is not very ideal. For example, the traditional Chinese medicine classical prescription cassia twig decoction can strengthen yang and promote qi circulation and harmonize ying and wei, but has no liver soothing and qi regulating effects in the process of treating anxiety; the four adverse qi dispersing, liver soothing, qi regulating and mind smoothing effects can not be achieved, but yin and yang can not be harmonized, so that the best effect of treating anxiety disorder can not be achieved.
In view of the above, the classical ancient prescription needs to be deeply excavated by combining the pathogenesis of anxiety disorder, and a safer and more effective novel traditional Chinese medicine composition for preventing and treating anxiety disorder is further developed.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine composition for preventing and treating anxiety disorder with better curative effect.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
In a first aspect, the invention provides a traditional Chinese medicine composition for preventing and treating anxiety disorder, which is prepared from the following raw materials in parts by weight: 1-20 parts of cassia twig, 5-30 parts of red paeony root, 5-25 parts of fructus aurantii, 1-20 parts of platycodon grandiflorum, 1-20 parts of costustoot and 1-10 parts of liquorice.
Preferably, in the above traditional Chinese medicine composition, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-10 parts of cassia twig, 10-20 parts of red paeony root, 10-15 parts of fructus aurantii, 5-10 parts of platycodon grandiflorum, 5-10 parts of costustoot and 3-5 parts of liquorice.
Preferably, in the above traditional Chinese medicine composition, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5 parts of cassia twig, 10 parts of red paeony root, 10 parts of bitter orange, 5 parts of platycodon root, 5 parts of costustoot and 3 parts of liquorice.
Preferably, in the above traditional Chinese medicine composition, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts of cassia twig, 20 parts of red paeony root, 15 parts of fructus aurantii, 10 parts of platycodon grandiflorum, 10 parts of elecampane and 5 parts of liquorice.
In a second aspect, the present invention provides a method for preparing the traditional Chinese medicine composition according to the first aspect, the method comprising the following steps: weighing the raw materials according to the parts by weight of the first aspect, pre-treating, extracting and concentrating.
Preferably, in the above preparation method, the Chinese medicinal composition of the present invention may be prepared by conventional methods well known in the art.
Preferably, the extraction method used in the present invention includes, but is not limited to, decoction extraction, reflux extraction, ultrasonic extraction, supercritical extraction, and the like.
Preferably, the treated drug substance may be extracted using water, an organic solvent commonly used in the art, or a mixture thereof.
Preferably, the organic solvent is methanol, ethanol, propanol, butanol, ethyl acetate, petroleum ether, or the like.
Preferably, the preparation method comprises the steps of weighing the raw materials, crushing, extracting and concentrating.
Preferably, the extraction method is decoction, and/or the extraction solvent is water or ethanol, and/or the times of decoction are 1-3 times, and/or the time of each decoction is 1-3 hours.
Preferably, water extraction (e.g., water decoction) is used.
In a third aspect, the invention provides a use of the traditional Chinese medicine composition described in the first aspect or the traditional Chinese medicine composition prepared by the preparation method described in the second aspect in preparation of medicines.
Preferably, in the above use, the medicament is for the prevention or treatment of anxiety disorders.
Preferably, in the above-mentioned use, the medicament has the efficacy of soothing liver-qi stagnation and regulating qi and blood.
Preferably, in the above use, the medicament is effective in regulating neurotransmitters, repairing nerve damage, and improving qi and blood circulation.
Preferably, in the above use, the medicament is in solid form, semi-solid form or liquid form.
Preferably, in the above use, the medicament is decoction pieces, decoction, powder, oral liquid, paste, granule, tablet or capsule.
Preferably, in the above use, the medicament is a decoction or a granule.
The prescription of the traditional Chinese medicine composition provided by the invention is as follows:
Anxiety disorder is a common mental disorder with excessive fear and anxiety as the primary manifestations, accompanied by abnormal gene regulation and synaptic dysfunction. Anxiety disorder belongs to the category of traditional Chinese medical mental diseases, and is similar to depression, mania, lily disease and other diseases. The pathogenesis of the disease is mainly emotional internal injury, liver and gall are lost and dredged, qi stagnation and unsmooth blood circulation are caused; the spleen stores the mind and is one of the emotional activities of the five zang organs, namely the spleen. Anxiety and anxiety can damage the spleen, cause deficiency of qi and blood, imbalance of yin and yang, yin failing to astringe yang, yang failing to enter yin, yang qi failing to warm the mind, and anxiety can also result. As described in the background art, the traditional Chinese medicine classic formula cassia twig decoction can strengthen yang and promote qi circulation and harmonize ying and wei, but has no liver soothing and qi regulating effects; the four-function dispersing stagnated liver qi and smoothing the mind, but can not harmonize yin and yang, and the two formulas can not achieve the best effect of treating anxiety disorder by singly using the four-function dispersing stagnated liver qi and smoothing the flow of qi, and the two formulas can achieve the effects of soothing liver, regulating spleen, nourishing blood, promoting blood circulation and harmonizing yin and yang by modifying and cutting the two formulas through clinical experience.
Therefore, the method takes soothing liver and relieving depression, regulating qi and blood as an legislation, wherein cassia twig has the effects of tonifying yang and qi, red paeony root has the effects of nourishing blood and activating blood, and costustoot has the effects of strengthening spleen and activating qi and activating blood, and harmonizing yin and yang; radix Platycodi, with pungent and dispersed nature, has effects of dispersing lung qi, increasing Tilly water, and ascending medicine; bitter orange can reduce bitter taste, reduce qi and relieve distention, and relieve chest stuffiness and promote qi circulation, and the two medicines are compatible, one ascending and one descending and one diffusing and one dispersing, so as to achieve the effects of promoting qi circulation and dispersing stagnation; licorice root, radix Glycyrrhizae Praeparata regulates the drugs. In conclusion, the whole formula can soothe liver and relieve depression and harmonize qi and blood, thereby achieving the effects of smoothening qi movement and restoring normal nerve function.
The ramulus Cinnamomi is twig of Cinnamomum cassia Presl of Lauraceae. Pungent, sweet and warm. Sweat and muscle release, warmth and unblock meridians, and strengthen yang and qi. It is indicated for wind-cold type common cold, cold congealing and blood stagnation type pain, phlegm retention, water retention and palpitation.
The radix Paeoniae Rubra is dry root of Paeonia lactiflora Pallas or Paeonia suffruticosa Pallas of Ranunculaceae. Bitter and slightly cold. Clear heat and cool blood, dissipate blood stasis and relieve pain. Can be used for treating heat-induced ying blood, toxic heat, speckle, hematemesis, epistaxis, conjunctival congestion, swelling and pain, liver Yu Xie pain, amenorrhea, dysmenorrhea, abdominal pain, traumatic injury, carbuncle, swelling and sore.
"Fructus Aurantii" is the dried immature fruit of Citrus aurantium and its cultivar. Bitter, pungent and sour, slightly cold. Regulate qi and relax middle energizer, promote the circulation of qi and relieve distention. Can be used for treating qi stagnation in chest and hypochondrium, distention and pain, food stagnation, phlegm retention, internal stagnation, and organ prolapse.
Radix Platycodi is dry root of radix Platycodi of Campanulaceae. Bitter and pungent. Disperse lung, relieve sore throat, eliminate phlegm and expel pus. Can be used for treating cough with excessive phlegm, chest distress, pharyngalgia, hoarseness, pulmonary abscess, and pus discharge.
The radix aucklandiae is dry root of radix aucklandiae of Compositae. Pungent and bitter, warm. Promoting qi circulation, relieving pain, invigorating spleen, and resolving food stagnation. Can be used for treating chest and hypochondrium pain, abdominal distention, diarrhea, dyspepsia, anorexia.
The Glycyrrhrizae radix is dry root and rhizome of Glycyrrhrizae radix of Leguminosae, glycyrrhiza uralensis Fisch. Or Glycyrrhiza glabra. Sweet and flat. Spleen invigorating, qi replenishing, heat and toxic materials clearing away, phlegm eliminating, cough relieving, pain relieving, and medicines regulating. Can be used for treating weakness of spleen and stomach, listlessness, debilitation, palpitation, short breath, cough with excessive phlegm, abdominal pain, limb spasm, carbuncle, swelling, sore and toxic materials, and relieving drug toxicity and intensity.
The invention has the following beneficial effects:
(1) The traditional Chinese medicine composition disclosed by the invention is modified and cut on the basis of traditional prescription cassia twig decoction and four-way powder for soothing liver and relieving depression and strengthening spleen and promoting qi, and further adopts a method of soothing liver and relieving depression and regulating qi and blood so as to obviously enhance the effect of the traditional Chinese medicine composition on preventing and treating anxiety.
(2) The traditional Chinese medicine composition has the effects of regulating neurotransmitters, repairing nerve injury and improving qi and blood circulation.
Drawings
Fig. 1: representative photographs (magnification X20) of the effect of the inventive traditional Chinese medicine composition on the expression of rat hippocampal tissue pyrosis related protein GSDMD.
Fig. 2: representative photographs (magnification X20) of the effect of the inventive traditional Chinese medicine composition on rat hippocampal tissue inflammatory body NLRP3 expression.
Fig. 3: representative photographs of the effects of the inventive herbal composition on rat neuronal cells (magnification X2500).
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, are all commercially available products.
Preparation examples:
Example 1:
10g of cassia twig, 20g of red paeony root, 15g of bitter orange, 10g of platycodon root, 10g of costustoot and 5g of liquorice.
Total amount: 70g of
Pulverizing all the materials into fine powder, decocting in distilled water 1000mL for 1.5 hr, filtering to obtain primary filtrate, decocting the residue in distilled water 1000mL for 1.5 hr, filtering to obtain secondary filtrate, mixing the primary filtrate and the secondary filtrate, and concentrating under reduced pressure to obtain concentrated solution 300 mL.
Example 2:
5g of cassia twig, 10g of red paeony root, 10g of bitter orange, 5g of platycodon root, 5g of costustoot and 3g of liquorice.
Total amount: 38g
The preparation method is the same as in example 1.
Comparative example 1:
9g of cassia twig, 9g of white paeony root, 6g of liquorice, 9g of ginger and 10g of jujube.
Total amount: 43g
The preparation method is the same as in example 1.
Comparative example 2:
27g of fructus aurantii, 18g of platycodon grandiflorum, 18g of elecampane and 9g of liquorice.
Total amount: 72g
The preparation method is the same as in example 1.
Comparative example 3:
radix Paeoniae alba 6g, fructus Aurantii Immaturus 6g, bupleuri radix 6g, glycyrrhrizae radix 6g.
Total amount: 24g
The preparation method is the same as in example 1.
Comparative example 4:
9g of cassia twig, 15g of white paeony root, 6g of immature bitter orange, 6g of radix bupleuri, 12g of liquorice, 9g of ginger and 10g of Chinese date.
Total amount: 67g
The preparation method is the same as in example 1.
Comparative example 5:
10g of cassia twig, 20g of white paeony root, 15g of immature bitter orange, 10g of platycodon root, 10g of costustoot and 5g of liquorice.
Total amount: 70g of
The preparation method is the same as in example 1.
Comparative example 6:
23g of cassia twig, 23g of white paeony root and 23g of liquorice.
Total amount: 69g
The preparation method is the same as in example 1.
Positive drug group:
Xiaoyao pill: purchased from Jilin tetracyclic Australian Kao pharmaceutical Co., ltd (national drug standard Z22020678). Dissolving XIAOYAO pill in physiological saline, and converting into SD rat stomach-lavage dosage according to 9 pills per day for adult.
Effect examples: pharmacodynamics research of the traditional Chinese medicine composition for preventing and treating anxiety disorder
1. Preparation of anxiety animal model establishment:
110 SPF-class SD male rats of 6-8 weeks old, body weight (160+ -10 g), purchased from Peking Violet laboratory animal technologies Co., ltd., animal production license number: SCXK (Beijing) 2016-0011. The animals were bred in Beijing, the animal experiment center of the company of the Uygur medicine science and technology, license number: SYXK (Beijing) 2022-0007, temperature (22+ -2deg.C), humidity (50+ -10)%, and 12h light/dark period. The animals are free to ingest and drink water.
2. Modeling, grouping and administration of animals
Adaptive feeding was performed for 3 days, and a rat anxiety model was established using 3 week empty bottle stress. The specific operation is as follows: training with regular feeding for 7 days, 9:00-9:10 and 21:00-21:10, animals are drunk for 10 minutes, and the water bottle is removed for water inhibition in the rest time. On day 21 of 8: 00-21:10 start the stress experiment, and the uncertain empty bottle stimulation was given according to the experimental procedure for the above 2 time periods, the administration of the stimulation was irregular, and maintained 1 or 2 times per day for 21 days.
SD rats were randomly divided into 10 groups after molding: example 1 group, example 2 group, comparative example 1 group, comparative example 2 group, comparative example 3 group, comparative example 4 group, comparative example 5 group, comparative example 6 group, model control group, positive control group; 10 untreated SD rats were also used as normal control groups. Example 1, example 2, comparative example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5, and comparative example 6 were administered with the concentrates prepared in the "preparation example" section described above, and the amount of the lavage was 1.6 mL/day to ensure consistency between the groups.
The control group and the model group were given 1.6 mL/day of physiological saline for gastric lavage. The stomach was irrigated 1 time a day for 21 consecutive days. The administration dosage is converted into the gastric lavage administration dosage of the rat according to the clinical recommended dosage of the daily oral administration of the adult of 70 kg and the body surface area conversion coefficient of the adult and the rat. The method of dosing can be found, for example, in Xu Shuyun, pharmaceutical Experimental methodologies, anhui province, anhui university of medical science, 2002-01-01; zhao Wei, sun Guozhi. Conversion of dosage between different experimental animals [ J ]. Animal husbandry and veterinary science and technology information, 2010 (05): 52-53.
3. Tissue specimen collection
After the last administration, the patients are fasted and not forbidden for 12 hours. 10% chloral hydrate was anesthetized by intraperitoneal injection at 4 mL/kg. The supine position is fixed on an operating table. The abdomen skin is clamped by a left hand-held pick-up, a small opening is cut under the xiphoid process by a right hand-held scissors, the xiphoid process is clamped by a hemostatic forceps to lift up, ribs are cut up to the lower collar at the positions of 2cm on two sides of the xiphoid process, the chest is turned up, and the heart is exposed. A perfusion needle was inserted from the apex of the heart into the ascending aorta and clamped with a hemostat. The right auricle was cut. The hemostatic forceps close the abdominal aorta. Normal saline was withdrawn through the needle tube and injected into the heart through the perfusion needle until the two forelimbs and the two lungs of the rat became white (about 100 mL). The perfusion needle is connected with a paraformaldehyde infusion bottle arranged at the high position, and the perfusion is continued under the action of gravity. When the anterior limb and the neck of the rat are stiff, the fixing effect is good, and the perfused brain tissue is white and hard. Cutting off the head between the rear and cervical vertebrae, cutting off the scalp along the middle of the frequency top to remove soft tissues, gradually removing the parietal bone from the occipital bone macropore by using hemostatic forceps, fully exposing the brain, cutting off the brain nerve, and taking out the whole brain. General observations were made to dissect structures such as hippocampus, striatum, etc. Or placing the extracted brain in 30% sucrose buffer solution, and preserving in a refrigerator at 4deg.C to make frozen slices.
4. Effect index inspection
(1) Evaluation of anxiety behavior in rats by elevated plus maze experiment
The elevated plus maze experiment is developed on the basis of an elevated Y-shaped maze. The overhead cross maze consists of two open arms and two closed arms, wherein the two open arms and the two closed arms are crossed, the joint part is a central area, and the height of the joint part is a certain height from the ground. The principle is that the open-arm rats facing new things can generate curiosity to explore, and have darkish nature (closed arms), and conflict behaviors of exploration and avoidance occur between the two, so that anxiety psychology is generated. The height of the whole maze from the ground is equivalent to the psychological that the human beings stand on the cliff side, and fear and anxiety of animals are easily caused. Anxiety behavior of rats can be evaluated by comparing the residence time and number of rats in the open and closed arms.
(2) Evaluation of anxiety behavior in rats in open field experiments
The open FIELD TEST analysis system is used for observing and researching various behaviors of experimental animals after neuropsychiatric changes and the experimental animals enter the open environment, such as fear of the animals to the new open environment and activities mainly in peripheral areas and less activities in central areas, but the exploratory characteristics of the animals promote the animals to generate the motivation of the animals to move in the central areas, and the anxiety psychology generated by the activities can be observed. The experimental animals were placed rapidly in the central region of the experimental box and immediately left. Animal behavioural analysis software was turned on to automatically record the animal's activity in the box, usually 15 minutes. Total distance, speed and movement time were recorded to evaluate anxiety behavior in rats.
(3) Immunohistochemical detection of expression of rat hippocampal tissue pyrosis-related protein GSDMD
Shaking and washing the frozen slices with pure water for 3 times, each time for 3min; the microwave oven, sodium citrate antigen repair liquid (pH6.8), preheat first, then put the flake into repair liquid, high fire boils to boiling, turn to the middle fire and keep 10min; then naturally cooling to room temperature; shaking and washing with PBS for 3 times, each time for 5min; grouping pen ring tissues; permeabilizing the membrane with 0.3% Triton X-100 (PBS as solvent) for 15min, and shaking with PBS; blocking endogenous peroxidase activity: incubating the mixture in 3% hydrogen peroxide solution at normal temperature for 15min; shaking and washing with PBS for 3 times, each time for 3min; serum blocking: sealing 10% secondary antibody homologous serum for 30min at normal temperature; incubating primary antibody: dripping primary antibody (GSDMD), taking out the whole tissue, and incubating for 12-18 hours at 4 ℃ in a wet box; rewarming for 30min: the wet box was taken out of the refrigerator and allowed to stand for 30min in a room temperature environment. Shaking and washing with PBS for 3 times, each time for 5min; incubating the secondary antibody: and (3) dripping biotin-labeled secondary antibody, and incubating for 15min at normal temperature. Shaking and washing with PBS for 3 times, each time for 5min; incubation of HRP: horseradish peroxidase-labeled streptavidin (diluted in PBS) was dropped and incubated at room temperature for 15min. Shaking and washing with PBS for 3 times, each time for 5min; DAB color development: DAB working solution is prepared at present, each slice is covered completely, the reaction time is counted by a stopwatch after observation under a mirror, and the same index controls the same reaction time as much as possible. Stopping the reaction with tap water after the color development is finished, and flushing with tap water for 5min; hematoxylin staining for 3min and running water washing for 10min; differentiation of 1% hydrochloric acid alcohol for 2s, and flushing with running water for 1min. After sealing, each slice in each group was randomly picked for at least 3 20-fold fields of view. When photographing, the tissue is filled with the whole visual field as much as possible, and the consistency of the background light of each photo is ensured. The same brown-yellow color was chosen as a unified standard for judging the positivity of all photographs using Image-Pro Plus 6.0 software, and each photograph was analyzed to obtain a cumulative optical density value (IOD) positive for each photograph.
(4) Immunofluorescence staining method for determining expression of rat hippocampal tissue inflammatory body NLRP3
The frozen sections were air-dried at room temperature for 15 minutes, the tissue to be stained was circled with a histochemical pen, and immersed in PBS for 10 minutes to remove OCT. Blocking the sections with PBS containing 10% normal goat serum for 1 hour at room temperature, washing with PBS 3 times for 10 minutes each; NLRP3 mab (1:200) was then added and incubated overnight at 4 ℃; the PBS was washed 3 times for 10 minutes each. IgG secondary antibody (1:50) of sheep anti-rat was added and incubated at 37C for 1 hour; washing with PBS for 3 times, each for 15 minutes; after sealing, the results were observed with a fluorescence microscope and photographed. At least 3 20-fold fields per slice within each group were randomly selected for taking pictures. When photographing, the tissue is filled with the whole visual field as much as possible, and the consistency of the background light of each photo is ensured. The Image-Pro Plus 6.0 software was used to convert the red fluorescence monochrome picture to a black and white picture and then the same black was chosen as a unified standard for judging the positivity of all pictures, and each picture was analyzed to obtain a cumulative optical density value (IOD) positive for each picture.
(5) Transmission electron microscope detection of rat neuronal cells
The fresh brain tissue is used for determining the material-taking part, mechanical injuries such as traction, contusion, extrusion and the like are reduced as much as possible, the tissue volume is generally not more than 1mm multiplied by 1mm, and the tissue is rapidly put into an electron microscope fixing solution for fixing for 2-4 hours at the temperature of 4 ℃. The solution was rinsed 3 times with 0.1M phosphate buffer PBS (pH 7.4) for 15min each. 1% osmium acid 0.1M phosphate buffer PBS (pH 7.4) was fixed at room temperature (20 ℃ C.) for 2 hours. The solution was rinsed 3 times with 0.1M phosphate buffer PBS (pH 7.4) for 15min each. The tissue is dehydrated in a gradient alcohol of 50% -70% -80% -90% -95% -100% -100% and acetone of 100% -100% in sequence in an upward way for 15min each time. Acetone:812 embedding agent=1:1, 2-4h, acetone:812 embedding agent=1:2 permeates overnight, pure 812 embedding agent is 5-8h, pure 812 embedding agent is poured into embedding plate, and sample is inserted into embedding plate and then oven is carried out at 37deg.C overnight. Polymerizing for 48h at 60 ℃. Ultrathin section of 60-80 nm. Uranium-lead double staining (2% uranium acetate saturated alcohol solution, lead citrate, 15min each), and slicing at room temperature overnight. And (5) observing under a transmission electron microscope, and collecting and analyzing images.
5. Statistical method
Statistical analysis and statistical mapping were performed using GRAPHPAD PRISM 8.0.0, experimental results were obtainedAnd (3) representing. The mean comparison between groups uses One-Way ANOVA analysis, and the difference represented by P <0.05 is statistically significant.
6. Results
(1) Overhead maze test
The results of the overhead plus maze experiments show that the proportion of times of entering the open arms and the proportion of residence time of the open arms can be increased in the groups of the embodiment 1 and the embodiment 2, and the exercise activity is improved and is superior to that of the comparative group and the positive drug group. The results are shown in Table 1.
Table 1: SD rat enters the open arm proportion of times and open arm residence time proportion, n=3）%
P < 0.05 compared with the control group; p < 0.05 compared with model group
(2) Evaluation of anxiety-like behavior in rats in open field experiments
The open field experimental results show that both example 1 and example 2 can increase the total distance, increase the speed and lengthen the movement time. The results are shown in Table 2.
Table 2: SD rat total distance, speed and movement time ratio in open field, n=3）
P < 0.05 compared with the control group; p < 0.05 compared with model group
(3) Immunohistochemical detection of expression of rat hippocampal tissue pyrosis-related protein GSDMD
The immunohistochemical results show that example 1 and example 2 can increase the expression of rat pyrosis related protein GSDMD. The results are shown in FIG. 1 and Table 3.
Table 3: cumulative optical density value (IOD) of SD rat hippocampal tissue GSDMD, n=6）
P < 0.05 compared with the control group; △ P < 0.05 compared to model group
(4) Immunofluorescent staining assay for expression of rat hippocampal tissue inflammatory bodies NLRP 3.
The immunofluorescence staining results suggest that example 1 and example 2 can reduce the expression of rat hippocampal tissue inflammatory body NLRP 3. The results are shown in FIG. 2 and Table 4.
Table 4: cumulative optical density value (IOD) of SD rat hippocampal tissue inflammatory body NLRP3, n=6）
P < 0.05 compared with the control group; p < 0.05 compared with model group
(5) Neuronal cells
The neuronal cells play an important role in the development of anxiety disorder, and the changes in the morphology and number of the neuronal cells were observed by transmission electron microscopy, and as a result, it was found that example 1 and example 2 can repair neuronal cells, reduce cellular edema and swelling of cellular organelle structures. The results are shown in FIG. 3.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. A traditional Chinese medicine composition for preventing and treating anxiety is characterized in that: the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 1-20 parts of cassia twig, 5-30 parts of red paeony root, 5-25 parts of fructus aurantii, 1-20 parts of platycodon grandiflorum, 1-20 parts of costustoot and 1-10 parts of liquorice.
2. The traditional Chinese medicine composition according to claim 1, wherein: the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-10 parts of cassia twig, 10-20 parts of red paeony root, 10-15 parts of fructus aurantii, 5-10 parts of platycodon grandiflorum, 5-10 parts of costustoot and 3-5 parts of liquorice.
3. The traditional Chinese medicine composition according to claim 2, wherein: the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5 parts of cassia twig, 10 parts of red paeony root, 10 parts of bitter orange, 5 parts of platycodon root, 5 parts of costustoot and 3 parts of liquorice.
4. The traditional Chinese medicine composition according to claim 2, wherein: the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts of cassia twig, 20 parts of red paeony root, 15 parts of fructus aurantii, 10 parts of platycodon grandiflorum, 10 parts of elecampane and 5 parts of liquorice.
5. The method for preparing a traditional Chinese medicine composition according to any one of claims 1 to 4, characterized in that: the preparation method comprises the following steps: the method for preparing the medicine according to the invention comprises the steps of weighing raw materials according to the parts by weight of any one of claims 1 to 4, pre-treating, extracting and concentrating.
6. Use of a traditional Chinese medicine composition according to any one of claims 1 to 4 or prepared by the preparation method of claim 5 in the preparation of a medicament.
7. Use according to claim 6, characterized in that: the medicament is used for preventing or treating anxiety disorder.
8. Use according to claim 6, characterized in that: the medicament is in solid form, semi-solid form or liquid form.
9. Use according to claim 8, characterized in that: the medicine is decoction pieces, decoction, powder, oral liquid, paste, granule, tablet or capsule.
10. Use according to claim 9, characterized in that: the medicine is decoction or granule.
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Citations (5)
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