Traditional Chinese medicine composition for treating cognitive disorder and sleep disorder and preparation method thereof

Abstract

The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for treating cognitive disorder and sleep disorder and a preparation method thereof. The traditional Chinese medicine composition is prepared from angelica sinensis, ligusticum wallichii, radix paeoniae alba, radix rehmanniae praeparata, uncaria, caulis spatholobi, selfheal, semen cassiae and corydalis tuber, has better effects of improving cognitive disorder and sleep disorder, and also has anti-inflammatory and anti-oxidation effects.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating cognitive impairment and sleep disorders and a preparation method thereof. Background Art

[0002] Cognitive disorder, also known as cognitive deficit, is a pathological process in which abnormalities occur in the brain's higher-level intellectual processing related to learning, memory, and thinking and judgment, leading to learning and memory disorders, accompanied by changes such as aphasia, apraxia, and agnosia. It can be caused by a variety of factors, including craniocerebral trauma, cerebrovascular disease, brain aging, chronic systemic diseases, environment, and mental and psychological abnormalities. The basis of cognition is the normal function of the cerebral cortex. Any factor that causes abnormalities in the function and structure of the cerebral cortex can lead to cognitive impairment. Due to the complexity of brain function and the interrelationship of different types of cognitive impairment, that is, cognitive problems in one aspect can cause cognitive abnormalities in another or multiple aspects, cognitive impairment is one of the most difficult problems in the diagnosis and treatment of brain diseases.

[0003] Sleep refers to a state of resting conscious activity with eyes closed. It is a crucial process for replenishing energy used during daytime activities and recovering from fatigue accumulated from physical activity. It is also the time when growth hormone, essential for human growth, is secreted at its highest levels. Sleep disorders are often manifested in sleep duration, sleep quality, rhythmic disturbances, and abnormal sleep behaviors. With the accelerating pace of life and increasing social pressures, the incidence of sleep disorders is gradually increasing, and the aging population is contributing to the increasing prominence of sleep disorders. Treatments for sleep disorders currently primarily include non-pharmacological and pharmacological therapies. Non-pharmacological treatments for sleep disorders include cognitive behavioral therapy for insomnia, sleep hygiene education, relaxation therapy, imagery, sleep restriction, and stimulus control therapy, and should be considered first-line treatments. Drug treatments for sleep disorders primarily include Western medications: benzodiazepines, antidepressants, barbiturates, other antipsychotics, and other drugs, all of which have varying degrees of side effects. Therefore, the development of traditional Chinese medicine compositions that can treat sleep disorders with minimal side effects is highly desirable.

[0004] Cognitive impairment typically presents as a mixed syndrome of underlying deficiency and superficial excess. Deficiency syndromes are generally due to kidney-essence or qi-blood deficiency, leading to malnutrition of the brain and marrow. Excess syndromes are generally caused by blood stasis, phlegm, and fluid retention. From the perspective of collateral stasis theory, this is a symptom of cerebral collateral blockage and the interplay of deficiency and stasis. Initially, qi stagnation slows and stagnates the flow of qi, blood, and body fluids, producing pathological products such as turbid phlegm and blood stasis. Simultaneously, turbid phlegm and blood stasis act as toxic pathogens, damaging tissues and the body, leading to a series of pathological changes such as microatherosclerotic plaques, microaneurysms, and lacunar infarctions, leading to cerebral collateral blockage. Blood deficiency, coupled with prolonged stasis and lack of new blood production, leads to malfunction of the brain's collaterals and a lack of nourishment for the spirit. Alternatively, prolonged stasis may transform into heat, toxicating the brain's collaterals, causing malfunction of the spirit and resulting in cognitive impairment.

[0005] The occurrence of sleep disorders is related to the blockage of brain meridians, insufficient marrow sea and malnutrition of the spirit, as well as the blockage of heart meridians and malnutrition of the mind. In addition, the ascending of liver fire can also disturb the spirit and cause insomnia and nightmares.

[0006] The existing technology lacks a safe and effective traditional Chinese medicine for treating cognitive impairment and sleep disorders. Summary of the Invention

[0007] In response to the above-mentioned problems and deficiencies, the present invention provides a traditional Chinese medicine composition (hereinafter referred to as the present composition). The composition comprises angelica sinensis, Chuanxiong rhizome, white peony root, Rehmannia root, Uncaria rhynchophylla, Millettia reticulata, Prunella vulgaris, Cassia seed, and Corydalis yanhusuo, and has a good effect in improving cognitive impairment and sleep disorders, and also has anti-inflammatory and antioxidant effects.

[0008] A traditional Chinese medicine composition, comprising the following raw materials in parts by weight:

[0009] 210 to 280 parts of Chinese Angelica sinensis,

[0010] 220 to 280 parts of Chuanxiong,

[0011] 160 to 230 parts of white peony root,

[0012] 160 to 250 parts of Rehmannia root,

[0013] 420-600 parts of Uncaria rhynchophylla,

[0014] 420-550 parts of Millettia reticulata,

[0015] 450 to 580 parts of Prunella Vulgaris,

[0016] 420 to 570 parts of Cassia seeds,

[0017] 220-290 parts of Corydalis yanhusuo.

[0018] Preferably, the Chinese medicine composition is made from the following raw materials in parts by weight:

[0019] 224.1 to 270.2 parts of Chinese Angelica sinensis,

[0020] 232.5 to 266.7 parts of Chuanxiong,

[0021] 178.1 to 217.2 parts of white peony root,

[0022] 181.0 to 233.0 parts of Rehmannia root,

[0023] 435.6 to 577.0 parts of Uncaria rhynchophylla,

[0024] 436.2 to 537.1 parts of Millettia reticulata,

[0025] 463.2 to 564.7 parts of Prunella Vulgaris,

[0026] 436.5 to 554.2 parts of Cassia seeds,

[0027] 232.0 to 275.6 parts of Corydalis yanhusuo.

[0028] Most preferably, the Chinese medicine composition is made from the following raw materials in parts by weight:

[0029] 253.5 parts of Chinese Angelicae Sinensis, 253.5 parts of Chuanxiong, 202.7 parts of White Peony Root, 202.7 parts of Rehmannia Glutinosa, 506.8 parts of Uncaria Rhynchophylla, 506.8 parts of Millettia Spatholobi, 506.8 parts of Prunella Vulgaris, 506.8 parts of Cassia Seed, and 253.5 parts of Corydalis yanhusuo.

[0030] In the above composition, the parts are parts by weight, and the weight is calculated based on the crude drug.

[0031] If the portion is in grams, a preparation having a dosage of 50-1000 times can be prepared. For example, if the preparation is made into tablets, 1000 tablets can be prepared, and each dosage can be 1-20 tablets, for a total of 50-1000 times. If the preparation is made into granules, 125 bags can be prepared, and each dosage can be 1-2 bags, for a total of 62.5-125 times.

[0032] The above composition is based on weight ratio, which can be increased or decreased according to the corresponding proportion during production. For example, large-scale production can be measured in kilograms or tons, and small-scale production can also be measured in milligrams. The weight can be increased or decreased, but the weight ratio of the raw medicinal materials between the components remains unchanged.

[0033] The above weight ratios are obtained through scientific screening. For special patients, such as those with severe or mild illnesses, obese or thin patients, the ratios of the components can be adjusted accordingly, with an increase or decrease of no more than 100%, and the efficacy remains unchanged.

[0034] The single Chinese herbal medicines in the above composition, especially the adjuvant and auxiliary medicines, can also be replaced by appropriate Chinese herbal medicines with the same medicinal properties, and the medicinal effects of the Chinese herbal preparations after replacement remain unchanged.

[0035] The Chinese medicine composition of the present invention is typically prepared in the form of a pharmaceutical preparation for oral administration. The Chinese medicinal raw materials of the above-mentioned formula are extracted or otherwise processed to produce a pharmaceutically active substance. This substance is then used as a raw material, and a pharmaceutically acceptable carrier is added as needed, in accordance with conventional pharmaceutical preparation techniques. The active substance can be obtained by extracting the Chinese medicinal raw materials separately, extracting them together, or by other methods, such as pulverization, pressing, calcining, grinding, screening, percolation, extraction, water extraction, alcohol extraction, ester extraction, ketone extraction, chromatography, or the like. These active substances can be in the form of extracts, either dry extracts or fluid extracts, and can be prepared in different concentrations according to the different needs of the formulation.

[0036] The pharmaceutically active substance of the present invention may comprise 0.1-99.9% by weight of the formulation, with the remainder being a pharmaceutically acceptable carrier. The pharmaceutical formulation of the present invention is in a unit dosage form, where the unit dosage form refers to a unit of the formulation, such as a tablet, a capsule, a bottle of oral solution, or a bag of granules.

[0037] The pharmaceutical preparation of the present invention can be any pharmaceutically acceptable dosage form, including tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, electuary preparations, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, and patches. The preparation of the present invention is preferably an oral dosage form, such as capsules, tablets, oral liquids, granules, pills, powders, pills, pills, ointments, and the like.

[0038] The pharmaceutical preparation of the present invention, for oral administration, may contain commonly used excipients such as binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavorings and wetting agents, and the tablets may be coated if necessary.

[0039] Suitable fillers include cellulose, mannitol, lactose, and other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone, and starch derivatives, such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate.

[0040] Solid oral compositions can be prepared by conventional methods of mixing, filling, tableting, etc. Repeated mixing can be used to distribute the active substance throughout those compositions using large amounts of fillers.

[0041] Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be dry products that can be reconstituted with water or other suitable vehicles before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, emulsifiers, for example, lecithin, sorbitan monooleate or gum arabic; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as glycerol esters, propylene glycol or ethanol; preservatives, for example, methylparaben or propylparaben or sorbic acid, and, if desired, conventional flavorings or coloring agents.

[0042] For injections, a liquid unit dosage form is prepared containing the active substance of the present invention and a sterile carrier. Depending on the carrier and concentration, the compound can be suspended or dissolved. Solutions are typically prepared by dissolving the active substance in a carrier, sterilizing by filtration before filling into a suitable vial or ampoule, and then sealing. Excipients such as a local anesthetic, preservatives, and buffers may also be dissolved in the carrier. To improve stability, the composition can be frozen after filling into the vial, and the water removed under vacuum.

[0043] The pharmaceutical preparation of the present invention can be selectively added with a suitable pharmaceutically acceptable carrier when it is prepared into a medicament. The pharmaceutically acceptable carrier is selected from the group consisting of mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, sodium calcium EDTA, carbonates, acetates, phosphates of monovalent alkali metals or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, and the like.

[0044] The pharmaceutical preparation of the present invention can be taken three times a day, with 1-20 doses each time, such as 1-20 bags, granules or tablets, to determine the dosage and administration according to the patient's condition.

[0045] The Chinese medicine preparation of the present invention can be prepared by the following method.

[0046] All the medicinal materials are decocted with water twice, each time for 1 to 2 hours, the extract is filtered, the filtrate is concentrated under reduced pressure, and dried to obtain a composite extract powder; appropriate amounts of auxiliary materials are added to prepare a pharmaceutical preparation that can be taken.

[0047] Preferably, the pharmaceutical preparation is a granule, and the preparation method is as follows: take all the medicinal materials according to the above weight, add water and boil twice, add 5 times the amount of water for the first time, boil for 2 hours, add 4 times the amount of water for the second time, boil for 1 hour, filter the extract, and concentrate the filtrate under reduced pressure to a relative density of 1.03-1.08 (60°C ± 2°C), spray dry to obtain a dry powder of the composition extract; add an appropriate amount of maltodextrin, mix well, dry-press and granulate to make 1000g.

[0048] Preferably, the spray drying equipment used in the spray drying is any equipment that can be used for drying fluid extracts, and the spray drying conditions are: the inlet air temperature is 140-190°C, the outlet air temperature is 80-105°C; the material temperature is maintained at 40-80°C during the spray drying operation;

[0049] More preferably, the spray drying conditions are as follows: the inlet air temperature is 170-180°C, the outlet air temperature is 95-105°C; and the material temperature is maintained at 50-60°C during the spray drying operation.

[0050] The traditional Chinese medicine composition of the present invention has the effects of nourishing blood, calming the liver, and promoting blood circulation and unblocking meridians, and can be used to prepare medicines for treating cognitive impairment and / or sleep disorders.

[0051] The Chinese medicine composition of the present invention can also be used to prepare anti-inflammatory and / or antioxidant drugs.

[0052] The Chinese medicine composition of the present invention can also be used to prepare drugs for resisting cell aging.

[0053] Explanation of the present invention's composition: Angelica sinensis nourishes and promotes blood circulation, primarily treating the primary symptom of liver blood deficiency; Chuanxiong rhizome ascends the head and eyes, relieves stagnation in the middle, regulates menstrual flow downward, and promotes blood circulation, qi circulation, and relieves pain, primarily treating the primary symptom of encephalopathy. Rehmannia glutinosa and white peony root nourish yin and blood, nourish the liver and kidneys, and reduce the hyperactivity of yang, assisting the main drug Angelica sinensis in strengthening its blood-tonifying effect. Uncaria rhynchophylla is sweet and cooling, and enters the liver and pericardium meridians. It clears heat and soothes the liver, suppresses yang, and relieves wind and spasms. It primarily treats the secondary symptom of hyperactivity of liver yang. Together with white peony root and Rehmannia glutinosa, it serves as a minister, assisting the main drug in nourishing blood, calming the liver, and relieving pain. Cassia seed and Prunella vulgaris are cold and cooling, clearing liver fire and suppressing hyperactivity of yang, serving as adjuvants to treat the secondary symptom of liver fire. They not only assist the main and minister drugs in treating dizziness and vertigo, but also prevent the excessive warming effects of yang-tonifying drugs such as Angelica sinensis, Chuanxiong, and Rehmannia glutinosa. In the prescription, Millettia reticulata has both blood-tonifying and blood-activating effects, while Corydalis yanhusuo activates blood circulation, promotes qi and relieves pain, and they are both guiding herbs. All the herbs work together to achieve the effects of nourishing blood, activating collaterals, calming the liver and clearing the liver.

[0054] Glossary:

[0055] The times are volume to weight ratios, such as L / kg, ml / g.

[0056] Beneficial technical effects of the present invention:

[0057] 1. The composition of the present invention adopts the whole-formula water extraction and dry pressing granulation technology, and adopts the spray drying method with specific parameters to obtain dry powder from the fluid extract through spray drying. It does not need to add auxiliary materials for drying at the same time, and does not add flavoring agents, and has a good bitterness reduction effect.

[0058] 2. The Chinese medicine composition of the present invention has good preventive and therapeutic effects on cognitive impairment and sleep disorders, and the preparation method of the Chinese medicine composition of the present invention is simple and suitable for large-scale production and promotion.

[0059] 3. The raw materials of this invention are all varieties listed in the Chinese Pharmacopoeia, have simple and safe medicinal flavors, do not contain medicinal materials marked as toxic according to legal standards or proven to be toxic by modern toxicology, and do not contain any of the 18 anti-toxic and 19 fear incompatibility taboos. Among them, Angelica sinensis, Prunella vulgaris, and Cassia seed are medicinal and edible varieties; Angelica sinensis, Chuanxiong, White Peony Root, Cassia seed, and Rehmannia root can be used in health care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Representative trajectory diagram of mice's activities in the target quadrant after the platform was removed in the water maze experiment of scopolamine model mice, A is the blank control group; B is the model group; C is the positive drug group; D is the low-dose group of the composition of the present invention; E is the high-dose group of the composition of the present invention.

[0061] Figure 2 The results of the effect of the composition of the present invention on PC12 cells induced by hydrogen peroxide: compared with the blank control group, ####p<0.0001, compared with the model group (drug concentration 0 group), *p<0.05, **p<0.01.

[0062] Figure 3A The effect of the composition of the present invention on the expression of interleukin-6 in BV2 cells induced by lipopolysaccharide; Figure 3B The effects of composition 1 and various concentrations of the composition of the present invention on the expression of interleukin-6 in BV2 cells induced by lipopolysaccharide; compared with the blank control group, ####p<0.0001; compared with the model group (0), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns: no significant difference.

[0063] Figure 4A The effect of composition 1 on HT-22 cells induced by galactose; Figure 4B The effect of the composition of the present invention on HT-22 cells induced by galactose; Figure 4CComparison of the effects of Composition 1 and the composition of the present invention at various concentrations on galactose-induced HT-22 cells. Compared with the blank control group, #### p < 0.0001; compared with the model group (drug concentration 0), *p < 0.05, **p < 0.01, ****p < 0.0001; ns: no significant difference.

[0064] Figure 5 Diagram of the rat taste preference experiment process.

[0065] Figure 6 Results of the rat taste preference experiment. * p<0.05. DETAILED DESCRIPTION

[0066] The present invention is further illustrated by the following examples, but is not intended to limit the present invention.

[0067] Example 1 Preparation of granules of the composition of the present invention

[0068] The composition of the present invention is prepared from the following raw materials in the following weight ratios:

[0069] Ratio 1

[0070] Angelica sinensis 253.5g, Chuanxiong 253.5g, White Peony Root 202.7g, Rehmannia Glutinosa 202.7g, Uncaria rhynchophylla 506.8g, Millettia reticulata 506.8g, Prunella vulgaris 506.8g, Cassia seed 506.8g, Corydalis yanhusuo 253.5g;

[0071] Ratio 2

[0072] Angelica 210g, Chuanxiong 220g, White Peony 160g, Rehmannia Glutinosa 160g, Uncaria 420g, Millettia Spatholobi 420g, Prunella Vulgaris 450g, Cassia Seed 420g, Corydalis 220g;

[0073] Ratio 3

[0074] Angelica 280g, Chuanxiong 280g, White Peony 230g, Rehmannia Glutinosa 250g, Uncaria 600g, Millettia Spatholobi 550g, Prunella Vulgaris 580g, Cassia Seed 570g, Corydalis 290g;

[0075] Ratio 4

[0076] Angelica sinensis 224.1g, Chuanxiong 232.5g, White Peony Root 178.1g, Rehmannia Glutinosa 181.0g, Uncaria rhynchophylla 435.6g, Millettia reticulata 436.2g, Prunella vulgaris 463.2g, Cassia seed 436.5g, Corydalis yanhusuo 232.0g;

[0077] Ratio 5

[0078] Angelica sinensis 270.2g, Chuanxiong 266.7g, White Peony Root 217.2g, Rehmannia Glutinosa 233.0g, Uncaria rhynchophylla 577.0g, Millettia reticulata 537.1g, Prunella vulgaris 564.7g, Cassia seed 554.2g, Corydalis yanhusuo 275.6g;

[0079] Preparation method:

[0080] The Chinese herbal medicine raw materials according to any of the above ratios are decocted with water twice, with 5 times the amount of water added for the first time and decocted for 2 hours, and with 4 times the amount of water added for the second time and decocted for 1 hour. The extract is filtered, and the filtrate is concentrated under reduced pressure to a relative density of 1.03-1.08 (60°C ± 2°C), spray-dried, and an appropriate amount of maltodextrin is added. The mixture is mixed and dry-pressed into granules to obtain 1000 g.

[0081] Example 2 Effect of the composition of the present invention on cognitive impairment in mice induced by scopolamine

[0082] Scopolamine (SCOP) was used to induce memory impairment in mice, thereby establishing a cognitive dysfunction model. The effect of the composition of the present invention on the behavior of the cognitive dysfunction model was evaluated to demonstrate the effect of the composition of the present invention in improving cognitive dysfunction.

[0083] 1. Experimental Materials:

[0084] 1.1 Experimental equipment

[0085] Open field test behavioral testing instrument, model XR-XZ301, Shanghai Xinruan Information Technology Co., Ltd.; Morris water maze test behavioral testing instrument, model XR-XM101, Shanghai Xinruan Information Technology Co., Ltd.; balance, model 390HA: manufacturer: Prism Company; centrifuge, model D3024R, manufacturer: American Cylojet Corporation.

[0086] 1.2 Experimental animals

[0087] Healthy, 6-week-old, SPF-grade KM mice weighing 18–22 g were purchased from Guangzhou University of Chinese Medicine (certificate number: 44005800014705, production license number: SYXK(粤)2018-0001). Animals were housed in the SPF-grade animal laboratory of the Experimental Animal Center of Guangzhou University of Chinese Medicine, with ample food and water, a temperature of 22 ± 2°C, a humidity of 40%–70%, and a 12-h light / 12-h dark cycle. All animal welfare and procedures were approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine and complied with the Regulations on the Administration of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China.

[0088] 1.3 Experimental Reagents

[0089] Experimental group: the composition of the application (ratio 1 in example 1), batch number: 20220801S, the clinical dosage is 12g / person / day, and the equivalent dosage of mice is 2.46g / kg. Positive drug: donepezil, batch number: C14567110, Shanghai Macklin Biochemical Technology Co., Ltd. Scopolamine: batch number C15676242, Shanghai Macklin Biochemical Technology Co., Ltd.

[0090] 2. Experimental method

[0091] 2.1 Experimental administration

[0092] After the mice were adaptively fed for one week, 0.9% normal saline, the composition of the application, and the clinical dosage of 12g / person / day were respectively administered by gavage according to the dosage conversion table. The equivalent dosage of mice is 2.46g / kg. The animals in each group were administered by gavage once a day, and the blank control group and the model group were administered by gavage with normal saline, for a total of 24 days; from the 16th day, 30 minutes after gavage with the above liquid, scopolamine (3mg / kg) was administered to induce memory impairment except for the blank control group, and the administration lasted until the 24th day. From the 16th day, the behavior test of mice was performed, the water maze experiment was performed from the 16th to the 22nd day, and the open field experiment was performed from the 23rd to the 24th day. Before the behavior test, the gavage administration and animal modeling were performed, that is, 30 minutes after gavage administration, the blank control group was injected intraperitoneally with normal saline, and the other groups were injected intraperitoneally with 3mg / kg / d of scopolamine, and 30 minutes later, the behavior test of mice was performed.

[0093] Table 1 Dosage and time of drug administration in scopolamine model of mice

[0094]

[0095] Dose conversion: the low-dose group of the composition of the application (clinical equivalent) is 2.46g / kg / d, and the high-dose group of the composition of the application (2 times the clinical equivalent) is set to 2 times the clinical dose, that is, 4.92g / kg. The intraperitoneal injection and gavage administration of mice are both calculated according to 0.1mL / 10g of body weight.

[0096] 2.2 Behavior test

[0097] 2.2.1 Water maze experiment

[0098] 1) Water maze adaptation training

[0099] On the 16th day, water maze adaptation training was performed. Each mouse was placed in the water from entry points 1 and 3 in turn. When placed in the water, the mouse faced the pool wall. The swimming time was recorded as 60 seconds. The mouse was allowed to swim freely in the pool. If the platform was not found within the specified time, the mouse was guided to the platform and stood for 20 seconds before being returned to the cage. If the mouse found the platform within the specified time, the actual time it took to find the platform (escape latency) was recorded and the system automatically stopped recording. The mouse was allowed to stand on the platform for 20 seconds, the moisture on the surface of the mouse was wiped off, and then returned to the cage.

[0100] 2) Positioning navigation experiment

[0101] Experiments were conducted on days 17-21, with each mouse undergoing four trials per day. Each time, the mouse was placed in the water from a different quadrant, facing the pool wall upon entry, and the mouse's escape latency was recorded. To prevent the mouse from finding the platform due to inertia, the order of entry points varied from day to day. If the mouse successfully found the escape platform within 60 seconds, the monitoring system automatically recorded the actual escape latency. If the mouse failed to find the escape platform within 60 seconds, the experimenter guided the mouse to the escape platform and allowed it to stand for 10 seconds, with the escape latency recorded as 60 seconds.

[0102] 3) Space exploration experiments

[0103] On the 22nd day, the platform was removed and a spatial exploration experiment was conducted. The mice were placed in the water at the junction of the second and third quadrants. The time, travel trajectory and average swimming speed of the experimental mice when they first crossed the platform within 60 seconds were recorded. The number of times they crossed the original platform position, the total distance and total time in the target quadrant were also recorded.

[0104] 2.2.2 Mine experiment

[0105] The open field test (OFT), also known as the open-box test, is a commonly used animal behavior experiment. The OFT can detect spontaneous movement and exploratory behavior in rats or mice. It is a method for evaluating the autonomous behavior, exploratory behavior, and stress levels of experimental animals in novel environments. For example, animals fearing new, open environments may focus on the peripheral areas and less on the central area, but their exploratory nature may also motivate them to move in the central area.

[0106] The experimental apparatus consists of an open-field reaction chamber and an automatic data acquisition and processing system. The rat open-field reaction chamber is 30 to 40 cm tall, with a base length of 100 cm. Its inner walls are painted black, and its bottom is evenly divided into 25 small 4 cm x 4 cm squares. A digital camera is mounted 2 meters above the chamber, with a field of view covering the entire open field. The open field is fully artificially illuminated, with adjustable "day" and "night" modes. During the day, four energy-saving lamps on either wall emit a simulated illumination of approximately 200 lux, while at night, an infrared light source on one wall provides illumination. Experimenters, computers, and other equipment are located in another room to minimize disturbance to the animals. Background noise in the laboratory is kept below 65 decibels.

[0107] The open field test is carried out on the 23rd-24th day in a quiet environment. The animal is placed in the center of the bottom of the box, and video and timing are carried out at the same time. Stop video recording after a certain period of observation. The observation time can be determined according to the experiment, usually 3 to 5 minutes. Clean the inner wall and bottom of the box to prevent the remaining information of the animal from the last time (such as the animal's defecation, urine, and odor) from affecting the results of the next test. Replace the animal and continue the experiment. The observable parameters vary depending on the design of the computer software, such as the time the animal stays in the central grid per unit time, the number of grids crossed by a certain limb is the horizontal score, the number of times the hind limbs stand is the vertical score, the number of grooming times, the number of urination and defecation times; movement speed, movement distance, rest time, side movement distance, central movement distance, etc.

[0108] 3. Statistical methods:

[0109] The data were expressed as mean ± standard error (Mean ± SEM). One-way analysis of variance (ANOVA) or non-parametric test was used to analyze the differences between the groups. P < 0.05 was used to judge the differences between the groups.

[0110] 4. Experimental Results

[0111] 4.1 Morris water maze test results

[0112] Table 2 shows the total distance traveled by mice in the target quadrant after the platform was removed. The longer the total distance traveled in the target quadrant, the stronger the spatial memory ability of the mice. Compared with the blank control group, the model group's activity distance in the target quadrant was significantly reduced (P<0.05); compared with the model group, the target quadrant activity distances of the positive drug group and the low-dose group of the composition of the present invention were significantly increased (P<0.05). The results of the time spent by mice in the target quadrant showed that compared with the blank control group, the model group's activity time in the target quadrant was significantly reduced (P<0.05); compared with the model group, the target quadrant activity time of the positive drug group, the low-dose group and the high-dose group of the composition of the present invention were significantly increased (P<0.05). From the above results, it can be seen that intraperitoneal injection of scopolamine can lead to learning and memory disorders in mice, and the composition of the present invention has an improving effect on the learning and memory of scopolamine-induced model mice. The activity trajectory of mice in the target quadrant is shown in Figure 2. Figure 1 The longer the trajectory in the target quadrant, the stronger the mouse's spatial memory ability.

[0113] Table 2 Results of the water maze test in scopolamine model mice

[0114]

[0115] Note: Compared with the blank control group, ## p<0.05, compared with the model group, * p<0.05, ** p<0.01, *** p<0.001

[0116] 4.2 Open field test results

[0117] Table 3 shows the effect of the composition of the present invention on the spontaneous movement of mice with scopolamine-induced memory impairment. Compared with the blank control group, after intraperitoneal injection of scopolamine in the model group mice, the spontaneous movement and exploratory activities of the mice decreased, and the activity distance in the central area was significantly reduced (P<0.05); compared with the model group, the activity distance in the central area of ​​the low-dose and high-dose groups of the composition of the present invention were significantly increased (P<0.05). In addition, compared with the blank control group, the activity time in the central area of ​​the model group was significantly reduced (P<0.001); compared with the model group, the activity time in the central area of ​​the low-dose and high-dose groups of the composition of the present invention were significantly increased (P<0.01). It shows that the composition of the present invention has an improving effect on the spontaneous movement and exploratory behavior of scopolamine model mice.

[0118] Table 3 Results of open field test in scopolamine model mice

[0119]

[0120] Note: Compared with the blank control group, #p<0.05, ### p<0.001, compared with the model group, * p<0.05, ** p<0.01

[0121] 5. Experimental Conclusion

[0122] The composition of the present invention can improve memory impairment and spontaneous movement behavior of a mouse model and improve cognitive impairment.

[0123] Example 3 Effect of the composition of the present invention on sleep in rats

[0124] A rat sleep model was established through a sodium pentobarbital-induced rat sleep experiment to evaluate the effect of the composition of the present invention on the sleep of rats, thereby demonstrating the sleep-improving effect of the composition of the present invention.

[0125] 1. Experimental equipment

[0126] Balance, model: BCE423l-1CCN, manufacturer: Sartorius Scientific Instruments Co., Ltd.; electronic balance, model: PL203, manufacturer: Mettler-Toledo Instruments Co., Ltd.; 50 ml drinking water bottle, model: ZK-SL-50, manufacturer: Henan Zhike Hongrun Environmental Protection Technology Co., Ltd.; rat gavage needle, model: ZK-GWZ-18Z, manufacturer: Henan Zhike Hongrun Environmental Protection Technology Co., Ltd.

[0127] 2. Experimental Animals

[0128] Four-week-old male SPF SD rats, weighing 100-120 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. (certificate number: 110322231103858115, production license number: SCXK (Beijing) 2019-0008). After purchase, the rats were placed in the animal room of Hebei University of Technology. The animal room environment maintained a temperature of 23±2°C, a humidity of 40-70%, and a 12-hour light and dark alternation. No more than 5 animals were kept in each cage, and the bedding was changed three times a week. In case of fighting, the rats were kept in a single cage. Rats were free to eat and drink during the breeding process, and subsequent experiments began after one week of adaptation to the animal room. This experiment followed the experimental operating procedures of the School of Life Sciences and Health Engineering of Hebei University of Technology and was approved by the Ethics Committee of Hebei University of Technology.

[0129] 3. Experimental reagents

[0130] The composition group of the present invention (ratio 1 in Example 1), batch number: 20220801S: 1.24 g / kg after conversion according to the clinical equivalent was administered by gavage;

[0131] Other reagents: Sodium pentobarbital, Beijing Solebao Technology Co., Ltd., catalog number: SW1098.

[0132] 4. Experimental methods

[0133] As shown in Table 4, 10 SD rats were divided into 2 groups: a blank control group and a composition group of the present invention. The rats were given the drug by gavage once a day for 7 consecutive days one week before modeling.

[0134] Table 4. Animal grouping and drug administration

[0135]

[0136] Dosage design: The proposed clinical dosage of the composition of the present invention is 12 g / person / day. The equivalent clinical dose for rats is 12 g / 60 kg (adult body weight) x 6.2 (conversion factor) = 1.24 g / kg. This experiment used a clinically equivalent dose of 1.24 g / kg.

[0137] 4.1 Effect of the composition of the present invention on sleep induced by sodium pentobarbital in mice

[0138] On the 8th day, 30 minutes after the drug treatment, all groups except the normal control group were intraperitoneally injected with a suprathreshold dose (40 mg / kg) of sodium pentobarbital. The sleep latency and sleep duration induced by the suprathreshold dose of sodium pentobarbital in rats were measured within 30 minutes.

[0139] 4.2 Data processing method:

[0140] Statistical methods: Data were expressed as mean ± standard deviation (Mean ± SEM). One-way analysis of variance (ANOVA) or non-parametric test was used to analyze the differences between the groups. P < 0.05 was used to judge the differences between the groups.

[0141] 5. Experimental Results

[0142] Effect of the composition of the present invention on sleep induced by sodium pentobarbital

[0143] As shown in Table 5, compared with the control group, the sleep latency of the rats in the composition of the present invention was reduced, and there was a significant difference (p<0.05).

[0144] Table 5 Sleep experiment data induced by sodium pentobarbital

[0145]

[0146] *p<0.05 compared with the blank control group.

[0147] 6. Experimental Conclusion

[0148] The experiment of observing the sleeping state of rats using a sleep model induced by intracavitary injection of sodium pentobarbital showed that the composition of the present invention has a certain effect of improving sleep.

[0149] Example 4 Protective Effect of the Composition on Hydrogen Peroxide-Induced Oxidative Damage in PC12 Cells

[0150] An in vitro oxidative damage cell model was established by inducing oxidative damage in PC12 cells by hydrogen peroxide (H2O2), and the effect of the composition of the present invention on the cell survival rate in the oxidative damage model was evaluated to reflect the antioxidant effect of the traditional Chinese medicine composition.

[0151] 1. Experimental Materials:

[0152] PC12 cells: The experiment used highly differentiated PC12 cells derived from pheochromocytoma of the adrenal medulla of adult rats (Gaining Biotechnology Co., Ltd., CM-R011).

[0153] The Chinese medicine composition of the present invention (ratio 1 in Example 1) (batch number: 20220801S).

[0154] PBS, Beijing Solebow Technology Co., Ltd., batch number: 20220609; fetal bovine serum (FBS), Zhejiang Tianhang Biotechnology Co., Ltd., batch number: 21050701; double-antibody, Beijing Solebow Technology Co., Ltd., batch number: 20220319; trypsin digestion solution, Beijing Solebow Technology Co., Ltd., batch number: 20220521; thiazole blue (MTT), Anhui White Shark Biotechnology Co., Ltd., batch number: 70080125; hydrogen peroxide solution (H2O2), Beijing Institute of Chemical Reagents Co., Ltd., batch number: 70080125.

[0155] 2. Experimental Methods

[0156] 0, 2, 10, 50, 250, and 500 μg / mL were selected as drug concentrations for subsequent experiments. The experiment used 400 μM hydrogen peroxide to treat cells. PC12 cells containing hydrogen peroxide were treated with the composition of the present invention for 24 hours, the supernatant was discarded, and thiazolyl blue solution was added. After 4 hours, the absorbance was measured using a microplate reader.

[0157] 3. Statistical processing:

[0158] The data were expressed as mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) or non-parametric test was used to analyze the differences between the groups. P < 0.05 was used to judge the differences between the groups.

[0159] 4. Experimental results:

[0160] The protective effect of the composition of the present invention on the oxidative damage of PC12 cells caused by hydrogen peroxide is expressed as cell survival rate. The higher the cell survival rate, the better the drug effect. The results are shown in Table 6 and Figure 2As shown, the composition of the present invention at a concentration of 50 μg / ml or above can increase the cell survival rate of PC12 cells after hydrogen peroxide-induced damage, and the difference is significant (P<0.05).

[0161] Table 6 Cell survival rate of the composition of the present invention on PC12 cells damaged by hydrogen peroxide

[0162]

[0163] Note: Compared with the blank control group, #### p<0.0001, compared with the model group (drug concentration was 0), * p<0.05, ** p<0.01

[0164] 5. Experimental conclusion:

[0165] The composition of the present invention has a protective effect on cell oxidative damage and an antioxidant effect.

[0166] Example 5 Effect of the Composition on Lipopolysaccharide-Induced Inflammatory Response of Bv2 Cells

[0167] An in vitro inflammatory cell model was established by inducing an inflammatory response in the in vitro cell line BV2 cells by lipopolysaccharide (LPS), and the effect of the traditional Chinese medicine composition of the present invention on the expression of interleukin-6 (IL-6) factor in the inflammatory model was evaluated to reflect the anti-inflammatory effect of the traditional Chinese medicine composition.

[0168] 1. Experimental Materials:

[0169] BV2 cells are derived from mouse microglia (CM-M059, manufactured by Gaining Biotechnology Co., Ltd.). The Chinese medicine composition of the present invention (Ratio 1 in Example 1) (Batch No.: 20220801S); Composition 1 (Batch No.: 210422).

[0170] Composition 1 is prepared from the following raw materials:

[0171] Angelica 253.5g, Chuanxiong 253.5g, White Peony 202.7g, Rehmannia Glutinosa 202.7g, Mother of Pearl 506.8g, Uncaria 506.8g, Millettia Spatholobi 506.8g, Prunella Vulgaris 506.8g, Cassia Seed 506.8g, Corydalis 253.5g, Asarum 50.5g.

[0172] Preparation method:

[0173] Above 11 flavors, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Corydalis, Semen Cassiae add 70% ethanol heating extraction twice, first time 2 hours, second time 1 hour, filter, reclaim ethanol and be concentrated into appropriate amount, standby. Radix Paeoniae Alba adds 60% ethanol heating extraction twice, first time 2 hours, second time 1 hour, filter, reclaim ethanol and be concentrated into appropriate amount, standby. Radix Rehmanniae Preparata, Rhizoma Uncariae, Caulis Spatholobi, Herba Prunellae, Concha Margarita, Herba Asari add water and decoct twice, first time 2 hours, second time 1 hour, filter, it is a clear paste of 1.06-1.10 (80 ℃) that the filtrate is concentrated into relative density, adds ethanol and makes alcohol content reach 65%~70%, leaves standstill 12~24 hours, filters, reclaim ethanol, be concentrated into appropriate amount, standby. Get above extract, add stevioside, dextrin appropriate, mix, granulate, dry, make 1000g, promptly.

[0174] DMEM high-glucose culture medium, Beijing Solebogen Technology Co., Ltd., batch number: 20220507; phosphate-buffered saline (PBS), Beijing Solebogen Technology Co., Ltd., batch number: 20220609; fetal bovine serum (FBS), Sijiqing, batch number: 21050701; double-stranded antibodies, Beijing Solebogen Technology Co., Ltd., batch number: 20220319; trypsin digestion solution, Beijing Solebogen Technology Co., Ltd., batch number: 20220521; methythiazolyl tetrazolium chloride (MTT), Anhui White Shark Biotechnology Co., Ltd., batch number: 70080125; interleukin-6 kit, Shanghai Yuanju Biotechnology Center, 202210; lipopolysaccharide (LPS), Sigma-Aldrich, batch number: 0000135214.

[0175] 2. Experimental Methods

[0176] Effects of the composition of the present invention on the expression of inflammatory factors in BV2 cells induced by lipopolysaccharide: 0, 5, 10, 50, and 250 μg / mL were selected as drug concentrations for subsequent experiments. The experiment used 0.1 μg / mL of lipopolysaccharide and treated cells. BV2 cells containing lipopolysaccharide were treated with the composition of the present invention and Composition 1, respectively. After 24 hours of treatment, the cell supernatant was collected and the interleukin-6 content in the supernatant was measured using an ELISA kit.

[0177] 3. Statistical processing:

[0178] The data were expressed as mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) or non-parametric test was used to analyze the differences between the groups. P < 0.05 was used to judge the differences between the groups.

[0179] 4. Experimental results:

[0180] The protective effect of the composition of the present invention on the inflammatory response of BV2 cells induced by lipopolysaccharide is expressed as the expression level of interleukin-6. The lower the expression level, the better the drug effect. As shown in Table 7, composition 1 and the composition of the present invention can inhibit the production of interleukin-6 in BV2 cells induced by lipopolysaccharide at a concentration of 50 μg / ml and above, and there is a significant difference (P<0.05). The composition of the present invention can inhibit the production of interleukin-6 in BV2 cells induced by lipopolysaccharide at a concentration of 5 μg / ml and above ( Figure 3A ), and there was a significant difference (P<0.0001). The composition of the present invention and composition 1 were compared with the same concentration ( Figure 3B ), and showed a stronger inhibitory effect on the lipopolysaccharide-induced interleukin-6 content in BV2 cells, with a significant difference (P < 0.05). Therefore, both Composition 1 and the present composition have the effect of inhibiting the lipopolysaccharide-induced inflammatory response in BV2 cells, and the present composition is more effective in inhibiting the production of interleukin-6.

[0181] Table 7 Effect of the composition of the present invention on interleukin-6 expression in BV2 cells induced by lipopolysaccharide (mean ± SD)

[0182]

[0183] Note: Compared with the blank control group, # p<0.05, ### p<0.001, #### p<0.0001; compared with the model group (drug concentration was 0), * p<0.05, ** p<0.01, *** p<0.001,

[0184] **** p<0.0001.

[0185] 5. Experimental conclusion:

[0186] Both the composition of the present invention and composition 1 have anti-inflammatory effects, and the composition of the present invention is better than composition 1 in inhibiting the production of interleukin-6 by BV2 cells induced by lipopolysaccharide.

[0187] Example 6 Effect of the composition of the present invention on the HT-22 cell response induced by D-galactose

[0188] An in vitro subacute senescence cell model was established by inducing the response of the in vitro cell line HT-22 cells using D-galactose (D-gal), and the effect of the Chinese medicine composition of the present invention on the aging damage model was evaluated to demonstrate the anti-aging effect of the Chinese medicine composition.

[0189] 1. Experimental Materials:

[0190] HT-22 cells, the experiment used mouse hippocampal neuronal cells derived from mice (Gai Ning Biotechnology Co., Ltd., CM-M055).

[0191] The Chinese medicine composition of the present invention (ratio 1 in Example 1) (batch number: 20220801S); composition 1 (described in Example 5) (batch number: 210422).

[0192] DMEM high-glucose culture medium, Beijing Solebogen Technology Co., Ltd., batch number: 20220507; phosphate-buffered saline (PBS), Beijing Solebogen Technology Co., Ltd., batch number: 20220609; fetal bovine serum (FBS), Sijiqing, batch number: 21050701; double-antibody, Beijing Solebogen Technology Co., Ltd., batch number: 20220319; trypsin digestion solution, Beijing Solebogen Technology Co., Ltd., batch number: 20220521; thiazolyl tetrazolium chloride (MTT), Anhui White Shark Biotechnology Co., Ltd., batch number: 70080125; D-galactose (D-gal), Anhui White Shark Biotechnology Co., Ltd., batch number: S11050.

[0193] 2. Experimental Methods

[0194] Effects of the composition of the present invention on D-galactose-induced cellular senescence in HT-22 cells: 0, 5, 10, 50, 250, and 500 μg / mL were selected as drug concentrations for subsequent experiments. Cells were treated with 200 mM D-galactose. HT-22 cells were also treated with the composition of the present invention and Composition 1, respectively, containing D-galactose. After 24 hours, the supernatant was discarded, and a thiazolyl blue solution was added. Four hours later, the absorbance was measured using a microplate reader.

[0195] 3. Statistical processing:

[0196] The data were expressed as mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) or non-parametric test was used to analyze the differences between the groups. P < 0.05 was used to judge the differences between the groups.

[0197] 4. Experimental results:

[0198] The protective effect of the composition of the present invention on the cell senescence of HT-22 cells induced by D-galactose is expressed as cell survival rate. The higher the cell survival rate, the better the drug effect. The results are shown in Table 8. Composition 1 at 50 μg / ml can increase the cell survival rate of HT-22 cells damaged by galactose (see Table 8). Figure 4A ), with significant differences; the composition of the present invention 50μg / ml and above can increase the cell survival rate of HT-22 cells induced by galactose damage, and there is a significant difference (see Figure 4B). Compared with the same concentration of composition 1, the effect of the composition of the present invention on the cell survival rate of HT-22 cells damaged by D-galactose below 50 μg / ml was not significantly different (see Figure 4C ); However, at concentrations of 250 μg / ml and 500 μg / ml, the cell survival rate of the composition of the present invention on HT-22 cells damaged by D-galactose was better than that of composition 1, and it had an anti-cell aging effect (see Figure 4C ).

[0199] Table 8. Protective effect of the composition of the present invention on aging damage of HT-22 cells induced by D-galactose

[0200]

[0201] Note: Compared with the blank control group, #### p<0.0001, compared with the model group (drug concentration 0 group), * p<0.05, ** p<0.01

[0202] 5. Experimental conclusion:

[0203] Both the composition of the present invention and composition 1 have anti-aging effects, and the anti-D-galactose-induced HT22 cell aging effect of the composition of the present invention is better than that of composition 1.

[0204] Example 7 Effect of the composition of the present invention on cognitive impairment in zebrafish induced by AlCl3

[0205] An in vivo cognitive impairment model was established by inducing the spatial memory response of zebrafish by aluminum chloride (AlCl3), and the effect of the Chinese medicinal composition of the present invention on the cognitive impairment model was evaluated to demonstrate the effect of the Chinese medicinal composition in improving cognitive impairment.

[0206] 1. Experimental Materials:

[0207] Six-month-old adult zebrafish of the AB strain, 3.07 ± 0.35 cm in length and 0.36 ± 0.12 g in weight, were purchased from the Nanjing Yishulihua Biotechnology Institute. They were maintained in a recirculating aquaculture system controlled at 28 ± 0.2°C with a 14:10 hr light:dark photoperiod. They were fed twice daily with hatched brine shrimp.

[0208] Experimental reagents

[0209] Experimental Group: Composition of the present invention (Ratio 1 in Example 1), Batch No. 20220801S. Composition 1 (described in Example 5) (Batch No. 210422). Positive Agent: Donepezil, Batch No. C14567110, Shanghai MacLean Biochemical Technology Co., Ltd. AlCl₃·6H₂O: Batch No. I2207229, Shanghai Aladdin Biochemical Technology Co., Ltd.

[0210] 2. Experimental Methods

[0211] 2.1 Experimental Grouping

[0212] Zebrafish were randomly divided into seven groups: a blank control group, a model group, a positive drug co-administration group, a high- and low-dose co-administration group of Composition 1, and a high- and low-dose co-administration group of the present invention's composition. Except for the blank control group, zebrafish in all other groups were immersed in AlCl₃·6H₂O solution and various drug solutions for 30 days, with half of the working solution replaced daily.

[0213] Table 9: Experimental groups and interventions

[0214]

[0215] 2.2 T-maze test

[0216] The T-maze consists of two arms of equal length and a vertical corridor. The right arm connects to the enriched chamber (EC), where the water level is 5 cm higher than in other areas. A beaker containing five zebrafish was placed within the EC to create a schooling effect. To prevent the camera from capturing the school's movements and interfering with the experiment, the beaker was covered with a white cloth with several small holes punched in it for ventilation. A suitable amount of brine shrimp was placed in a perforated transparent bag and attached to the outside of the beaker to attract the zebrafish.

[0217] The day before the experiment, the zebrafish were given an acclimatization period and allowed to move freely in the T-maze for 25 minutes. On the day of the experiment, the zebrafish were placed at the starting point of the corridor and allowed to move freely in the T-maze for 30 seconds. The experiment lasted for 6 minutes. The latency time from the starting point to the zebrafish finding the nutrient-rich area was observed and recorded (the zebrafish entered the nutrient-rich area only after finding the zebrafish school and staying there for 30 seconds). Food was rewarded in the nutrient-rich area. Fish that did not find the nutrient-rich area were guided into the nutrient-rich area after 6 minutes and stayed there for 1 minute. Food was also rewarded. The experiment was conducted once a day for a total of 3 days, and the latency time was recorded each time.

[0218] Testing phase: The testing phase begins on the second day after three days of training. Each zebrafish swims freely to find a nutrient-rich area (there is no food at this time). Software is used to connect the camera of the T-maze to automatically record the time required by the zebrafish, path selection and other indicators to evaluate its spatial learning and memory ability.

[0219] 3. Statistical processing:

[0220] The data were expressed as mean ± standard error (Mean ± SEM). One-way analysis of variance (ANOVA) or non-parametric test was used to analyze the differences between the groups. P < 0.05 was used to judge the differences between the groups.

[0221] 4. Experimental results:

[0222] Table 10 shows the latency of zebrafish to first reach the nutrient-rich area. The shorter the time, the better the zebrafish's spatial memory and cognitive function. Compared with the blank control group, the latency of zebrafish in the model group to first reach the nutrient-rich area increased (P < 0.05); compared with the model group, the latency of zebrafish in the low- and high-dose groups of Composition 1 and the low- and high-dose groups of the present composition to first reach the nutrient-rich area was significantly reduced (P < 0.05), indicating that the present composition and Composition 1 have a significant improvement effect on AlCl3-induced cognitive dysfunction in zebrafish (P < 0.05), and the improvement effect of the present composition is better than that of Composition 1.

[0223] Table 10. Protective effect of the composition of the present invention on cognitive impairment in zebrafish caused by AlCl3

[0224]

[0225] Note: Compared with the blank control group, # p<0.05, compared with the model group, * p<0.05, ** p<0.01, *** p<0.001

[0226] 5. Experimental conclusion:

[0227] Both the composition of the present invention and composition 1 have the effect of improving cognitive impairment, and the improvement effect of the composition of the present invention is better than that of composition 1.

[0228] Example 8 Population Taste Evaluation Experiment

[0229] An experiment was conducted to observe people's taste preference for the Chinese medicine composition of the present invention and composition 1.

[0230] 1. Experimental materials and reagents

[0231] Volunteer screening: Our research team screened the recruited volunteers, including bitterness sensitivity, and finally selected 20 healthy volunteers as subjects.

[0232] Sample reagents:

[0233] The composition of the present invention (ratio 1 in Example 1) (batch number: 20220801S) and composition 1 (described in Example 5) (batch number: 210422): 12 g of composition 1 and the composition of the present invention were dissolved in 300 ml of water and stirred to fully dissolve.

[0234] 2. Experimental methods

[0235] Taste preference evaluation: A double-blind randomized experiment was conducted, and 20 volunteers were invited to taste two test samples. They were asked to rinse their mouths and rest for 2 to 3 minutes between evaluating different samples, and record which sample they preferred.

[0236] 3. Calculation method:

[0237] Calculate the preference index: Preference index of the composition of the present invention = n B / (n A +n B );n A Number of people who prefer combination 1, n B is the number of people who prefer the composition of the present invention.

[0238] 4. Experimental results

[0239] As shown in Table 11, under the experimental conditions, the 20 volunteers' preference index for Composition 1 was 0.1, while the preference index for the present composition was 0.9, indicating that the volunteers preferred the present composition. Regarding the preparation method, Composition 1 was filtered and concentrated, and then the extract, stevia, and an appropriate amount of dextrin were used as a slurry. The remaining dextrin was used as a base, which was then granulated and dried. In contrast, the present composition was filtered, concentrated, and dried, and then an appropriate amount of maltodextrin was added to the dry extract powder, mixed, granulated, and dried. Because the present composition did not contain the flavoring agent stevia, it was preferred by the volunteers.

[0240] Table 11. Volunteers' preference index for the combination of the present invention

[0241]

[0242] 5. Experimental Conclusion

[0243] Under the conditions of this experiment, compared with composition 1, the volunteers preferred the taste of the composition of the present invention.

[0244] Example 9 Taste preference test of rats on the composition of the present invention

[0245] The taste preference experiment of the composition of the present application and composition 1 was observed.

[0246] 1. Experimental materials and experimental animals:

[0247] The composition of the present application (ratio 1 in Example 1) (batch number: 20220801S) and composition 1 (described in Example 5) (batch number: 210422): take 12 g of composition 1 and the composition of the present application respectively, dissolve in 300 ml of water, and stir to fully dissolve.

[0248] Balance, model: BCE423l-1CCN, manufacturer: Sartorius Scientific Instruments Co., Ltd.; electronic balance, model: PL203, manufacturer: Mettler-Toledo Instruments Co., Ltd.; 50 ml drinking bottle, model: ZK-SL-50, manufacturer: Henan Zhike Hongrun Environmental Protection Technology Co., Ltd.;

[0249] 8-week-old male SPF SD rats, weighing 180-220 g, purchased from Beijing Huafukang Biotechnology Co., Ltd. (qualification certificate number: 110322231103858115, production license number: SCXK (Jing) 2019-0008). After purchasing, the rats were placed in the animal room of Hebei University of Technology for feeding. The animal room environment maintained a temperature of 23±2℃, humidity of 40-70%, and 12 hours of light and dark alternation. Each cage housed no more than 5 animals, and the bedding was changed three times a week, or the animals were housed individually if they fought or the like. The rats could freely eat and drink during feeding, and after one week of adaptation in the animal room, the subsequent experiments began. This experiment followed the experimental operation procedures of the College of Life Sciences and Health Engineering of Hebei University of Technology and was approved by the Hebei University of Technology Ethics Committee.

[0250] 2. Experimental method

[0251] 2.1 Grouping and administration

[0252] After one week of adaptation in the animal room, the rats were subjected to the taste preference experiment. During the taste preference experiment, the rats were housed individually. The taste preference experiment consisted of two stages. The first stage was the adaptation stage: in this stage, each cage of rats was given two bottles of distilled water for 48 hours. The second stage was the taste preference test: after the adaptation stage, the rats were deprived of water and food for 12 hours, and then each cage of rats was given one bottle of composition 1 solution and one bottle of the composition of the present application, with the left and right positions being random (see Figure 5 ). The intake of the composition 1 solution and the composition of the present application was recorded after the rats freely drank for 2 hours (milliliters), and the taste preference degree of the composition of the present application was converted.

[0253] 3. Data processing:

[0254] The calculation formula is: preference of the present invention composition = intake of the present invention composition solution / ((intake of the present invention composition solution + intake of composition 1 solution)) × 100%

[0255] 4. Experimental Results

[0256] As can be seen from Table 12, the preference index of rats for the composition of the present invention is 0.78, which is greater than 0.5, indicating that rats prefer the composition of the present invention (see Figure 6 ).

[0257] Table 12. Statistical analysis of the volume of solution consumed by rats before and after administration

[0258]

[0259] 5. Experimental Conclusion

[0260] Compared with composition 1, rats preferred the composition of the present invention.

[0261] Example 10 Preparation of tablets of the composition of the present invention

[0262] The composition of the present invention is prepared from the following raw materials in the following weight ratios:

[0263] Angelica sinensis 270.2g, Chuanxiong 266.7g, White Peony 217.2g, Rehmannia glutinosa 233.0g, Uncaria rhynchophylla 577.0g, Millettia reticulata 537.1g, Prunella vulgaris 564.7g, Cassia seed 554.2g, Corydalis yanhusuo 275.6g.

[0264] Preparation method:

[0265] All the medicinal materials are decocted twice with water, 5 times the amount of water is added for the first time and decocted for 2 hours, and 4 times the amount of water is added for the second time and decocted for 1 hour. The extract is filtered, and the filtrate is concentrated under reduced pressure to a relative density of 1.06 (60°C), spray-dried, and appropriate amounts of starch and magnesium stearate are added. The mixture is mixed, tableted, and coated with sugar to obtain the product.

[0266] Example 11 Preparation of capsules of the composition of the present invention

[0267] The composition of the present invention is prepared from the following raw materials in the following weight ratios:

[0268] Angelica sinensis 224.1g, Chuanxiong 232.5g, White Peony 178.1g, Rehmannia glutinosa 181.0g, Uncaria rhynchophylla 435.6g, Millettia reticulata 436.2g, Prunella vulgaris 463.2g, Cassia seed 436.5g, Corydalis yanhusuo 232.0g.

[0269] Preparation method:

[0270] All the medicinal materials are decocted twice with water, 5 times the amount of water is added for the first time and decocted for 2 hours, and 4 times the amount of water is added for the second time and decocted for 1 hour. The extract is filtered, and the filtrate is concentrated under reduced pressure to a relative density of 1.03 (60°C), spray-dried, granulated, and encapsulated to obtain the product.

[0271] Example 12 Preparation of granules of the composition of the present invention

[0272] Angelica sinensis 253.5g, Chuanxiong 253.5g, White Peony 202.7g, Rehmannia glutinosa 202.7g, Uncaria rhynchophylla 506.8g, Millettia reticulata 506.8g, Prunella vulgaris 506.8g, Cassia seed 506.8g, Corydalis yanhusuo 253.5g.

[0273] Preparation method:

[0274] All the medicinal materials were taken by weight and decocted twice with water. The first time, 5 times the amount of water was added and decocted for 2 hours. The second time, 4 times the amount of water was added and decocted for 1 hour. The extract was filtered, and the filtrate was concentrated under reduced pressure to a relative density of 1.05 (60°C). The extract was spray-dried under the following conditions: the inlet air temperature was 180°C and the outlet air temperature was 100°C. During the spray-drying operation, the material temperature was maintained at 60°C to obtain a dry extract powder of the composition. An appropriate amount of maltodextrin was added, mixed, and dry-pressed into granules to obtain 1000g.

Claims

1. A Chinese medicine composition, prepared from the following raw materials in parts by weight: 210 to 280 parts of Chinese Angelica sinensis, 220 to 280 parts of Chuanxiong, 160 to 230 parts of white peony root, 160 to 250 parts of Rehmannia root, 420-600 parts of Uncaria rhynchophylla, 420-550 portions of Millettia reticulata, 450 to 580 parts of Prunella Vulgaris, 420 to 570 parts of Cassia seeds, 220-290 parts of Corydalis yanhusuo.

2. The Chinese medicine composition according to claim 1, is prepared from the following raw materials in parts by weight: 224.1 to 270.2 parts of Chinese Angelica sinensis, 232.5 to 266.7 parts of Chuanxiong, 178.1 to 217.2 parts of white peony root, 181.0 to 233.0 parts of Rehmannia root, 435.6 to 577.0 parts of Uncaria rhynchophylla, 436.2 to 537.1 parts of Millettia reticulata, 463.2 to 564.7 parts of Prunella Vulgaris, 436.5 to 554.2 parts of Cassia seeds, 232.0 to 275.6 parts of Corydalis yanhusuo.

3. The Chinese medicine composition according to claim 1 is prepared from the following raw materials in parts by weight: 253.5 parts of Chinese angelica, 253.5 parts of Chuanxiong, 202.7 parts of white peony root, 202.7 parts of Rehmannia root, 506.8 parts of Uncaria rhynchophylla, 506.8 parts of Millettia reticulata, 506.8 parts of Prunella vulgaris, 506.8 parts of Cassia seed, and 253.5 parts of Corydalis yanhusuo.

4. The Chinese medicine composition according to claim 1, which is prepared into any pharmaceutical preparation for administration.

5. The pharmaceutical preparation according to claim 4, in any pharmaceutically acceptable dosage form, including: Tablets, capsules, hard capsules, oral liquids, lozenges, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, sprays, drops, and patches.

6. The method for preparing the Chinese medicine composition according to claim 1, comprising extracting or otherwise processing the Chinese medicine raw materials of the above formula to prepare a pharmaceutically active substance, and then using the substance as a raw material and adding a pharmaceutically acceptable carrier as needed to prepare the composition according to conventional pharmaceutical techniques.

7. The preparation method according to claim 6, comprising the following steps: All the medicinal materials are decocted with water twice, each time for 1 to 2 hours, the extract is filtered, the filtrate is concentrated under reduced pressure, and dried to obtain a composite extract powder; appropriate amounts of auxiliary materials are added to prepare a pharmaceutical preparation that can be taken.

8. The preparation method according to claim 7, wherein the pharmaceutical preparation is a granule, and the preparation method is as follows: taking all the medicinal materials, adding water and boiling twice, adding 5 times the amount of water for the first time, boiling for 2 hours, and adding 4 times the amount of water for the second time, boiling for 1 hour, filtering the extract, and concentrating the filtrate under reduced pressure to a relative density of 1.03-1.08 (60°C ± 2°C), spray drying to obtain a dry powder of the composition extract; adding an appropriate amount of maltodextrin, mixing, dry-pressing and granulating to prepare 1000g of granules, wherein, The spray drying equipment used in the spray drying is any equipment that can be used for drying fluid extracts. The spray drying conditions are: the inlet air temperature is 140-190°C, the outlet air temperature is 80-105°C; the material temperature is maintained at 40-80°C during the spray drying operation. Preferably, the spray drying conditions are as follows: the inlet air temperature is 170-180°C, the outlet air temperature is 95-105°C; and the material temperature is kept at 50-60°C during the spray drying operation.

9. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a medicament for treating cognitive impairment and / or sleep disorders.

10. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of anti-inflammatory and / or anti-oxidative and / or anti-cell aging drugs.

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