Use of a sleep-aiding and tranquilizing composition in the preparation of a product for preventing and treating Alzheimer's disease

Abstract

The application discloses application of a sleep-aiding and tranquilizing composition in preparation of a product for preventing and treating Alzheimer's disease. Through animal experiment observation of the sleep-aiding and tranquilizing composition, the experiment rats are modeled by D-galactose, and the experiment results show that, compared with a blank control group, the number of times of crossing the platform, the time of staying in the platform quadrant, the new object recognition coefficient and the target quadrant swimming path percentage of the D-GAL model group of rats are significantly reduced, which indicates that the AD modeling is successful; compared with the D-GAL model group, the number of times of crossing the platform, the time of staying in the platform quadrant, the new object recognition coefficient and the target quadrant swimming path percentage of the sleep-aiding and tranquilizing composition administration group are significantly increased. The results show that the sleep-aiding and tranquilizing composition has the effect of treating Alzheimer's disease, and the sleep-aiding and tranquilizing composition plays the positive role in treating AD through multiple targets and multiple pathways, thereby providing a new idea for clinically treating AD.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, more particularly, to application of a sleep-aiding and tranquilizing composition in preparation of a product for preventing and treating Alzheimer's disease. BACKGROUND

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with insidious onset, accompanied by neuron degeneration and death, and AD patients gradually decline in memory, cognitive function and language function. AD has a high morbidity rate, and the quality of life of patients will gradually decline, and even in the late stage, the patients will be unable to take care of themselves and need to rely on others; most AD patients have poor prognosis, so AD also has a high mortality rate. The damage caused by AD is not limited to the patients themselves, but also to their families and society, causing heavy economic burden and different degrees of psychological pressure, depression and other health problems for the family members of the patients.

[0003] The specific pathogenesis of AD is not completely clear at present and is complex and diverse, but there are various hypotheses, such as cholinergic neuron hypothesis, neuroinflammation hypothesis, free radical damage hypothesis, Tau protein abnormality hypothesis, Aβ toxicity hypothesis, insulin hypothesis, energy metabolism hypothesis, etc. The therapeutic drugs currently used in AD clinically are mostly single-target drug molecules, which only act on a single target, which may be the main reason for their relatively large toxic side effects and less than ideal clinical efficacy, and they cannot fundamentally reverse the pathological process of AD, therefore, seeking drugs for treating AD by targeting multiple targets will become the mainstream of developing AD treatment drugs.

[0004] Patent CN 110772595 A discloses a composition with sleep-aiding and tranquilizing effects, which is a combination of food and medicine, and the main components are Chinese date kernel, ginseng and polygonatum, etc. The combination of the drugs has the effects of dynamic and static compatibility, cold and warm compatibility, promoting sleep, improving sleep quality and prolonging sleep time. In clinical application, it can significantly improve the sleep disorders of patients. However, there is no report on the effect of the composition in the treatment of AD and learning and memory disorders. SUMMARY

[0005] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and to provide application of a sleep-aiding and tranquilizing composition in preparation of a product for preventing and / or treating Alzheimer's disease.

[0006] Still another object of the present application is to provide application of a sleep-aiding and tranquilizing composition in preparation of a product for improving learning and memory disorders related to aging.

[0007] The above object of the present application is achieved by the following technical solution:

[0008] The subject group of the present application discloses a composition with sleep-aiding and tranquilizing effects, i.e. a sleep-aiding and tranquilizing formula, which comprises the following components in mass parts: 5-10 parts of ginseng, 10-15 parts of rhizoma polygonati, 10-15 parts of mulberry fruit, 10-15 parts of poria cocos, 10-15 parts of lotus seed, 15-30 parts of spina date, 10-20 parts of lily, 10-15 parts of longan arillus, 15-30 parts of hui wheat, and 4-8 parts of dried tangerine or orange peel. The sleep-aiding and tranquilizing formula is used for AD treatment test, and the experimental rats are modeled by D-galactose. The experimental results show that, compared with the normal control group, the number of crossing platform, the platform quadrant time of the D-galactose model rats are obviously reduced, and the new object recognition coefficient and the target quadrant swimming distance percentage are also obviously decreased, indicating that the AD disease modeling is successful. Compared with the D-galactose model group, the number of crossing platform, the platform quadrant time of the model rats after sleep-aiding and tranquilizing formula drug intervention are significantly increased, the new object recognition coefficient and the target quadrant swimming distance percentage are significantly increased, and the sleep-aiding and tranquilizing formula can improve the cell morphology and arrangement of hippocampal neurons of the model rats, improve the loss of Nissl bodies, reduce the relative expression of BAX protein, and improve the relative expression of BCL-2, indicating that the sleep-aiding and tranquilizing formula can improve the neuron apoptosis of the D-galactose model rats, improve the learning and memory impairment related to aging, and improve the learning and memory ability, indicating that the sleep-aiding and tranquilizing formula has the effect of treating Alzheimer's disease.

[0009] In addition, the prediction analysis by network pharmacology shows that the effective components of the sleep-aiding and tranquilizing formula for resisting AD are 60 in total, in which 10 components such as β-sitosterol, kaempferol, quercetin, naringenin, baicalein, β-carotene, wogonoside, nobiletin, glycyrrhizin, and diosgenin play a major key role; and the five potential core target points of ZAF for resisting AD are AKT1, GAPDH, ALB, BCL2, and CASP3, which are closely related to cell apoptosis. It is shown that the sleep-aiding and tranquilizing formula has the advantages of multi-target and multi-pathway, and shows the unique advantages and infinite potential of traditional Chinese medicine in treating AD in the face of the complex pathogenesis of AD, thereby providing a new idea for the clinical treatment of AD.

[0010] Therefore, the present application provides the use of the sleep-aiding and tranquilizing composition in the preparation of products for preventing and / or treating Alzheimer's disease, which comprises the following components in mass parts: 5-10 parts of ginseng, 10-15 parts of rhizoma polygonati, 10-15 parts of mulberry fruit, 10-15 parts of poria cocos, 10-15 parts of lotus seed, 15-30 parts of spina date, 10-20 parts of lily, 10-15 parts of longan arillus, and 15-30 parts of hui wheat.

[0011] The term "preventing and / or treating" refers to the treatment, prevention, alleviation and / or relief of the disease or disorder described in the present application in a subject.

[0012] The application also provides use of the sleep-aiding and tranquilizing composition in preparation of a product for improving learning and memory impairment related to aging, wherein the sleep-aiding and tranquilizing composition comprises the following components by mass: 5-10 parts of ginseng, 10-15 parts of rhizoma polygonati, 10-15 parts of mulberry fruit, 10-15 parts of poria cocos, 10-15 parts of lotus seed, 15-30 parts of spina date, 10-20 parts of lily, 10-15 parts of longan arillus, and 15-30 parts of hui wheat.

[0013] As a preferred embodiment, the sleep-aiding and tranquilizing composition comprises the following components by mass: 10 parts of ginseng, 15 parts of rhizoma polygonati, 15 parts of mulberry fruit, 10 parts of poria cocos, 10 parts of lotus seed, 15 parts of spina date, 10 parts of lily, 10 parts of longan arillus, and 15 parts of hui wheat.

[0014] Further, the sleep-aiding and tranquilizing composition further comprises the following components by mass: 4-8 parts of citrus reticulata.

[0015] As a preferred embodiment, the sleep-aiding and tranquilizing composition comprises the following components by mass: 10 parts of ginseng, 15 parts of rhizoma polygonati, 15 parts of mulberry fruit, 10 parts of poria cocos, 10 parts of lotus seed, 15 parts of spina date, 10 parts of lily, 10 parts of longan arillus, 15 parts of hui wheat, and 6 parts of citrus reticulata.

[0016] Further, the sleep-aiding and tranquilizing composition further comprises the following components by mass: 8-12 parts of bergamot.

[0017] As a preferred embodiment, the sleep-aiding and tranquilizing composition comprises the following components by mass: 10 parts of ginseng, 15 parts of rhizoma polygonati, 15 parts of mulberry fruit, 15 parts of poria cocos, 15 parts of lotus seed, 15 parts of spina date, 15 parts of lily, 15 parts of longan arillus, 15 parts of hui wheat, and 10 parts of bergamot.

[0018] Further, the sleep-aiding and tranquilizing composition further comprises the following components by mass: 4-8 parts of citrus reticulata and 3-7 parts of gardenia.

[0019] As a preferred embodiment, the sleep-aiding and tranquilizing composition comprises the following components by mass: 10 parts of ginseng, 10 parts of rhizoma polygonati, 10 parts of mulberry fruit, 15 parts of poria cocos, 15 parts of lotus seed, 30 parts of spina date, 15 parts of lily, 15 parts of longan arillus, 30 parts of hui wheat, 6 parts of citrus reticulata, and 5 parts of gardenia.

[0020] Further, the sleep-aiding and tranquilizing composition further comprises the following components by mass: 8-12 parts of fructus amomi rotundus and 8-12 parts of malt.

[0021] As a preferred embodiment, the sleep-aiding and tranquilizing composition comprises the following raw material components by mass fraction: 5 parts of ginseng, 15 parts of rhizoma polygonati, 15 parts of mulberry fruit, 15 parts of poria cocos, 15 parts of lotus seed, 20 parts of spina date, 20 parts of lily, 10 parts of longan arillus, 15 parts of wheat, 10 parts of fructus alpiniae oxyphyllae, and 10 parts of malt.

[0022] Further, the product is a medicine or a health product. Since all the herbs in the sleep-aiding and tranquilizing composition are "medicinal food" and no obvious dose dependence is shown in the experimental detection, the sleep-aiding and tranquilizing composition can be used for preparing a medicine or can be applied in daily diet in a small dose for anti-aging (i.e. for health care) and prevention of Alzheimer's disease.

[0023] The medicine or health product can be used for patients or other animals receiving the medicine or health product of the present application for treating, preventing, reducing and / or relieving the diseases or conditions described in the present application. In a preferred embodiment, the medicine or health product is used for mammals. The mammals include but are not limited to humans, cows, horses, sheep, pigs, goats, rabbits, cats, dogs, mice and any other mammals having a liver and suffering from damage.

[0024] In a preferred embodiment, the medicine or health product is used for humans.

[0025] The administration dose of the medicine or health product of the present application depends on many factors, such as the nature and severity of the disease to be prevented or treated, the gender, age, weight and individual response of the patient or animal, the administration route and administration frequency, etc. The medicine or health product can be administered in a single dose form or divided into several, for example, two, three or four dose forms. The dose level should be selected according to the specific administration route, the severity of the condition to be treated and the condition and medical history of the patient to be treated, etc.

[0026] However, it should be recognized that the total daily amount of the medicine or health product of the present application should be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specifically therapeutically effective dose level should be determined according to various factors, including the disorder to be treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health condition, gender and diet; the administration time, administration route and excretion rate; the treatment duration; the drugs used in combination or simultaneously; and similar factors known in the medical field.

[0027] Further, the present application also provides a preparation method of the sleep-aiding and tranquilizing composition, which comprises mixing the raw material components according to the mass fraction, soaking in water, decocting for several times, combining the filtrates, concentrating, freeze-drying to obtain the freeze-dried powder of the sleep-aiding and tranquilizing composition.

[0028] Preferably, the concentration is rotary evaporation concentration.

[0029] Preferably, the product further comprises a pharmaceutically or nutraceutically acceptable excipient.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] The application provides application of a sleep-aiding and tranquilizing composition in preparation of a product for preventing and treating Alzheimer's disease, and the sleep-aiding and tranquilizing composition is observed for a pharmacodynamic effect through animal experiments. D-galactose modeling is carried out on experimental rats, and the experimental results show that, compared with a blank control group, the number of times of crossing the platform, the time of staying in the platform quadrant, the new object recognition coefficient and the percentage of the target quadrant swimming path of the D-GAL model group of rats are significantly reduced; compared with the model group, the number of times of crossing the platform, the time of staying in the platform quadrant, the new object recognition coefficient and the percentage of the target quadrant swimming path of the sleep-aiding and tranquilizing composition administration group are significantly increased; the sleep-aiding and tranquilizing composition can improve the cell morphology and arrangement of neurons in the hippocampus of model rats, improve the loss of Nissl bodies, reduce the relative expression of BAX protein and improve the relative expression of BCL-2, which indicates that the sleep-aiding and tranquilizing composition can improve neuron apoptosis of D-galactose model rats and improve learning and memory impairment related to aging, and indicates that the sleep-aiding and tranquilizing prescription has the effect of treating Alzheimer's disease. The sleep-aiding and tranquilizing prescription plays an active role in treating AD through multiple targets and multiple pathways, and provides a new idea for clinical treatment of AD. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The figure is a comparison of the average speed of rats in each experimental group. CTRL: normal model group; D-GAL: D-gal model group; ZAF-L: ZAF low-dose group; ZAF-H: ZAF high-dose group; DNP: donepezil group.

[0033] Figure 2 The figure is a comparison of the new object cognition index (%) of rats in each experimental group. CTRL: normal model group; D-GAL: D-gal model group; ZAF-L: ZAF low-dose group; ZAF-H: ZAF high-dose group; DNP: donepezil group.

[0034] Figure 3 The figure is a comparison of the escape latency of rats in the water maze experiment in each experimental group. CTRL: normal model group; D-GAL: D-gal model group; ZAF-L: ZAF low-dose group; ZAF-H: ZAF high-dose group; DNP: donepezil group.

[0035] Figure 4 The figure is a water maze movement trajectory graph of rats in each experimental group.

[0036] Figure 5For the comparison of spatial exploration ability of rats in each experimental group. CTRL: normal model group; D-GAL: D-gal model group; ZAF-L: ZAF low-dose group; ZAF-H: ZAF high-dose group; DNP: donepezil group.

[0037] Figure 6 For hippocampal neurons (HE staining, x40).

[0038] Figure 7 For hippocampal neurons (Nissl staining, x40).

[0039] Figure 8 For the protein expression of P-PI3K and P-AKT of rats in each experimental group. 1: normal model group; 2: D-gal model group; 3: ZAF low-dose group; 4: ZAF high-dose group; 5: donepezil group. CTRL: normal model group; D-GAL: D-gal model group; ZAF-L: ZAF low-dose group; ZAF-H: ZAF high-dose group; DNP: donepezil group.

[0040] Figure 9 For hippocampal neurons (TUNEL staining, x40). 1: normal model group; 2: D-gal model group; 3: ZAF low-dose group; 4: ZAF high-dose group; 5: donepezil group.

[0041] Figure 10 For the protein expression of Bax, Bcl-2 and Cleave Caspase-3 of rats in each experimental group. 1: normal model group; 2: D-gal model group; 3: ZAF low-dose group; 4: ZAF high-dose group; 5: donepezil group. CTRL: normal model group; D-GAL: D-gal model group; ZAF-L: ZAF low-dose group; ZAF-H: ZAF high-dose group; DNP: donepezil group.

[0042] Figure 11 For the intersection of ZAF target and AD target.

[0043] Figure 12 For the potential target of ZAF target.

[0044] Figure 13 For the visualization analysis of potential core target of ZAF treating AD.

[0045] Figure 14 For the histogram of GO enrichment analysis results.

[0046] Figure 15 For the KEGG enrichment analysis path bubble chart.

[0047] Figure 16 For the network diagram of drug-component-potential target protein of ZAF. DETAILED DESCRIPTION

[0048] The present application will be further described by the following description of the drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Example 1 Preparation of Zhisuananshenfang freeze-dried powder

[0051] The extraction method of the Zhisuananshenfang freeze-dried powder continues the previous extraction method of traditional Chinese medicine by the members of the research group. According to the dosage of the prescription, 10 g of ginseng, 15 g of rhizoma polygonati, 15 g of mulberry fruit, 10 g of poria cocos, 10 g of lotus seed, 15 g of semen ziziphi spinosae, 10 g of lily, 10 g of longan meat, 15 g of wheat, and 6 g of dried tangerine or orange peel are weighed and uniformly mixed (the total weight of each dose of medicine is 116 grams, and 5 doses of medicine are decocted each time, i.e., the total weight is 580 g). 10 times the weight of distilled water (about 5.8 liters) is added to the total amount of the prescription medicine to soak for 1 hour. The medicine is placed in a thermostat and heated until it boils for 1 hour. After filtering through multiple layers of cotton gauze, the traditional Chinese medicine liquid is collected. 8 times the weight of distilled water (about 4.6 liters) is added to the decocted traditional Chinese medicine residue, and the heating is continued. After boiling for 1 hour, the liquid is filtered through multiple layers of cotton gauze. The traditional Chinese medicine liquids from the two decoctions are mixed, and vacuum filtration is performed using a vacuum pump device to obtain a relatively clear traditional Chinese medicine liquid for use.

[0052] The above liquid is concentrated by evaporation using a rotary evaporator to make the liquid more concentrated. The concentrated liquid is poured into a flat container, sealed, and placed in a -80°C freezer for 12 hours. Then it is placed in a freeze-drying machine for dehydration treatment to prepare Zhisuananshenfang freeze-dried powder for use.

[0053] Example 2 Zhisuananshenfang treatment of AD

[0054] I. Experimental materials

[0055] 1. Experimental animals

[0056] Fifty-five SD rats were selected for the experiment (Rat information: SPF level, male, 7-8 weeks old, body weight range 200±20g, purchased from Guangdong Medical Laboratory Animal Center, Experimental Animal Production License No: SCXK(yue)2022-0002; Certificate No: 44007200108544. Raising in the barrier environment of Guangzhou University of Chinese Medicine Experimental Animal Center, Experimental Animal Use License No: SYXK(yue)2018-0001, maintaining free diet, free water, feeding temperature 26±2℃, constant indoor humidity, good ventilation, repeated 12-hour light, dark, and changing bedding 2-3 times per week. This study disposes animals in accordance with the requirements of the Guidelines for the Humane Treatment of Laboratory Animals, obtains animal ethics approval and complies with relevant welfare requirements, and is supervised by the ethics committee.

[0057] After one week of adaptive feeding, the rats were randomly divided into normal control group (CTRL), D-gal model group (D-GAL), ZAF low-dose group (ZAF-L), ZAF high-dose group (ZAF-H), and positive drug (donepezil) group (DNP). Each group has 11 rats, divided into 2 cages, 5-6 rats per cage. The feed and water conditions of the rats in each cage are the same except for the necessary drug administration.

[0058] II. Experimental methods

[0059] 1. Drug dosage

[0060] (1) ZAF dosage

[0061] The equivalent dose conversion formula used in this experiment is based on the formula provided by the FDA in the Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. This formula is based on the body surface area conversion of different species, and the formula is: X (adult daily dose) / 60 kg (body weight of humans) x 6.2. The calculated drug dosage is set as the daily drug dosage for rats in the ZAF high-dose group, so the ZAF high-dose and low-dose groups of rats are given ZAF for sleep and tranquilization at a dose of 12, 6 g / kg / d (calculated based on the weight of crude drugs).

[0062] (2) Positive drug (donepezil) preparation and dosage

[0063] Referring to the adult donepezil dosage of 5mg / day, the calculated donepezil dosage for rats is 3mg / kg / d based on the animal and human equivalent dose conversion formula. The donepezil used in this experimental study is a powder, the donepezil solvent is distilled water, and the administration method is intragastrically administered.

[0064] (3) Model drug (D-galactose, referred to as D-gal) preparation and dosage

[0065] Reference to the invention of the task group in the past about the modeling method of aging model with cognitive impairment, this experiment D-gal administration dose is 150mg / kg / day, D-gal solvent is sterile saline, the administration mode is intraperitoneal injection.

[0066] 2, animal modeling and test drug intervention method

[0067] (1) aging-related cognitive impairment rat modeling

[0068] After the end of the adaptive feeding, start the abdominal injection of D-galactose modeling, the modeling time is ten weeks, except that the normal control group is injected with the same volume of saline every day, the rest of the test drug groups are injected with D-gal solution 150mg / kg / d.

[0069] (2) Test drug intervention

[0070] In the third week of modeling, rats in each test drug group began to be given corresponding dose of drugs by gavage, for a total of 8 weeks, with a dose of 1mL / 100g. The normal control group and the D-gal model group were given pure water by gavage, the low-dose group of the sleep-aiding and tranquilizing prescription was given 6g / kg / day, and the high-dose group was given 12g / kg / day. The positive drug group was given donepezil 3mg / kg / day.

[0071] 3, behavioral experiment

[0072] Behavioral experiments were conducted 10 days before the end of test drug intervention, including open field test, novel object recognition test, and water maze test. During this period, modeling and drug intervention were still given.

[0073] (1) Open field test

[0074] One day before the experiment, the rats were moved to the behavioral laboratory, where they were maintained in a 12-hour light-dark cycle to adapt to the test environment and avoid stress and pressure caused by changes in experimental environment. The test was conducted in a quiet and stable light intensity behavioral laboratory. The rats were quickly placed in the experimental box (size: 40cm x 40cm x 35cm), and the free activity of the rats was recorded within 5 minutes. The open box was divided into 16 grids on average, and the time spent in the 4 central quadrants was considered as the central residence time. The total distance, central distance, and central time of the rats were recorded and analyzed using SuperMaze animal behavior video analysis software. The experimental box must be cleaned and sprayed with 75% alcohol and wiped dry between two rat experiments to avoid interference from the residual odor of the previous experimental rat. The video content was analyzed by SuperMaze animal behavior video analysis software V2.0.

[0075] (2) Novel object recognition test

[0076] The new object recognition experiment was performed on the second day after the open field experiment, for two days, using the new object recognition experiment instrument and software from Shanghai Xinsoft Company. Since the experimental rats had been freely active in the current experimental box the day before the new object recognition experiment, the new object recognition experiment was directly carried out without the need for further adaptation. On the first day of the experiment, two identical objects A (A1 and A2) were placed in the experimental box at relative positions. The rats were placed in the experimental box with their backs to the objects A1 and A2, and timed for 5 minutes. The distance from the placement point of the rats to the objects A1 and A2 should be consistent. The animals were taken out and placed back in the animal cage after the timing ended. On the second day, one of the two identical objects A (A1 or A2) was replaced with another object B of similar size but different shape, so that the objects placed in the experimental box at relative positions on the second day of the experiment were the old object A and the new object B. The rats were placed in the experimental box with their backs to the two objects, and the real-time analysis software was used to record the data of the rats' exploration behavior towards the new and old objects, with a test time of 5 minutes. The placement of the rats into the experimental box was as uniform as possible, with their backs to the objects and equal distances from the two objects. In order to exclude the influence of residual information such as odor and excrement, the objects and experimental box were cleaned in time and sprayed with 75% alcohol and wiped dry.

[0077] (3) Morris water maze experiment

[0078] Water maze device: black circular pool (diameter 1.6 meters, height 50 centimeters), platform (diameter 10 centimeters, height 35 centimeters), camera system, with light blue curtain to isolate external direct light. During the experiment, the water in the pool was dyed black with edible carbon powder, and colorful and different shaped pictures were pasted on the wall as maze outside hints. Before the experiment, the pool surface was divided into 4 equal quadrants using real-time analysis software, with the platform located in the 1st quadrant, and the 2nd, 3rd, and 4th quadrants in a clockwise direction.

[0079] Maze adaptation training: During this training period, the platform was located 1 cm above the water. The water maze adaptation training lasted for 1 day. First, the rats were placed on the platform for 30 seconds (to facilitate the rats' memory of the platform's location based on the maze outside hints), and then the water training began. The rats were placed into the pool from the midpoint of the 1st quadrant pool wall facing the wall, timed for 60 seconds. If the rats found the platform within 60 seconds, they were allowed to stand on the platform for 15 seconds, their bodies were mostly dried, and then they were placed back in the cage. Warm air and a warm stove were used for warming to avoid stress reactions caused by long-term low temperature.

[0080] Position navigation experiment: During this experiment, the platform was placed 1 cm under water. The position navigation experiment lasted for 5 days, and each day, the rats were trained 3 times, and randomly entered the water from 3 different entry points (the entry points of the first 5 days were 2, 3, and 4; 1, 4, and 3; 4, 1, and 2; 3, 2, and 1; and 2, 4, and 3, respectively). The maximum time limit for each rat to swim in the pool was 60 seconds. If the rat successfully found and climbed onto the platform within the specified time range, the time from entering the pool to successfully climbing onto the platform was recorded as the escape latency. If the rat did not find the platform within 60 seconds of entering the water, the tester would use a climbable tool to pull the rat to the platform. The rat was allowed to stay on the platform for 15 seconds to help the rat better remember the location of the platform. If the rat did not climb onto the platform within the time limit, the escape latency was recorded as 60 seconds.

[0081] Spatial exploration experiment: During this experiment, the hidden platform under water was removed. The rats were placed in the water from the entry point of the 3rd quadrant, facing the wall of the pool. The number of times the rats crossed the original platform location, the number of times they entered the quadrant where the platform was located, the time percentage of the quadrant where the platform was located, the first time they passed through the platform, and the swimming trajectory were recorded within 60 seconds.

[0082] 4. Material preparation

[0083] After the behavioral index test, the experimental rats in each group were subjected to fasting without water restriction for 12 hours. Physiological saline, PBS solution, and 4% paraformaldehyde solution (solvent: PBS) were prepared in advance and subjected to ice bath treatment. An appropriate amount of chloral hydrate was weighed and dissolved in physiological saline to prepare a 10% chloral hydrate solution.

[0084] Three rats from each group were randomly selected and given intraperitoneal anesthesia with 10% chloral hydrate solution, and their weights were measured. After 10 minutes of anesthesia, the rats were fixed on the operation board in a supine position. The chest was opened, and the heart was exposed by pulling with forceps. Perfusion was performed through the left ventricle. The right atrium was incised, and PBS treated with ice bath was first perfused. When the liver and lungs turned white, and the right atrium flowed with clear liquid, 4% paraformaldehyde treated with ice bath was perfused. When the rat's limbs and tail became stiff, the perfusion was immediately terminated, the head was removed, and the brain was placed in 4% paraformaldehyde fixative. After 24 hours, the fixative was replaced with new 4% paraformaldehyde. The remaining 7 rats in each group were anesthetized, and the brain tissue was quickly removed after the animal was sacrificed, rinsed gently in ice-cold physiological saline to remove blood, dried with absorbent paper, and separated into hippocampus and cortex parts, then placed in EP tubes and temporarily stored in liquid nitrogen before being transferred to a -80°C refrigerator for storage.

[0085] 5. Detection of cerebral cortex and hippocampal neurons in rats of each experimental group

[0086] (1) Embedding and sectioning

[0087] The fixed 48 hours of rat brain tissue was placed in a brain mold to divide the brain tissue into two halves in the sagittal plane, and appropriate trimming was performed. The brain tissue was placed in a centrifuge tube containing a 30% sucrose solution (4% paraformaldehyde solution) for dehydration treatment. After about 48 hours, the brain tissue sank to the bottom of the centrifuge tube, and the next step was performed (the temperature of the freezing microtome was adjusted to -20°C in advance). The dehydrated brain tissue was removed and washed with PBS. After the water on the surface of the brain tissue was absorbed with a water-absorbing paper, it was placed flat in a silicone mold. Sakura OCT cryo-embedding agent was dripped and ensured that the tissue was completely soaked with the embedding agent. Then the mold was placed on a freezing table for freezing treatment. The frozen brain tissue block was removed and fixed flat on a tissue support, clamped on the microtome holder, and then sectioned. The cut frozen sections were collected in a container containing PBS. An appropriate amount of PBS was dripped on a pathological glass slide, and the collected brain sections were selected for appropriate size for mounting. The glass slide with the brain sections was dried at room temperature, and then stored in a -80°C refrigerator for standby.

[0088] (2) Nissl staining

[0089] The frozen sections were removed from the -80°C refrigerator and placed in a wet box. They were allowed to warm up and dry naturally at room temperature. 80 μl of Nissl staining solution was dropped onto the tissue sections. An appropriate amount of water was added to the wet box, the lid was closed, and it was placed in a 37°C oven for 10 minutes. The Nissl staining solution on the glass slide was poured off, and it was placed in a staining jar containing distilled water for washing. Then it was placed in 95% ethanol for rapid differentiation (about 5 seconds). After differentiation, it was placed in 95% ethanol staining jar I and staining jar II for 2 minutes each for dehydration. After dehydration, it was placed in staining jar I and staining jar II containing biological transparent agent TO for 5 minutes each for transparency. It was removed and slightly dried, and then it was ready for neutral resin sealing and mounting.

[0090] Detection: Under a microscope, the morphological changes of neurons in the hippocampal CA1 and CA3 regions of the brain were observed, as well as the number of Nissl bodies.

[0091] (3) Hematoxylin-eosin staining (HE staining)

[0092] The frozen section was taken out from the -80℃ refrigerator and placed in a wet box, and was allowed to re-warm and naturally dry at room temperature. 40 μl of hematoxylin staining solution was dropped on the tissue for staining treatment for 5 minutes. The stained slide was placed in a staining jar and washed under flowing water for 10 minutes. After washing with distilled water, 40 μl of eosin staining solution was dropped on the brain tissue for staining treatment for 30 seconds. Then, the slide was sequentially placed in 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 10 seconds each for gradient dehydration treatment. Then, the slide was placed in a biological transparent agent TO staining jar I and a staining jar II for 5 minutes each for transparency. After slight drying, the slide was sealed with neutral gum.

[0093] Detection: The cell nucleus was blue-violet, and the cytoplasm and extracellular matrix were purple-red.

[0094] 6. Detection of PI3K / AKT signaling pathway related indicators

[0095] (1) Western blot (WB) detection

[0096] Western blot is a method of separating proteins according to their molecular weight by polyacrylamide gel electrophoresis (PAGE), then transferring them to a polyvinylidene fluoride membrane (PVDF membrane) or a nitrocellulose membrane (NC membrane), and finally detecting and analyzing the expression level of the protein by immunology.

[0097] 1) Protein sample extraction

[0098] 20 mg of hippocampus tissue was weighed using a one-hundredth electronic analytical balance, cut into small pieces and placed in a 2 mL centrifuge tube. The centrifuge tube was added with a lysis solution containing protease inhibitors (brain tissue mass (mg): lysis solution volume (μL) = 1:9 ratio). The hippocampus tissue was placed in a high-speed low-temperature tissue grinder for homogenization. After homogenization, the EP tube was placed on ice for 30 minutes and then centrifuged (conditions: 15 minutes, 4℃, 12000 rmp). The supernatant was aspirated and centrifuged again (conditions: 10 minutes, 4℃, 12000 rmp). The supernatant was obtained, which was the protein sample to be tested. The EP tube was used for sub-packaging and placed in a -80℃ refrigerator for testing.

[0099] 2) Protein quantification

[0100] The operation method according to the BCA kit instruction is performed. First, the protein standard sample is diluted to a final concentration of 0.5 mg / mL protein standard sample solution. Then, the BCA working solution is prepared. The protein standard sample with a concentration of 0.5 mg / mL is added to the standard sample wells of the 96-well plate in the order of 0, 1, 2, 4, 8, 12, 6, 20 μL, and the standard sample diluent of 0.9% NaCl is added to each well to a solution volume of 20 μL. 20 μL of protein sample is added to the sample wells of the 96-well plate, followed by the addition of 200 μL of BCA working solution to each well. After shaking for 30 seconds to mix thoroughly, the plate is incubated in a 37°C oven for 0.5 hours. After removing the plate from the oven, it is shaken again for 30 seconds, and the absorbance is measured at a wavelength of 560 nm. The protein standard curve is calculated based on the absorbance, and the concentration of the test protein sample is estimated according to the formula.

[0101] 3) Protein denaturation

[0102] According to the protein concentration of each sample calculated in the previous step, the test protein sample obtained in 2.2.6.1.1 is diluted with RIPA lysis buffer without protease inhibitor to obtain a test protein sample with uniform protein concentration. Then, loading buffer is added (test protein volume: loading buffer volume = 1:4), and the sample is denatured in a 100°C constant temperature metal bath for 10 minutes, and then loaded. The unused sample can be stored at -80°C.

[0103] 4) Electrophoresis

[0104] The separation gel and 5% concentrated gel are prepared according to the molecular weight of the protein. After the concentrated gel is solidified, the comb is carefully removed. The prepared gel plate is fixed in the electrophoresis tank, and the prepared electrophoresis solution is added. No sample is added to the two outermost holes on both sides of the gel plate. 1.5 μL of protein Marker is added to the first and last holes except the outermost holes, and 3 μL of denatured protein sample is added to the remaining holes in order (the order of sample addition is: normal control group, D-gal model group, ZAF low dose group, ZAF high dose group, and donepezil group). After the sample addition is completed, the power of the multifunctional electrophoresis instrument is turned on. The initial voltage is set to 80 V, and the electrophoresis time is maintained for about 30 minutes. When the sample and Marker enter the separation gel, the voltage is increased to 120 V, and the process is stopped when the bromophenol blue migrates to a position about 0.5 cm from the bottom of the gel. This process takes about 1 hour and 20 minutes.

[0105] The concentration of the separation gel is selected according to the following table when preparing the separation gel. The concentration of the separation gel refers to the percentage of acrylamide in the separation gel, as shown in Table 1.

[0106] Table 1 Separation gel concentration formulation reference table

[0107]

[0108] 5) Transfer

[0109] The transfer buffer must be pre-cooled in an ice bath or placed in a 4°C refrigerator. Cut the PVDF membrane according to the size of the gel, and soak the cut PVDF membrane in methanol for 3-5 minutes for activation, then wash it with distilled water, and place it in the transfer buffer for standby. Soak two filter papers, a sponge pad, and the PVDF membrane in the transfer buffer for standby. During the transfer preparation, place the negative electrode (blackboard) at the bottom of the tray containing the transfer buffer, and then place the sponge pad, filter paper, and gel on the filter paper in turn. Then place the activated PVDF membrane on the gel, and then place the filter paper and sponge pad on it in turn. Carefully remove the air bubbles in each layer, cover the positive electrode (transparent plate), and then fix the transfer plate in the electrophoresis transfer slot for transfer preparation. Connect the power supply to the side of the transfer plate close to the PVDF membrane, set the constant current to 300 mA, and set the time to 1 hour (according to the specific protein molecular weight). Heat will be generated in the slot during the transfer, so place an appropriate size ice box on one side of the transfer slot for cooling. The excitation wavelength of Cy3 is 550 nm, and the emission wavelength is 570 nm (red fluorescence).

[0110] 6) Blocking and antibody incubation

[0111] After the transfer, take out the PVDF membrane and directly soak it in a 5% blocking solution (BSA / milk, TBST solution) for blocking for about 1 hour. After blocking, pour out the blocking solution, add TBST, and quickly wash it on a shaker for 3 times, each for 10 minutes. Soak the PVDF membrane in the primary antibody (1:1000, diluted in 5% BSA solution (TBST)) and place it on a shaker for incubation overnight (4°C). Recover the primary antibody, and first wash the PVDF membrane with TBST on a shaker for 3 times, each for 10 minutes. Soak the PVDF membrane in the secondary antibody diluted 1:8000, and incubate it on ice for 1.5 hours. Add TBST and wash it on a shaker for 3 times, each for 10 minutes.

[0112] 7) Blot luminescence imaging

[0113] Prepare the ECL luminescence solution (Luminol:Peroxide solution = 1:1) immediately before use. Place the PVDF membrane in the dark room of the imager, and evenly drop the luminescence solution on the membrane to obtain the image. Use the Image J (developed by the National Institutes of Health) software to analyze the band image obtained by development.

[0114] 7. Tunel apoptosis detection

[0115] The frozen section was taken out from the -80℃ refrigerator and placed in a wet box. It was rewarmed and naturally dried at room temperature. 50 μL of immunostaining strong penetration liquid was added to the brain tissue, and incubated at room temperature for 5 minutes. The TUNEL detection liquid was prepared according to the instructions. The permeabilized section was washed with PBS twice, and 50 μL of TUNEL detection liquid was added to the brain tissue section. The appropriate amount of water was added, the cover was covered, and it was incubated in a 37℃ oven for 60 minutes. The incubated section was washed with PBS three times, then naturally dried, and then sealed with anti-fluorescence quenching sealing liquid. The above operations were all carried out in the dark environment.

[0116] Detection: TUNEL positive cell nuclei were labeled with TUNEL reaction Cyanine 3 (Cy3) probe red fluorescence; DAPI stained cell nuclei emitted blue fluorescence.

[0117] 8. Statistical processing

[0118] The data was analyzed and processed by SPSS 26.0 statistical software. When the experimental data as a whole obeyed normal distribution, the experimental data was expressed in the form of Mean ± SE, and the experimental data between groups was compared by one-way analysis of variance. P<0.05 indicates that the data results have significant statistical difference, P<0.001 indicates that the data results have significant statistical difference, and P<0.001 indicates that the results between groups have extremely significant difference. The experimental results of Western blotting were analyzed by Image J.

[0119] III. Results

[0120] 1. Effect of ZAF on the behavior of rats

[0121] (1) Open field test

[0122] When studying the autonomous behavior of experimental animals, the open field test is often used to reflect the autonomous motor behavior of rats. The average speed, i.e. the ratio of total distance and time, horizontal score, etc. The results of this experimental index- average speed are shown in Table 2, Figure 1The average speed of D-gal model group was less than that of normal control group (P<0.01), indicating that the autonomous activity ability of rats was reduced after D-gal modeling; the average speed of ZAF low-dose group was obviously improved compared with that of D-gal model group (P<0.05); the average speed of ZAF high-dose group and that of donepezil group were improved compared with that of D-gal model group, but there was no statistical difference. This shows that the autonomous activity ability of D-gal model rats can be improved after low-dose drug intervention of ZAF, but the improvement is not proportional to the dose of the drug.

[0123] Table 2: Effect of ZAF on the average speed of rats in the open field (Mean ± SE, n = 8)

[0124]

[0125] Note: The data is in normal distribution, expressed as Mean ± SE, single factor analysis of variance is used, the result of variance homogeneity test is 0.095, which meets the variance homogeneity, multiple comparison uses LSD test, ## P<0.01 compared with normal control group; * P<0.05 compared with D-gal model group.

[0126] (2) Novel object recognition test

[0127] The novel object recognition test (NOR) is a simple and easy-to-operate experimental method that can verify whether the cognitive memory of rats is impaired. This experiment uses the instinct of rats to explore new things. The animals do not need to be contacted in advance before the experiment, which can avoid individual differences in the learning and training process. The novel object cognition index = new object exploration time / (new object exploration time + old object exploration time) x 100%. The results of the novel object cognition index of rats in each experimental group are shown in Table 3. In the experiment, compared with the normal control group, the novel object cognition index of the D-gal model group was significantly reduced (P<0.05); compared with the D-gal model group, the novel object cognition index of the ZAF high-dose group was significantly increased (P<0.05), and the cognition index of the ZAF low-dose group and the donepezil group was also improved compared with the model group, but there was no statistical difference. This shows that the cognitive ability of rats is impaired after D-gal modeling, and the high-dose treatment of ZAF significantly improves the cognitive ability of rats.

[0128] Table 3: Effect of ZAF on the novel object cognition index of rats in each experimental group (%) (Mean ± SE, n = 8)

[0129]

[0130] Note: The data accord with normal distribution, expressed as Mean ± SE, single factor analysis of variance was used, the result of variance homogeneity test was 0.095, which met the variance homogeneity, multiple comparisons used LSD test, # p<0.05 compared with the normal control group; * p<0.05 compared with the D-gal model group.

[0131] (3) Morris water maze experiment

[0132] The indicators of Morris water maze experiment can reflect the success of modeling of aging-related cognitive impairment from the perspective of behavior. It can also evaluate the therapeutic effect of drug intervention on experimental animals and verify the results of subsequent biochemical and pathological experiments. Compared with other maze experiments, water maze experiment has higher sensitivity to age-related learning and memory impairment, and does not require fasting before the experiment. Therefore, Morris water maze experiment was selected to evaluate the learning and memory of experimental rats in this study.

[0133] The escape latency of rats in each experimental group in the 5-day positioning and navigation experiment is shown in Tables 4, 5 and Figure 3 、 4 During the first 2 days of the experiment, there was no statistical difference in the escape latency of rats in each experimental group. On the 3rd, 4th and 5th days, the escape latency of rats in the D-gal model group was significantly longer than that in the normal control group, with a significant statistical difference (P<0.05), indicating that the modeling of D-gal was successful, and the spatial memory ability of rats was significantly decreased after injection of D-gal solution. After drug intervention with donepezil and ZAF, the escape latency of rats in the ZAF low-dose group and donepezil group was shorter than that in the D-gal model group on the 3rd day (P<0.05), and the escape latency of rats in the ZAF high-dose group was significantly shorter than that in the model group (P<0.001). On the 4th and 5th days, there was still a statistical difference in the escape latency between the ZAF high-dose group and the D-gal model group (P<0.05), and the escape latency of rats in the ZAF low-dose group and the donepezil group was shorter than that in the model group, but no statistical difference was observed. This indicates that ZAF can improve the spatial memory ability of D-gal model rats, and the effect may be positively correlated with the dose.

[0134] Table 4 Effect of ZAF on the escape latency of rats (seconds) (Mean ± SE, n = 8)

[0135]

[0136] Table 5 Effect of ZAF on the escape latency of rats (seconds)

[0137]

[0138] Note: The P value of normality test of each group on the first day was 0.2; the P value of normality test of each group on the second day was 0.2; the P value of normality test of each group on the third day was 0.056; the P value of normality test of each group on the fourth day was 0.011; the P value of normality test of each group on the fifth day was 0.076. Therefore, the data on the first, second, third and fifth days met the normal distribution, and were expressed as Mean ± SE, and single factor analysis of variance was used. The results of variance homogeneity test on the first, second, third and fifth days were 0.338, 0.801, 0.421 and 0.183, respectively, which met the homogeneity of variance, and LSD test was used for multiple comparisons. # p<0.05, ## p<0.01 compared with the normal control group; * p<0.05, *** p<0.001 compared with the D-gal model group. The normality test on the fourth day showed P<0.05, which did not meet the normal distribution, and therefore the median (quartile value) was used, and Kruskal-Wallis test was used for comparison among multiple groups, and the results of pairwise comparison showed that compared with the blank group,

[0139] The results of the experimental indicators of the spatial exploration test of the rats in each experimental group, i.e., the number of crossing the platform, the time in the target quadrant (i.e., the quadrant where the platform was located), and the target quadrant distance / total distance, are shown in Tables 6, 7 and 8, Figure 5 (A, B and C). Compared with the normal control group, the number of entering the target quadrant of the rats in the D-gal model group was significantly reduced, the time of staying in the target quadrant was significantly shortened, and the ratio of target quadrant distance / total distance was reduced (P<0.05), which was consistent with the behavioral results described above, indicating that the use of D-gal modeling would cause certain damage to the learning and memory of the rats; after the intervention of the sleep-aiding and spirit-soothing prescription, the number of crossing the platform of the low-dose group was significantly increased, the ratio of target quadrant distance / total distance was significantly improved (P<0.05), and the time of staying in the target quadrant was significantly increased (P<0.01); the time of staying in the target quadrant of the high-dose group was also significantly increased, and the ratio of target quadrant distance / total distance was significantly improved (P<0.01); the learning and memory improvement effect of donepezil on the rats was not obvious, and no significant difference was shown. The results of the positioning navigation experiment suggested that the sleep-aiding and spirit-soothing prescription could improve the learning and memory ability of the rats.

[0140] Table 6 Comparison of the number of entering the target quadrant of the rats in each experimental group (Mean ± SE, n=8)

[0141]

[0142] Table 7 Comparison of the time of staying in the target quadrant of the rats in each experimental group (Mean ± SE, n=8)

[0143]

[0144] Table 8 Comparison of target quadrant distance / total distance of rats in each experimental group (Mean ± SE, n = 8)

[0145]

[0146] Note: The data conforms to normal distribution, expressed as Mean ± SE, single factor analysis of variance is used, variance homogeneity test results are shown in the table, three indexes meet the homogeneity of variance, multiple comparisons use LSD test, # p < 0.05 compared with the normal control group; * p < 0.05, ** p < 0.01 compared with the D-gal model group.

[0147] 2, The effect of ZAF on hippocampal neurons of rats in each experimental group

[0148] HE staining results show that Figure 6 In the normal control group, the hippocampal CA3 region neurons are arranged in order and close, with clear edges, normal morphology, clear nuclei, blue-violet color, uniform staining, cytoplasm is peach red, uniform staining; compared with the normal control group, the D-gal model group of rats showed cell swelling, sparse and disordered arrangement; compared with the model group, the ZAF and donepezil administration groups can improve the sparse and disordered arrangement of hippocampal neurons and cell swelling. The shape, size and number of Nissl bodies can reflect the damage state of neurons in each region of the hippocampus. Nissl staining results show that Figure 7 Compared with the normal control group, the D-gal model group showed loose cell arrangement, partial cell swelling, and a significant decrease in intracellular Nissl bodies; compared with the model group, the ZAF treatment group showed a significant increase in Nissl bodies, and the neurons were arranged in order and close, without swollen cells, and the neurons in the donepezil administration group were also arranged in order compared with the model group. This indicates that ZAF may have a protective and repair effect on hippocampal cells in D-gal model rats.

[0149] 3, The effect of ZAF on the PI3K / AKT signaling pathway

[0150] (1) The results of P-PI3K, P-AKT, and AKT protein expression in the hippocampus of rats in each experimental group

[0151] In this experiment, Western Blot was used to detect the expression level of PI3K / AKT pathway related proteins in the hippocampus of rats in each experimental group, as shown in Table 9, Figure 8The protein expression level of P-AKT / AKT was lower than that of the normal control group, indicating that the role of PI3K / AKT pathway was weakened in the model group after D-gal drug modeling; the expression of P-AKT increased after the intervention of ZAF, indicating that ZAF might play a role in resisting AD by activating the PI3K / AKT pathway.

[0152] Table 9 Comparison of P-AKT relative expression of rats in each experimental group (Mean ± SE, n = 8)

[0153]

[0154] Note: The data conforms to normal distribution, expressed as Mean ± SE, single factor analysis of variance was used, the results of variance homogeneity test are shown in the table, which meets the homogeneity of variance, LSD test was used for multiple comparisons, # p < 0.05 compared with the normal control group.

[0155] 4. Effect of ZAF on neuronal apoptosis

[0156] (1) TUNEL method was used to detect hippocampal neuronal apoptosis

[0157] The results of TUNEL staining in the CA3 and CA1 regions of the hippocampus of rats in each experimental group are shown in Table 9, and the red fluorescence expression of the D-gal model group was significantly more than that of the normal control group, indicating that the apoptosis of hippocampal cells was more than that of normal rats after modeling; after the intervention of high and low doses of ZAF, the apoptosis of cells was significantly improved compared with the model group, indicating that ZAF can inhibit cell apoptosis. Figure 9

[0158] (2) WB was used to detect the protein expression of Bax, Bcl-2 and Cleaved Caspase-3 in each experimental group

[0159] Bax and Bcl-2 are key proteins related to apoptosis, and the ratio of Bax / Bcl-2 can reflect the condition of apoptosis. The expression results of apoptosis-related proteins (Bax, Bcl-2, Cleaved Caspase-3) of rats in each experimental group are shown in Table 9. Figure 10 ​The expression levels of Bax and Caspase-3 proteins in the hippocampus of the D-gal model group were significantly higher than those in the normal model group, and the expression level of BCL-2 was significantly lower than that in the normal model group; the protein expression level of Bcl-2 in the high-dose Zhenmian Anshen Decoction group and the donepezil group was significantly increased, and the protein expression level of Bcl-2 in the low-dose Zhenmian Anshen Decoction group was relatively small; the relative expression level of Bax protein in the rats intervened by Zhenmian Anshen Decoction and donepezil was significantly lower than that in the D-gal model group. It is indicated that Zhenmian Anshen Decoction can effectively improve the impaired learning and memory of D-gal model rats, which may be related to its inhibition of hippocampal neuron apoptosis.

[0160] Table 10 Comparison of the relative expression levels of Bax / Bcl-2 in rats in each experimental group (Mean ± SE, n = 8)

[0161]

[0162] Note: The data conforms to normal distribution, expressed as Mean ± SE, single factor analysis of variance is used, the results of variance homogeneity test are shown in the table, which meets the variance homogeneity, multiple comparisons are performed by LSD test, ### p < 0.001 compared with the normal control group; * p < 0.05, ** p < 0.01 compared with the D-gal model group.

[0163] The animal experiment of this embodiment verifies the use of the classical D-galactose modeling method, which is a mature, recognized and most commonly used aging model. The D-gal-induced aging model produces a series of pathological characteristics such as learning and cognitive impairment, cholinergic dysfunction, neuroinflammation, oxidative stress, mitochondrial dysfunction and abnormal neuron structure, and even AD-like pathological characteristics such as amyloid plaque deposition, neuron fiber tangles and Tau protein hyperphosphorylation. The D-galactose modeling method has the advantages of simple operation, high repeatability, low mortality and comprehensive damage to the body. AD is an age-related degenerative disease, so it is quite reasonable to choose D-gal model rats to study AD.

[0164] In the behavioral experiment, the results of behavioral experiment showed that the average speed of D-gal model rats in the open field experiment, the new object recognition index in the new object recognition test were lower than those of the normal control group; in the Morris water maze test, compared with the normal control group, the escape latency of D-gal model rats was longer, the number of crossing the platform was less, the time of staying in the target quadrant was shorter, and the ratio of target quadrant path / total path was smaller, indicating that the model rats had learning ability impairment and memory impairment. After the intervention of the sleep-aiding and tranquilizing prescription, the autonomous motor ability, learning ability and spatial memory of the model rats were improved to different degrees.

[0165] The results of Nissl staining and HE staining showed that the neurons in the hippocampal CA1 and CA3 regions of D-gal model rats appeared swelling, increased intercellular space, disordered arrangement, and obvious pathological changes such as loss of Nissl bodies and vacuoles. The TUNEL staining results showed that the apoptosis of hippocampal cells in D-gal model rats was increased compared with normal rats. After treatment with the sleep-aiding and tranquilizing prescription, the pathological damage and apoptosis in the hippocampal region of D-gal model rats were significantly improved.

[0166] The expression levels of PI3K / AKT signaling pathway and apoptosis-related proteins were detected by Western Blot. The results showed that the relative expression levels of P-AKT and P-PI3K in D-gal model rats were reduced, indicating that D-galactose could cause the transmission of PI3K / AKT signaling pathway to be blocked, which would cause the apoptosis of hippocampal neurons; the ratio of Bax / Bcl-2 was significantly increased. After the intervention of the sleep-aiding and tranquilizing prescription, the relative expression levels of P-AKT and P-PI3K were increased; the ratio of Bax / Bcl-2 was significantly reduced. It is indicated that the sleep-aiding and tranquilizing prescription can improve the apoptosis of hippocampal neurons, and may play a role by activating the PI3K / AKT signaling pathway.

[0167] Example 3 Network pharmacology experiment

[0168] Network pharmacology is a frontier discipline that can more systematically and comprehensively study the pharmacological mechanism of traditional Chinese medicine and can explain the complex network relationship among "drug-target-disease". Traditional Chinese medicine has the advantages of multi-target and multi-pathway, and it has unique advantages and unlimited potential in the treatment of AD with complex pathogenesis. Therefore, in this embodiment, the method of network pharmacology is used to screen, predict and analyze the multi-component, multi-target and multi-pathway mechanism of the sleep-aiding and tranquilizing prescription for preventing and treating AD. The specific steps are as follows:

[0169] I. Method

[0170] 1. Screening of active ingredients and target genes of ZAF

[0171] (1) ZAF effective component screening: log in to the Herb website, respectively input "jujube kernel", "ginseng", "Huangjing" and so on, collect the name, alias and other information of 10 kinds of effective components; log in to the STRING11.0 database, according to the name, alias, molecular formula and other information of the collected effective components, obtain the CAS number of the effective components without MOL ID; log in to the TCMSP database, according to the obtained CAS number, convert the herb ID of the effective components into MOL ID, and collect the OB value, DL value and other data information of the effective components, remove the effective components without target by taking OB value≥30, DL≥0.18 as the screening condition, screen the effective components and their target names. Log in to PubChem, input the molecular structure of the effective components in the sleep-aiding and tranquilizing prescription without MOL ID and CAS number, obtain the related information of the effective components, and then according to the Lipinski five principles (molecular weight less than 500Da; the number of hydrogen bond donors not more than 5; the number of hydrogen bond acceptors not more than 10; the logarithmic value (logP) of the lipid water partition coefficient of the compound not more than 5; the number of rotatable bonds in the compound not more than 10), the Isomeric SMILES of the screened effective components are recorded.

[0172] (2) ZAF effective component target protein official name correction: the Isomeric SMILES collected in (1) are searched and the pharmacokinetic information of the related components is obtained through SwissADME, and screened according to the standards of GI absorption (High) and at least three Yes, and the target prediction is carried out by using Swiss Target prediction, the targets of each effective component are integrated, and the target protein name is corrected to its official name.

[0173] 2、ZAF treatment AD target screening

[0174] Log in to GeneCards, OMIM and DisGeNET respectively, input "Alzheimer's disease" as the keyword to search for all targets for treating AD, export the search results, combine and delete duplicate data of the search results of each database, and take the intersection of the final results and the targets corresponding to the ZAF effective components to obtain the related target data of ZAF for treating AD. The target data of ZAF for treating AD is arranged, the microbioinformatics cloud platform is logged in, the Wayne diagram is drawn for the data results, and the STRING11.0 database is displayed.

[0175] 3、Protein-protein interaction (PPI) network construction

[0176] Import the corrected ZAF-related target information for AD treatment, select "homo sapiens" as the species, and obtain and export the tsv file containing the target protein-target interaction data. Import the tsv file into Cytoscape.

[0177] Software 3.9.1 is used to modify and enhance the PPI graph, and the Centiscape 2.2 plugin is used to perform topological analysis on the PPI graph. Finally, the differences in node color size are displayed according to their degree values, with the intensity of the hue reflecting the magnitude of the combine score between nodes. Potential core targets are then screened based on the degree values.

[0178] 4. Geneontology (GO) functional enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis

[0179] Log in to the DAVID database and input the potential targets for treating AD from the ZAF active ingredient collected in step 2. Complete the GO and KEGG pathway enrichment analysis for all potential targets and export the enrichment results. Delete invalid data and sort the target protein counts in descending order. Plot the results using an online bioinformatics platform.

[0180] 5. Construction of network diagrams of drug components and target proteins, and target proteins and pathways.

[0181] Data on ZAF drugs, active ingredients, and ZAF anti-AD therapeutic targets were compiled. Using Cytoscape 3.9.1 software, the compiled data was entered, and a "drug-ingredient-target" network diagram was constructed. The CytoHubba plugin was then used to analyze the degree values ​​of each node in the network diagram and the relationships between nodes. Based on the degree value results, the main active ingredients that play a role in the prevention and treatment of AD were identified.

[0182] II. Results

[0183] 1. Screening results of the effective components and targets of ZAF in the fight against AD

[0184] Finally, 1087 effective components of ZAF were collected by Herb, and 60 effective components were obtained by screening related information. The action gene target points of ZAF were 985, which were collected by the effective components. The potential action target points of AD were 14956, which were collected by GeneCards and DisGeNET. After median screening, 1605 potential action target points of AD were obtained. The intersection of the effective component target points of ZAF and the potential action target points of AD was 328, which were related to the potential target points of ZAF against AD (see Figure 11 )

[0185] 2. PPI network construction

[0186] The target points of ZAF against AD were input into STRING11.0 database, and the network interaction data was saved as tsv format file and imported into Cytoscape3.9.1 software after PPI network analysis. The PPI network diagram obtained is shown in the left of Figure 12 The nodes in the diagram represent target proteins, and each edge represents the interaction between them. The PPI network interaction diagram shows that there are 328 proteins that interact with each other, and the number of edges is 7926. According to centiscape 2.2, the topological analysis was performed, and three parameters of betweenness, closeness and degree were selected and screened according to the median of the three parameters. The key potential target points that meet the three conditions of betweenness≥328.63, closeness≥0.0016 and degree≥48.32 were obtained. The top five core target points in degree are AKT1, GAPDH, ALB, BCL2 and CASP3, respectively. Figure 13 ).

[0187] 3. Gene ontology (GO) function enrichment analysis

[0188] The 328 target points of ZAF against AD were input into DAVID database for GO function analysis, and the data were sorted according to the involved gene number. A total of 1179 biological processes, 168 cellular components and 271 molecular functions were obtained. Among them, the biological processes mainly involve signal transduction, protein phosphorylation, positive regulation of RNA polymerase II promoter transcription, negative regulation of apoptosis, etc.; the cellular components mainly involve plasma membrane, cytoplasm and cytosol, etc.; the molecular functions involve protein binding, identical protein binding, ATP binding, protein serine / threonine / tyrosine kinase activity, etc. The top 10 of the count of related target genes in GO function analysis were imported into microbioinformatics platform for visual analysis (see Figure 14), BP is represented by bamboo green, CC by pumpkin green, and MF by blue-purple.

[0189] 4. KEGG pathway enrichment analysis

[0190] The 328 target sites of the sleep-aiding and calming formula against AD were input into the DAVID database. After enrichment analysis, the pathways were sorted according to the number of proteins involved, resulting in 195 pathways. Based on the number (count) of target genes enriched in the signaling pathways and their significance, this embodiment screened the top 20 pathways related to AD. Next, this embodiment used the KEGG Pathway to generate a bubble diagram using the MicroBio platform. The diagram shows that the larger the bubble, the more genes enriched in that pathway, which means the higher the importance of that pathway (see...). Figure 15 Among these signaling pathways, those closely related to Alzheimer's disease (AD) include the AD pathway, the PI3K / Akt pathway, and neurodegenerative pathways.

[0191] 5. Construction of the network diagram of ZAF drug-component-potential target protein

[0192] Based on the aforementioned screening data, this embodiment visualizes information such as the drug, its active ingredients, and the target proteins of the sleep-aiding and calming formula for AD. Using Cytoscape 3.9.1 software, this embodiment constructs a drug-ingredient-potential target network diagram, which illustrates the interactions between the drug, active ingredient, and AD-related target protein pathways in ZAF (see [link to documentation]). Figure 16 Using the CytoHubba plugin for... Figure 16 Analysis of the main active ingredients in ZAF revealed the following ten components: Beta-sitosterol, Kaempferol, Quercetin, Naringenin, Baicalein, Beta-carotene, Wogonin, Nobiletin, DFV, and Diosgenin. These ten components all scored highly and can be considered key components of ZAF in the prevention and treatment of Alzheimer's disease (AD).

[0193] The above results indicate that the sleep-aiding and calming formula has the advantages of multiple targets and multiple pathways. In the face of AD with its complex pathogenesis, it demonstrates the unique advantages and unlimited potential of traditional Chinese medicine in the treatment of AD, and provides new ideas for the clinical treatment of AD.

[0194] Example 4

[0195] The sleep-aiding and spirit-calming formula: 10 g of ginseng, 15 g of rhizoma polygonati, 15 g of mulberry fruit, 15 g of poria, 15 g of lotus seed, 15 g of spina date seed, 15 g of lily, 15 g of longan arillus, and 15 g of hui wheat are weighed according to the prescription and uniformly mixed. The sleep-aiding and spirit-calming formula lyophilized powder is prepared according to the method described in Example 1, and the AD treatment test is carried out according to the method described in Example 2. The results show that the sleep-aiding and spirit-calming formula has the effect of treating Alzheimer's disease and improving the learning and memory impairment related to aging.

[0196] Example 5

[0197] The sleep-aiding and spirit-calming formula: 10 g of ginseng, 15 g of rhizoma polygonati, 15 g of mulberry fruit, 15 g of poria, 15 g of lotus seed, 15 g of spina date seed, 15 g of lily, 15 g of longan arillus, and 15 g of hui wheat are weighed according to the prescription and uniformly mixed. The sleep-aiding and spirit-calming formula lyophilized powder is prepared according to the method described in Example 1, and the AD treatment test is carried out according to the method described in Example 2. The results show that the sleep-aiding and spirit-calming formula has the effect of treating Alzheimer's disease and improving the learning and memory impairment related to aging.

[0198] Example 6

[0199] The sleep-aiding and spirit-calming formula: 10 g of ginseng, 10 g of rhizoma polygonati, 10 g of mulberry fruit, 15 g of poria, 15 g of lotus seed, 30 g of spina date seed, 15 g of lily, 15 g of longan arillus, 30 g of hui wheat, 6 g of tangerine peel, and 5 g of gardenia are weighed according to the prescription and uniformly mixed. The sleep-aiding and spirit-calming formula lyophilized powder is prepared according to the method described in Example 1, and the AD treatment test is carried out according to the method described in Example 2. The results show that the sleep-aiding and spirit-calming formula has the effect of treating Alzheimer's disease and improving the learning and memory impairment related to aging.

[0200] Example 7

[0201] The sleep-aiding and spirit-calming formula: 5 g of ginseng, 15 g of rhizoma polygonati, 15 g of mulberry fruit, 15 g of poria, 15 g of lotus seed, 20 g of spina date seed, 20 g of lily, 10 g of longan arillus, 15 g of hui wheat, 10 g of yizhi, and 10 g of malt are weighed according to the prescription and uniformly mixed. The sleep-aiding and spirit-calming formula lyophilized powder is prepared according to the method described in Example 1, and the AD treatment test is carried out according to the method described in Example 2. The results show that the sleep-aiding and spirit-calming formula has the effect of treating Alzheimer's disease and improving the learning and memory impairment related to aging.

Claims

1. Use of a sleep-aiding and nerve-calming composition in the preparation of a medicament for the treatment of Alzheimer's disease, characterized in that, The sleep-aiding and tranquilizing composition is prepared from the following raw material components in mass parts: 5-10 parts of ginseng, 10-15 parts of rhizoma polygonati, 10-15 parts of mulberry fruit, 10-15 parts of poria cocos, 10-15 parts of lotus seed, 15-30 parts of spina date, 10-20 parts of lily, 10-15 parts of longan arillus, 15-30 parts of wheat, and 4-8 parts of dried tangerine or orange peel.

2. Use according to claim 1, characterized in that, The sleep-aiding and tranquilizing composition is prepared from the following raw material components in mass parts: 5-10 parts of ginseng, 10-15 parts of rhizoma polygonati, 10-15 parts of mulberry fruit, 10-15 parts of poria cocos, 10-15 parts of lotus seed, 15-30 parts of spina date, 10-20 parts of lily, 10-15 parts of longan arillus, 15-30 parts of wheat, and 4-8 parts of dried tangerine or orange peel.

3. Use according to claim 2, characterized in that, The concentration is rotary evaporation concentration.

4. The use according to claim 1, characterized in that, The medicine also comprises pharmaceutically acceptable adjuvants.

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