Application of sleep-aiding and nerve-calming composition in preparation of product for preventing and treating Alzheimer disease

Abstract

The invention discloses application of a sleep-aiding and nerve-calming composition in preparation of a product for preventing and treating Alzheimer's disease. The drug effect of the sleep-aiding and nerve-calming composition is observed through animal experiments, experimental rats are subjected to D galactose modeling, and experimental results show that compared with a blank control group, the number of times of platform crossing and platform quadrant staying time of rats in a D-GAL model group are remarkably reduced, the new object recognition coefficient and the target quadrant distance percentage are remarkably reduced, and the experimental results show that the number of times of platform crossing and platform quadrant staying time of rats in the D-GAL model group are remarkably reduced. AD modeling is successful; compared with a D-GAL model group, the number of times of platform crossing and platform quadrant staying time of an administration group of the sleep-aiding and nerve-soothing composition are remarkably increased, and the new object recognition coefficient and the target quadrant swimming distance percentage are remarkably increased. Results show that the sleep-aiding and nerve-soothing composition has the effect of treating the Alzheimer's disease, the active effect of the sleep-aiding and nerve-soothing composition on treating the AD is achieved through multiple targets and multiple ways, and a new thought is provided for clinical treatment of the AD.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to the use of a sleep-aiding and tranquilizing composition in the preparation of a product for preventing and treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset. Along with the degeneration and death of neurons, the memory, cognitive function and language function of AD patients gradually decline. AD has the pathogenic characteristics of a high disability rate. The quality of life of patients will gradually decline, and even in the late stage, they will be unable to take care of themselves and need to rely on others for care. Most AD patients have a poor prognosis, so AD also has a high mortality rate. The harm caused by AD is not limited to the patients themselves, but also harms their families and society, bringing a heavy economic burden, and causing psychological stress, depression and other health problems to family members of patients to varying degrees.

[0003] The specific pathogenesis of AD is not yet fully understood and is complex and diverse, but there are many hypotheses, such as the cholinergic neuron hypothesis, the neuroinflammation hypothesis, the free radical damage hypothesis, the Tau protein abnormality hypothesis, the Aβ toxicity hypothesis, the insulin hypothesis, the energy metabolism hypothesis, etc. At present, most of the therapeutic drugs used in clinical treatment of AD are single-target drug molecules, which only work on a single target, which may be the main reason for their unsatisfactory clinical efficacy and relatively large toxic side effects, and they cannot fundamentally reverse the pathological process of AD. Therefore, seeking drugs for the treatment of AD with multiple targets will become the mainstream of the research and development of AD therapeutic drugs.

[0004] Patent CN 110772595 A discloses a medicine-food combination that has a sleep-inducing and tranquilizing effect. Its main ingredients are Chinese date seeds, ginseng, and polygonatum. The combination of these medicines has the effects of both movement and stillness, cold and warm, and can strengthen the spleen and nourish blood, nourish the heart and liver, promote sleep, improve sleep quality, and prolong sleep time. In clinical applications, it can significantly improve patients' sleep disorders. However, there is no report on the effect of this combination in the treatment of AD and learning and memory disorders. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a sleep-aiding and tranquilizing composition for use in the preparation of a product for preventing and / or treating Alzheimer's disease.

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

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The research group of the present invention previously disclosed a composition with sleep-inducing and calming effects, namely, a sleep-inducing and calming prescription, which comprises the following raw material components in parts by weight: 5-10 parts of ginseng, 10-15 parts of polygonatum, 10-15 parts of mulberry fruits, 10-15 parts of poria cocos, 10-15 parts of lotus seeds, 15-30 parts of spiny jujube seeds, 10-20 parts of lilies, 10-15 parts of longan pulp, 15-30 parts of Huai wheat, and 4-8 parts of tangerine peel. The invention adopts the sleep-inducing and tranquilizing prescription to carry out AD treatment test, and D-galactose modeling is carried out on experimental rats. The experimental results show that compared with the normal control group, the number of times the D-galactose model rats cross the platform and the platform quadrant time are significantly reduced, and the new object recognition coefficient and the target quadrant distance percentage are also significantly reduced, indicating that the AD disease modeling is successful; compared with the D-galactose model group, after the drug intervention of the sleep-inducing and tranquilizing prescription, the number of times the model rats cross the platform and the platform quadrant time are significantly increased, and the new object recognition coefficient and the target quadrant swimming distance percentage are significantly increased, and the sleep-inducing and tranquilizing prescription can improve the cell morphology and arrangement of neurons in the hippocampus of the model rats, improve the loss of Nissl bodies, reduce the relative expression of BAX protein, and increase the relative expression of BCL-2, indicating that the sleep-inducing and tranquilizing prescription can improve the neuronal apoptosis of the D-galactose model rats and improve the learning and memory damage related to aging, and can improve the learning and memory ability, indicating that the sleep-inducing and tranquilizing prescription has the effect of treating Alzheimer's disease.

[0009] In addition, through the prediction and analysis of network pharmacology, it was found that the anti-AD effective ingredients of the sleep-control and tranquilizing prescription were 60 in total, of which 10 ingredients played a major key role, including β-sitosterol, kaempferol, quercetin, naringenin, baicalin, β-carotene, wogonin, nobiletin, licorice, diosgenin, etc.; the five potential core targets of ZAF against AD were AKT1, GAPDH, ALB, BCL2, and CASP3, and these five core targets were closely related to cell apoptosis. This shows that the sleep-control and tranquilizing prescription has the advantages of multiple targets and multiple pathways. In the face of AD with complex pathogenesis, it demonstrates the unique advantages and unlimited potential of traditional Chinese medicine in treating AD, and provides new ideas for the clinical treatment of AD.

[0010] Therefore, the present invention provides the use of the sleep-aiding and calming composition in the preparation of a product for preventing and / or treating Alzheimer's disease, wherein the sleep-aiding and calming composition comprises the following raw material components in parts by weight: 5 to 10 parts of ginseng, 10 to 15 parts of polygonatum, 10 to 15 parts of mulberry fruits, 10 to 15 parts of poria cocos, 10 to 15 parts of lotus seeds, 15 to 30 parts of spiny jujube seeds, 10 to 20 parts of lilies, 10 to 15 parts of longan pulp, and 15 to 30 parts of Huai wheat.

[0011] The term "prevention and / or treatment" means that the disease or disorder described in the present invention can be treated, prevented, alleviated and / or alleviated in a subject.

[0012] The present invention also provides an application of the sleep-aiding and tranquilizing composition in the preparation of a product for improving aging-related learning and memory disorders. The sleep-aiding and tranquilizing composition comprises the following raw material components in parts by weight: 5 to 10 parts of ginseng, 10 to 15 parts of polygonatum, 10 to 15 parts of mulberry fruits, 10 to 15 parts of poria cocos, 10 to 15 parts of lotus seeds, 15 to 30 parts of spiny jujube seeds, 10 to 20 parts of lilies, 10 to 15 parts of longan pulp, and 15 to 30 parts of Huai wheat.

[0013] As a preferred implementation method, the sleep-aiding and calming composition comprises the following raw material components in parts by mass: 10 parts of ginseng, 15 parts of polygonatum, 15 parts of mulberry fruit, 10 parts of poria, 10 parts of lotus seed, 15 parts of spiny jujube kernel, 10 parts of lily, 10 parts of longan pulp, and 15 parts of Huai wheat.

[0014] Furthermore, the sleep-aiding and calming composition also comprises the following raw material components in parts by mass: 4 to 8 parts of tangerine peel.

[0015] As a preferred implementation method, the sleep-aiding and calming composition comprises the following raw material components in parts by mass: 10 parts of ginseng, 15 parts of polygonatum, 15 parts of mulberry fruit, 10 parts of poria, 10 parts of lotus seeds, 15 parts of spiny jujube seeds, 10 parts of lily, 10 parts of longan pulp, 15 parts of Huai wheat, and 6 parts of tangerine peel.

[0016] Furthermore, the sleep-aiding and calming composition also comprises the following raw material components in parts by mass: 8 to 12 parts of bergamot.

[0017] As a preferred implementation method, the sleep-aiding and calming composition comprises the following raw material components in parts by mass: 10 parts of ginseng, 15 parts of polygonatum, 15 parts of mulberry fruits, 15 parts of poria, 15 parts of lotus seeds, 15 parts of spiny jujube seeds, 15 parts of lilies, 15 parts of longan pulp, 15 parts of Huai wheat, and 10 parts of citron.

[0018] Furthermore, the sleep-aiding and calming composition also comprises the following raw material components in parts by mass: 4 to 8 parts of dried tangerine peel and 3 to 7 parts of gardenia.

[0019] As a preferred implementation method, the sleep-aiding and calming composition comprises the following raw material components in parts by mass: 10 parts of ginseng, 10 parts of polygonatum, 10 parts of mulberry fruit, 15 parts of poria, 15 parts of lotus seeds, 30 parts of spiny jujube seeds, 15 parts of lily, 15 parts of longan pulp, 30 parts of Huai wheat, 6 parts of tangerine peel, and 5 parts of gardenia.

[0020] Furthermore, the sleep-aiding and calming-mind composition also comprises the following raw material components in parts by weight: 8 to 12 parts of Alpinia oxyphylla kernels and 8 to 12 parts of malt.

[0021] As a preferred implementation method, the sleep-aiding and calming composition contains the following raw material components in parts by mass: 5 parts of ginseng, 15 parts of polygonatum, 15 parts of mulberry fruits, 15 parts of poria, 15 parts of lotus seeds, 20 parts of spiny jujube seeds, 20 parts of lilies, 10 parts of longan pulp, 15 parts of Huai wheat, 10 parts of alpinia oxyphylla seeds, and 10 parts of malt.

[0022] Furthermore, the product is a medicine or health product. Since the Chinese medicine in the sleep-aiding and tranquilizing composition is both a medicine and food, and does not show obvious dose dependence in experimental tests, it can be used to prepare medicines or can be used in small doses in daily diets 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 that receive the medicine or health product of the present invention to treat, prevent, alleviate and / or relieve the diseases or symptoms of the present invention. In a preferred embodiment, the medicine or health product is used for mammals, including but not limited to humans, cattle, horses, sheep, pigs, goats, rabbits, cats, dogs, mice and any other mammals that have livers and can cause damage.

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

[0025] The dosage of the drug or health product of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the route of administration and the number of administrations, etc. It can be administered in a single dose or divided into several, such as two, three or four doses. The dosage level must be selected based on the specific route of administration, the severity of the condition to be treated, and the condition and previous medical history of the patient to be treated.

[0026] However, it should be recognized that the total daily dosage of the drug or health product of the present invention must be determined by the attending physician within the scope of sound medical judgment. For any specific patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health, sex and diet; administration time, route of administration and excretion rate; duration of treatment; drugs used in combination or concurrently; and similar factors known in the medical field.

[0027] Furthermore, the present invention also provides a method for preparing the sleep-aiding and tranquilizing composition, which comprises mixing the raw material components according to their mass fractions, soaking them in water, decocting them several times, combining the filtrate, concentrating them, and freeze-drying them to obtain a freeze-dried powder of the sleep-aiding and tranquilizing composition.

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

[0029] Preferably, the product further comprises excipients acceptable in pharmaceutical or health products.

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

[0031] The present invention provides an application of a sleep-aiding and tranquilizing composition in the preparation of a product for preventing and treating Alzheimer's disease. The present invention observes the pharmacodynamic effect of the sleep-aiding and tranquilizing composition through animal experiments, and performs D-galactose modeling on experimental rats. The experimental results show that compared with the blank control group, the number of times the D-GAL model group rats cross the platform and the time they stay in the platform quadrant are significantly reduced, and the new object recognition coefficient and the percentage of the target quadrant distance are significantly reduced; compared with the model group, the number of times the sleep-aiding and tranquilizing composition administration group crosses the platform and the time they stay in the platform quadrant are significantly increased, and the new object recognition coefficient and the percentage of the target quadrant swimming distance are significantly increased, and the sleep-aiding and tranquilizing composition can improve the cell morphology and arrangement of neurons in the hippocampus of the model rats, improve the loss of Nissl bodies, reduce the relative expression of BAX protein, and increase the relative expression of BCL-2, indicating that the sleep-aiding and tranquilizing composition can improve the neuronal apoptosis of D-galactose model rats and improve the learning and memory damage related to aging, indicating that the sleep-aiding and tranquilizing prescription has the effect of treating Alzheimer's disease. And it is to play its positive role in treating AD through multiple targets and multiple pathways, providing new ideas for the clinical treatment of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 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 Comparison of novel object recognition 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 Comparison of the escape latency of the water maze test in rats of 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 Figure 2 is the water maze movement trajectory diagram of rats in each experimental group.

[0036] Figure 5Comparison 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 The hippocampal neurons (HE staining, ×40).

[0038] Figure 7 The hippocampal neurons (Nissl staining, ×40).

[0039] Figure 8 The protein expressions of P-PI3K and P-AKT in rats of 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] Fig. 9 Figure 1 shows neurons in the hippocampus (TUNEL staining, ×40). 1: normal model group; 2: D-gal model group; 3: low-dose ZAF group; 4: high-dose ZAF group; 5: donepezil group.

[0041] Fig.10 The protein expressions of Bax, Bcl-2, and Cleave Caspase-3 in rats of 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] Fig.11 It is the intersection of ZAF targets and AD targets.

[0043] Fig.12 It is a potential target of ZAF.

[0044] Fig.13 Visual analysis of potential core targets of ZAF for treating AD.

[0045] Fig.14 This is a histogram of GO enrichment analysis results.

[0046] Fig.15 This is a bubble diagram of the KEGG enrichment analysis pathway.

[0047] Fig.16 This is a network diagram of ZAF's drugs, ingredients, and potential target proteins. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention 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 Lyophilized Powder of Sleep-Assisting and Anxiety-Calming Recipe

[0051] The method for extracting freeze-dried powder of the sleep-aiding and tranquilizing prescription continues the previous Chinese medicine extraction method of the research team members. According to the dosage of the prescription, 10g of ginseng, 15g of polygonatum, 15g of mulberry fruit, 10g of poria, 10g of lotus seed, 15g of spiny jujube seed, 10g of lily, 10g of longan meat, 15g of Huai wheat, and 6g of tangerine peel were weighed. 10 herbs are mixed evenly (the total weight of each dose is 116 grams, and 5 doses of medicine are decocted each time, i.e., the total weight is 580g), and distilled water (about 5.8 liters) 10 times the weight of the total amount of the prescription drugs is added to fully soak for 1 hour; the medicine is placed in a thermostat for heating, and after boiling for 1 hour, the Chinese medicine liquid is collected after filtering through multiple layers of cotton gauze; distilled water (about 4.6 liters) 8 times the weight of the original prescription drugs is added to the Chinese medicine residue after one decoction, and heating is continued. After boiling for 1 hour, the liquid is collected after filtering through multiple layers of cotton gauze; the Chinese medicine liquids after two decoctions are mixed, and after filtering with a vacuum pump equipment, a relatively clear Chinese medicine liquid is obtained for use.

[0052] The above-mentioned medicinal liquid is evaporated and concentrated using a rotary evaporator to make the medicinal liquid become a relatively thick state; the concentrated medicinal liquid is poured into a flat container, sealed, and placed in a -80°C refrigerator for 12 hours, and then placed in a freeze dryer for dehydration to prepare a lyophilized powder of the sleep-aiding and tranquilizing prescription for use.

[0053] Example 2: Treatment of AD with a sleep-aiding and tranquilizing prescription

[0054] 1. Experimental Materials

[0055] 1. Experimental Animals

[0056] Fifty-five SD rats were selected for the experiment (rat information: SPF grade, male, 7-8 weeks old, body weight range of 200±20 g, purchased from the Guangdong Provincial Medical Experimental Animal Center, experimental animal production license number: SCXK(Yue)2022-0002; certificate number: 44007200108544. They were housed in the barrier environment of the Experimental Animal Center of Guangzhou University of Chinese Medicine, experimental animal use license number: SYXK(Yue)2018-0001, maintained with free access to diet and water, the breeding temperature was 26±2 °C, the indoor humidity was constant, the ventilation was good, with a 12-hour light-dark cycle, and the bedding was changed regularly 2-3 times a week. In this study, animals were treated in accordance with the requirements of the "Guiding Opinions on the Humane Treatment of Laboratory Animals", obtained animal ethics approval and complied with relevant welfare requirements, and were supervised by the ethics committee.

[0057] After 1 week of adaptive feeding, the rats were randomly divided into a normal control group (CTRL), a D-gal model group (D-GAL), a low-dose ZAF group (ZAF-L), a high-dose ZAF group (ZAF-H), and a positive drug (donepezil) group (DNP). There were 11 rats in each group, divided into 2 cages, with 5-6 rats in each cage. Except for the necessary drug administration operations, the feed and drinking water conditions of the rats in each cage were the same during daily life.

[0058] II. Experimental methods

[0059] 1. Drug dosage

[0060] (1) Dosage of the Zhumian Anshen formula (ZAF)

[0061] In this experiment, the equivalent dose conversion formula was referred to the formula provided by the FDA in the "Guidance on the Estimation of the Maximum Safe Starting Dose in Initial Clinical Trials of Therapeutic Drugs 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 (human body weight) × 6.2. The calculated administration dose was set as the daily administration dose of the rats in the high-dose ZAF group. Therefore, the doses of the Zhumian Anshen formula administered by gavage to the rats in the high-dose and low-dose ZAF groups were 12 and 6 g / kg / d (calculated by the weight of crude drug), respectively.

[0062] (2) Preparation and dosage of the positive drug (donepezil)

[0063] Referring to the adult donepezil use dose of 5 mg / day, the drug administration dose of the rats in the donepezil group was calculated as 3 mg / kg / d according to the animal-human equivalent dose conversion formula. The donepezil used in this experimental study was a powdery drug, the solvent of donepezil was distilled water, and the administration method was gavage.

[0064] (3) Preparation and dosage of the modeling drug (D-galactose, abbreviated as D-gal)

[0065] The drug administration was carried out with reference to the previous modeling method of the inventor's research group on aging models with cognitive impairment. In this experiment, the D-gal dosage was 150 mg / kg / day, the solvent of D-gal was sterile saline, and the administration method was intraperitoneal injection.

[0066] 2. Animal modeling and experimental drug intervention methods

[0067] (1) Rat model of aging-related cognitive impairment

[0068] After the adaptive feeding was completed, D-galactose was injected into the abdomen to establish the model. The modeling lasted for ten weeks. Except for the normal control group, which was injected with an equal volume of normal saline every day, the other experimental drug groups were injected with D-gal solution 150 mg / kg / d in the abdomen.

[0069] (2) Trial drug intervention

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

[0071] 3. Behavioral Experiment

[0072] Behavioral experiments were started 10 days before the end of the experimental drug intervention, including open field test, novel object recognition test and water maze test, during which the model was still established and drug intervention was given.

[0073] (1) Open field test

[0074] The rats were moved to the behavioral laboratory one day before the experiment, and a 12-hour light-dark cycle was maintained during the experiment to adapt to the test environment and avoid stress and pressure on the experimental animals caused by the change of the experimental environment. The test experiment was carried out in a quiet behavioral laboratory with weak and stable light intensity. The rats were quickly placed in the experimental box (specifications: 40cm×40cm×35cm), and the free activities of the rats were recorded within 5 minutes. The open box was divided into 16 grids by SuperMaze animal behavior video analysis software. The time spent in the 4 central quadrants was considered as the central residence time, and the analysis software was used to record and analyze the total distance, central distance, central time, etc. of the rats' movement. The experimental box must be cleaned between the experiments of two rats, sprayed with 75% alcohol and wiped dry. Avoid the interference of the residual smell of the previous experimental rat on the experimental animal placed this time. The video content was analyzed by SuperMaze animal behavior video analysis software V2.0.

[0075] (2) New object recognition experiment

[0076] The new object recognition experiment was conducted on the second day after the open field experiment, which lasted for two days. The new object recognition experiment instrument and software of Shanghai Xinruan Company were used. Since the experimental rats had been free in the current experimental box one day before the new object recognition experiment, the new object recognition experiment was carried out directly without adaptation. On the first day of the experiment, two identical objects A (A1 and A2) were placed in the relative positions in the experimental box. The rats were placed in the experimental box with their backs to objects A1 and A2, and the time was counted for 5 minutes. The distance between the placement point of the rats and objects A1 and A2 should be consistent. After the time is over, the animals were taken out and put back into the animal cage. On the second day, one of the two identical objects A (A1 or A2) was replaced with another object (B) of the same size and different shape. Therefore, on the second day of the experiment, the objects placed in the relative positions in the experimental box 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 various data of the experimental rats' exploration behavior of the new and old objects. The test time was 5 minutes. The position of the rats placed in the experimental box each time should be as consistent as possible, with their backs facing the objects and at equal distances from the two objects. In order to eliminate the influence of residual information such as odor and excrement, the objects and experimental box should be cleaned in time, sprayed with 75% alcohol and wiped dry.

[0077] (3) Morris water maze test

[0078] Water maze device: black circular pool (1.6 meters in diameter, 50 centimeters in height), platform (10 centimeters in diameter, 35 centimeters in height), camera system, light blue curtain to block direct light from the outside. During the experiment, the water in the pool was dyed black with edible carbon powder, and pictures of bright colors and different shapes were posted on the wall as hints outside the maze. Before the experiment began, the pool surface was divided into 4 equal quadrants using real-time analysis software. The quadrant where the platform was located was the first quadrant, and the second, third, and fourth quadrants were clockwise.

[0079] Maze adaptation training: During this training period, the platform is located 1 cm above the water. Water maze adaptation training lasts for 1 day. First, place the rat on the platform for 30 seconds (so that the rat can remember the location of the platform based on the hints outside the maze), and then start water training. Put the rat into the pool from the midpoint of the first quadrant pool wall facing the pool wall, and count for 60 seconds. If the rat finds the platform within 60 seconds, let it stand on the platform for 15 seconds, wipe off most of the water on its body, and then put it back into the cage. Each rat is trained once. Use a heater and a stove for heating to avoid stress reactions caused by long-term low temperature.

[0080] Positioning and navigation experiment: During this experiment, the platform was placed 1 cm underwater. The positioning and navigation experiment lasted for 5 days, with 3 training sessions per day. The rats randomly entered the water from 3 water entry points (the 3 water entry quadrants on days 1-5 were: 2, 3, 4; 1, 4, 3; 4, 1, 2; 3, 2, 1; 2, 4, 3). The maximum time limit for each rat to enter the pool and swim was 60 seconds. If the rats successfully found and climbed onto the platform within the specified time frame, the time recorded from entering the pool to successfully climbing onto the platform would be considered as the escape latency. If the rats did not find the platform within 60 seconds after entering the water, the tester would use a clinging tool to pull the rats to the platform. The rats stayed on the platform for 15 seconds so that they could better remember the location of the platform. For rats that did not climb onto the platform within the time limit, the escape latency was recorded as 60 seconds.

[0081] Spatial exploration test: During this experimental period, the platform hidden in the water was removed. The rats were placed in the water from the entry point in the third quadrant, facing the pool wall. The number of times the rats crossed the original platform position, the number of times they entered the quadrant where the platform was located, the percentage of time in the quadrant where the platform was located, the time of first passing the platform, and the swimming trajectory were recorded within 60 seconds.

[0082] 4. Obtain materials

[0083] After the behavioral index test, the experimental rats in each group were fasted but not watered for 12 hours. Physiological saline, PBS solution, and 4% paraformaldehyde solution (solvent: PBS) were prepared in advance and placed in an ice bath. An appropriate amount of chloral hydrate was weighed and prepared into a 10% chloral hydrate solution with physiological saline.

[0084] Three rats were randomly selected from each experimental rat group, and given intraperitoneal anesthesia with 10% chloral hydrate solution, and weighed. After anesthesia for 10 minutes, the rats were fixed on the operating board in a supine position. The chest cavity was opened, fixed and pulled with forceps to expose the heart, and the perfusion was inserted through the left ventricle. The right auricle was cut open, and PBS treated with ice bath was first perfused. When the liver and lungs turned white and clear liquid flowed out of the right atrium, 4% paraformaldehyde after ice bath could be perfused. When the rats showed stiffness in the limbs and tail, the perfusion was stopped immediately, the brain was removed by decapitation and placed in 4% paraformaldehyde fixative. After 24 hours, the fixative was replaced with new 4% paraformaldehyde. After anesthesia, the remaining 7 rats in each group were killed and the brain tissue was quickly removed, placed in frozen physiological saline for slight rinsing, blood was removed, and dried with absorbent paper. The hippocampus and cortex were separated, placed in EP tubes, temporarily stored in liquid nitrogen, and then transferred to a -80℃ refrigerator for storage.

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

[0086] (1) Embedding and sectioning

[0087] The rat brain tissue fixed for 48 hours was placed in a brain mold, the sagittal plane of the brain tissue was divided into two halves, and it was properly trimmed and placed in a centrifuge tube containing 30% sucrose solution (4% paraformaldehyde) for dehydration. After about 48 hours, the brain tissue sank to the bottom of the centrifuge tube and the next step could be performed (the temperature of the freezing slicer was adjusted to -20°C in advance); the dehydrated brain tissue was taken out and washed with PBS, the surface of the brain tissue was dried with absorbent paper, and it was placed flat in a silicone mold, and Sakura OCT was dropped into it. Freeze the embedding agent and ensure that the tissue is completely immersed in the embedding agent, then put the mold on the freezing table for freezing, take out the frozen brain tissue block and fix it flat on the tissue support, clamp it on the slicer holder, and then you can start slicing. Collect the cut frozen slices in a container filled with PBS, drop an appropriate amount of PBS on the pathology slide, select the collected brain slices of appropriate size for patch processing, and the slides with brain slices need to dry at room temperature, and then the slides can be placed in a -80℃ refrigerator for storage.

[0088] (2) Nissl staining

[0089] Take out the frozen sections from the -80℃ refrigerator and place them in a humidified box. Let them warm up and dry naturally at room temperature. Take 80μl of Nissl staining solution and drop it on the tissue sections. Add an appropriate amount of water to the humidified box, cover it, and place it in a 37℃ oven for 10 minutes. Pour off the Nissl staining solution on the slides, put them into a dyeing jar filled with distilled water to wash them slightly, and then put them into 95% ethanol for rapid differentiation (about 5 seconds). After differentiation, put them into 95% ethanol dyeing jar I and dyeing jar II for dehydration, each for 2 minutes. After dehydration, put them into dyeing jar I and dyeing jar II filled with biological transparent agent TO for 5 minutes each to make them transparent. Take them out and let them dry slightly, then you can use neutral gum to seal the slides.

[0090] Detection: Microscopic observation, observation of neuronal tissue morphology and changes in the number of Nissl bodies in the CA1 and CA3 regions of the hippocampus.

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

[0092] The frozen sections were taken out of the -80℃ refrigerator and placed in a wet box. They were allowed to warm up and dried naturally at room temperature. 40μl of hematoxylin staining solution was dropped on the tissue for staining for 5 minutes. The stained slides were placed in a staining jar and rinsed under running water for 10 minutes. The slides were then rinsed with distilled water and 40μl of eosin staining solution was dropped on the brain tissue for staining for 30 seconds. The slides were then placed in 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 10 seconds each for gradient dehydration. The slides were then placed in staining jars I and II containing biological transparent agent TO for 5 minutes each for transparency. After drying slightly, the slides were sealed with neutral gum.

[0093] Detection: The cell nucleus is blue-purple, and the cytoplasm and extracellular matrix are purple-red.

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

[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 polyvinylidene fluoride membrane (PVDF membrane) or nitrocellulose membrane (NC membrane), and finally analyzing the expression level of the proteins by immunological detection.

[0097] 1) Protein sample extraction

[0098] Weigh 20 mg of hippocampal tissue using a 1 / 10,000 electronic analytical balance, cut it into pieces and put it into a 2 mL centrifuge tube. Add lysis solution containing protease inhibitors to the centrifuge tube (brain tissue weight (mg): lysis solution volume (μL) = 1:9 ratio). Put the hippocampal tissue into a high-speed low-temperature tissue grinder for homogenization. After the homogenization, place the EP tube on ice for 30 minutes and then centrifuge it (conditions: 15 minutes, 4°C, 12000rmp), aspirate the supernatant, and centrifuge it again (conditions: 10 minutes, 4°C, 12000rmp) to obtain the supernatant, which is the protein sample to be tested. Use EP tubes to divide it and put it into a -80°C refrigerator for testing.

[0099] 2) Protein quantification

[0100] According to the operating method of the BCA kit manual, first dilute the protein standard with the standard diluent to a final concentration of 0.5 mg / mL of protein standard solution. Then prepare the BCA working solution for use. Add the protein standard with a concentration of 0.5 mg / mL to the standard wells of the 96-well plate one by one at a volume of 0, 1, 2, 4, 8, 12, 6, and 20 μL, and use the standard diluent of 0.9% NaCl to supplement the solution volume of each well to 20 μL. Add 20 μL of protein sample to the sample well of the 96-well plate, then add 200 μL of BCA working solution to each well, place it on the microplate reader and shake it for 30 seconds to mix it thoroughly, then place it in an oven at 37 ° C and incubate it for 0.5 hours, take it out and shake it again on the microplate reader for 30 seconds, measure the absorbance at a wavelength of 560 nm, and calculate the standard curve of the protein based on this, and then estimate the concentration of the protein sample to be tested based on these formulas.

[0101] 3) Protein denaturation

[0102] According to the protein concentration of each sample calculated in the previous step, dilute the protein sample to be tested obtained in 2.2.6.1.1 with RIPA lysis buffer without protease inhibitors to a protein sample to be tested with the same protein concentration, then add loading buffer (volume of protein to be tested: volume of loading buffer = 1:4), place in a 100℃ constant temperature metal bath for denaturation for 10 minutes, and then load the sample. Unused samples can be stored at -80℃.

[0103] 4) Electrophoresis

[0104] Prepare separation gel and 5% concentrated gel of corresponding concentration according to the molecular weight of the protein. After the concentrated gel solidifies, carefully pull out the comb. Fix the prepared gel plate in the electrophoresis tank, add the configured electrophoresis solution, do not add samples to the two outermost holes on both sides of the gel plate, add 1.5μL protein Marker to the first and last holes except the outermost holes, and add 3μL denatured protein samples to the remaining holes in sequence (the order of adding samples is: normal control group, D-gal model group, ZAF low-dose group, ZAF high-dose group, donepezil group). After completing the sample addition operation, start the power supply of the multifunctional electrophoresis instrument, set the voltage to 80V in the initial stage, and keep the electrophoresis time for about 30 minutes. When the sample and Marker enter the separation gel, increase the voltage to 120V until bromophenol blue migrates to a position about 0.5 cm from the bottom of the gel, then stop the electrophoresis, and this process takes about 1 hour and 20 minutes.

[0105] When preparing the separation gel, refer to the table below to select the concentration of the separation gel. The concentration of the separation gel refers to the percentage of acrylamide in the volume of the separation gel, as shown in Table 1.

[0106] Table 1 Separation gel concentration preparation reference table

[0107]

[0108] 5) Transfer

[0109] The electrotransfer 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. The cut PVDF membrane must be immersed in methanol for 3-5 minutes for activation, then washed with distilled water, and then placed in the electrotransfer buffer for use. Then soak 2 filter papers, sponge pads and PVDF membranes in the electrotransfer buffer for later use. In the process of preparing for the transfer membrane, place the negative electrode (blackboard) on the tray containing the electrotransfer buffer as the bottom, and place the sponge pad and filter paper in turn. The gel is carefully separated from the glass plate and placed on the filter paper. Then place the activated PVDF membrane on the gel, and then cover it with filter paper and sponge pads in turn. Each layer is carefully drained of bubbles, and the positive electrode (transparent plate) is covered. After the transfer plate is fixed and securely inserted into the electrophoresis transfer tank for electrotransfer preparation. Let the side of the transfer plate close to the PVDF membrane face the anode and connect the power supply, the constant current is 300mA, and the time is set to 1 hour (according to the specific setting of the protein molecular weight). During electroporation, heat will be generated in the tank. An ice box of appropriate size can be placed on one side of the electroporation tank for cooling. The excitation wavelength of Cy3 is 550nm and the emission wavelength is 570nm (red fluorescence).

[0110] 6) Blocking and antibody incubation

[0111] After the transfer, take out the PVDF membrane and directly immerse it in a 5% blocking solution (BSA / milk, prepared with TBST solution) for about 1 hour; after the blocking is completed, pour out the blocking solution, add TBST and wash quickly on a shaker for 3 times, 10 minutes each time; immerse the PVDF membrane in the primary antibody (1:1000, 5% BSA solution dilution (TBST preparation)), and place it on a shaker to incubate overnight (4°C). Recover the primary antibody, and wash the PVDF membrane with TBST on a shaker for 3 times, 10 minutes each time. Immerse the PVDF membrane in a secondary antibody diluted 1:8000 and incubate on ice for 1.5 hours; add TBST and place it on a shaker to wash 3 times, 10 minutes each time.

[0112] 7) Imaging of blot luminescence

[0113] ECL luminescent solution was prepared and used immediately (Luminol:Peroxide solution=1:1). A PVDF membrane was placed in the darkroom of the imager and the luminescent solution was evenly dropped on the membrane to obtain images. The developed band images were analyzed using Image J (developed by National Institutes of Health) software.

[0114] 7. Tunel apoptosis detection

[0115] Take the frozen sections out of the -80°C refrigerator and place them in a wet box. Rewarm and dry them naturally at room temperature. Drop 50 μL of strong immunostaining permeabilization solution on the brain tissue and incubate at room temperature for 5 minutes. Prepare TUNEL detection solution according to the configuration ratio in the instructions for use. Wash the permeabilized sections twice with PBS, drop 50 μL of TUNEL detection solution on the brain tissue sections, add an appropriate amount of water, cover the lid, and incubate in a 37°C oven for 60 minutes. Wash the incubated sections three times with PBS, then dry them naturally and seal them with anti-fluorescence quenching sealing solution. All the above operations are performed in a light-proof environment.

[0116] Detection: TUNEL-positive cell nuclei are labeled with red fluorescence using the TUNEL reaction Cyanine 3 (Cy3) probe; cell nuclei stained with DAPI emit blue fluorescence.

[0117] 8. Statistical processing

[0118] SPSS26.0 statistical software was used to analyze and process the data. When the experimental data obeyed the normal distribution as a whole, the experimental data were expressed in the form of Mean±SE. The experimental data among the groups were compared by one-way analysis of variance. P<0.05 indicated that the data results had significant statistical differences, P<0.001 indicated that the data results had significant statistical differences, and P<0.001 indicated that the differences between the groups were extremely significant. Western blotting experimental results were analyzed using Image J.

[0119] 3. Results

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

[0121] (1) Open field test

[0122] When studying the autonomous behavior of experimental animals, the experimental indicators of the open field test are often used to reflect the autonomous movement behavior ability of rats, such as average speed, which is the ratio of the total movement distance and time, level score, etc. The results of this experimental indicator - average speed are shown in Table 2. Figure 1As shown. The average speed of the D-gal model group was lower than that of the normal control group (P < 0.01), indicating that the autonomous activity ability of rats was reduced after D-gal modeling; the average speed of the low-dose ZAF group was significantly improved compared with the D-gal model group (P < 0.05); the average speed of the high-dose ZAF group and the nepezil group was improved compared with the D-gal model group, but there was no statistical difference. This shows that the autonomous movement ability of D-gal model rats can be improved after low-dose drug intervention of the Auxiliary Sleeping and Anshen Recipe, but this improvement is not proportional to the dose of the drug.

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

[0124]

[0125] Note: The data conform to normal distribution and are expressed as Mean ± SE. One-way ANOVA was used, and the result of variance homogeneity test was 0.095, which met the variance homogeneity. LSD test was used for multiple comparisons. ## p<0.01 compared with the normal control group; * p<0.05 compared with the 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. There is no need to contact the animals in advance before the experiment, which can avoid individual differences in the learning and training process. Novel object recognition index = novel object exploration time / (new object exploration time + old object exploration time) × 100%. The results of the novel object recognition index of rats in each experimental group of this experiment are shown in Table 3. In the experiment, compared with the normal control group, the novel object recognition index of the D-gal model group was significantly reduced (P<0.05); compared with the D-gal model group, the novel object recognition index of the high-dose ZAF group was significantly increased (P<0.05), and the cognitive index of the low-dose ZAF group and the donepezil group was also improved compared with the model group, but there was no statistical difference. It shows that the cognitive ability of rats was impaired after D-gal modeling, and the high-dose treatment of the sleep-controlling recipe significantly improved the cognitive ability of rats.

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

[0129]

[0130] Note: The data conform to normal distribution and are expressed as Mean ± SE. One-way ANOVA was used, and the result of variance homogeneity test was 0.095, which met the variance homogeneity. LSD test was used for multiple comparisons. # p<0.05 compared with the normal control group; * p<0.05 compared with the D-gal model group.

[0131] (3) Morris water maze test

[0132] The indicators of the Morris water maze experiment can provide feedback from a behavioral perspective on the success of modeling aging-related cognitive impairment. It can also evaluate the efficacy of experimental animals after drug intervention, and can also be verified with the results of subsequent biochemical pathology experiments. Compared with other maze experiments, the advantage of the water maze experiment is that it has a higher sensitivity to age-related learning and memory impairment, and fasting is not required before the experiment. Therefore, this experimental study chose the Morris water maze experiment to evaluate the learning and memory of experimental rats.

[0133] During the 5-day localization and navigation experiment, the results of the escape latency of rats in each experimental group are shown in Tables 4, 5 and Figure 3 , 4 As shown. In the first two days of the experiment, the escape latency of rats in each experimental group showed no statistical difference. 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, and there was a significant statistical difference (P < 0.05), indicating that the D-gal modeling was successful, and the injection of D-gal solution significantly reduced the spatial memory ability of rats; after the drug intervention of donepezil and the sleep-control formula, the escape latency of the low-dose ZAF group and the donepezil group was shorter than that of the D-gal model group on the 3rd day (P < 0.05), and the escape latency of the high-dose ZAF group was significantly shorter than that of the model group (P < 0.001); on the 4th and 5th days, there was still a statistical difference in the escape latency between the high-dose ZAF group and the D-gal model group (P < 0.05), and the escape latency of the low-dose ZAF group and the donepezil group was shorter than that of the model group, but there was no statistical difference. This indicates that the sleep-aiding and tranquilizing prescription can improve the spatial memory ability of D-gal model rats, and it may be positively correlated with the size of the dosage.

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

[0135]

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

[0137]

[0138] Note: The P value of normality test for each group on the first day is 0.2; the P value of normality test for each group on the second day is 0.2; the P value of normality test for each group on the third day is 0.056; the P value of normality test for each group on the fourth day is 0.011; the P value of normality test for each group on the fifth day is 0.076. Therefore, the data on the first, second, third, and fifth days conform to the normal distribution, expressed as Mean ± SE, and one-way analysis of variance was used. The results of the 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 variance homogeneity, and the 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. On the fourth day, the normality test P<0.05 did not conform to the normal distribution, so the median (quartile value) was used to express it, and the Kruskal-Wallis test was used for comparison among multiple groups. The results of pairwise comparison showed that compared with the blank group.

[0139] The experimental indicators of the spatial exploration test of rats in each experimental group - the number of times crossing the platform, the time in the target quadrant (i.e., the quadrant where the platform is located), and the distance in the target quadrant / total distance are shown in Tables 6, 7, 8, Figure 5 (A, B, C). Compared with the normal control group, the D-gal model group rats entered the target quadrant significantly less times, stayed in the target quadrant for a significantly shorter time, and the ratio of target quadrant distance to total distance decreased (P < 0.05), which was consistent with the behavioral results mentioned above, indicating that the use of D-gal modeling would cause certain damage to the learning and memory of rats; after drug intervention of the Auxiliary Sleeping and Anshen Recipe, the low-dose group crossed the platform significantly more times, the ratio of target quadrant distance to total distance was significantly improved (P < 0.05), and the target quadrant stay time was significantly increased (P < 0.01); the high-dose group also stayed in the target quadrant for a significantly longer time, and the ratio of target quadrant distance to total distance was significantly improved (P < 0.01); Donepezil had no obvious effect on the improvement of rats' learning and memory, and no significant difference was shown. The results of the positioning navigation experiment suggest that the Auxiliary Sleeping and Anshen Recipe can improve the learning and memory abilities of rats.

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

[0141]

[0142] Table 7 Comparison of the target quadrant residence time of 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 conform to normal distribution and are expressed as Mean ± SE. One-way ANOVA was used. The results of the variance homogeneity test are shown in the table. All three indicators meet the variance homogeneity. LSD test was used for multiple comparisons. # p<0.05 compared with the normal control group; * p<0.05, ** p<0.01 compared with the D-gal model group.

[0147] 2. Effects of ZAF on hippocampal neurons of rats in each experimental group

[0148] HE staining results showed that ( Figure 6 ), the neurons in the CA3 region of the hippocampus in the normal control group were arranged neatly and tightly, with clear edges and normal morphology, clear cell nuclei, bluish purple, and evenly stained, and the cytoplasm was pink and evenly stained; compared with the normal control group, the cells in the D-gal model group were swollen and arranged sparsely and disorderly; compared with the model group, both the sleep-control and donepezil groups could improve the sparse and disordered arrangement of hippocampal neurons and cell swelling. Changes in the shape, size, and number of Nissl bodies can reflect the damaged state of neurons in various regions of the hippocampus. The results of Nissl staining showed ( Figure 7 ), compared with the normal control group, the cells in the D-gal model group were not arranged compactly, some cell bodies were swollen, and the number of Nissl bodies in the cells was significantly reduced; compared with the model group, the number of Nissl bodies in the Zhumian Anshen Fang treatment group was significantly increased, and the neurons were arranged more neatly and compactly, and no swollen cell bodies were found. The neurons in the donepezil group were also arranged more neatly than those in the model group. This shows that the Zhumian Anshen Fang may have a protective and repairing effect on the hippocampal cells of D-gal model rats.

[0149] 3. Effect of ZAF on PI3K / AKT signaling pathway

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

[0151] In this experiment, Western Blot was used to detect the expression levels 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 effect of PI3K / AKT pathway in the model group was weakened after D-gal drug modeling; the expression of P-AKT increased after the intervention of Zhumian Anshen Fang, indicating that Zhumian Anshen Fang may play an anti-AD role by activating PI3K / AKT pathway.

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

[0153]

[0154] Note: Data conform to normal distribution, expressed as Mean ± SE, and one-way ANOVA was used. The results of variance homogeneity test are shown in the table. The variance homogeneity was satisfied, and 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 assay 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 as follows Fig. 9 As shown, the red fluorescence expression of rats in the D-gal model group was significantly higher than that in the normal control group, indicating that the cell apoptosis in the hippocampus after modeling was more than that in normal rats; after high and low dose intervention of the Auxiliary Sleep and Anshen Decoction, the cell apoptosis was significantly improved compared with the model group, indicating that the Auxiliary Sleep and Anshen Decoction can inhibit cell apoptosis.

[0158] (2) Western blot analysis of the expression of Bax, Bcl-2, and Cleaved Caspase-3 proteins in rats of each experimental group

[0159] Bax and Bcl-2 are key proteins related to cell apoptosis. The ratio of Bax / Bcl-2 can reflect the status of cell apoptosis. The expression results of apoptosis-related proteins (Bax, Bcl-2, Cleaved Caspase-3) in rats of each experimental group are shown in Figure 2. Fig.10As shown in Table 10. The expression levels of Bax and Caspase-3 proteins in the hippocampus of rats in the D-gal model group were significantly higher than those in the normal model group, and the expression of BCL-2 was significantly lower than that in the normal model group; the protein expression of Bcl-2 in rats in the high-dose Zhumian Anshen Fang group and the donepezil group was significantly increased, and the protein expression of Bcl-2 in the low-dose Zhumian Anshen Fang group was relatively small; the relative expression of Bax protein in rats after drug intervention with Zhumian Anshen Fang and donepezil was significantly lower than that in the D-gal model group. This shows that Zhumian Anshen Fang can effectively improve the impaired learning and memory of D-gal model rats, which may be related to its ability to inhibit the apoptosis of hippocampal neurons.

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

[0161]

[0162] Note: Data conform to normal distribution, expressed as Mean ± SE, and one-way ANOVA was used. The results of variance homogeneity test are shown in the table. The variance homogeneity was satisfied, and LSD test was used for multiple comparisons. ### 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 verification of this embodiment adopts the classic D-galactose modeling method, which is a mature, recognized and most commonly used aging model. The aging model caused by D-gal will produce a series of pathological characteristics such as learning and cognitive impairment, cholinergic dysfunction, neuroinflammation, oxidative stress, mitochondrial dysfunction and abnormal neuronal structure, and even pathological characteristics of AD-like diseases such as amyloid plaque deposition, neurofibrillary tangles and hyperphosphorylation of Tau protein. The D-galactose modeling method has the advantages of simple operation, high repeatability, low mortality rate and comprehensive damage to the body. AD is an aging degenerative disease that is closely related to age, so it is quite reasonable to choose D-gal model rats to study AD.

[0164] In the behavioral experiment, the results showed that the average speed of D-gal model rats in the open field test and 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 D-gal model rats had a longer escape latency, fewer platform crossings, shorter target quadrant stay time, and a smaller target quadrant distance / total distance ratio, indicating that the model group rats had learning impairment and memory disorders. After the intervention of the sleep-aiding and tranquilizing prescription, the model rats' autonomous movement ability, learning ability, and spatial memory were improved to varying degrees.

[0165] The results of Nissl staining and HE staining showed that the neurons in the CA1 and CA3 regions of the hippocampus of the D-gal model rats were swollen, the intercellular spaces were enlarged, the arrangement was disordered, and the Nissl bodies were lost and the cells appeared vacuolated. The results of TUNEL staining showed that the apoptosis of the hippocampus of the D-gal model rats was more than that of the normal rats. After the treatment of the Zhumian Anshen prescription, the pathological damage and apoptosis of the hippocampus of the D-gal model rats were significantly improved.

[0166] Western Blot was used to detect the expression of PI3K / AKT signaling pathway and apoptosis-related proteins. The results showed that the relative expression of P-AKT and P-PI3K in D-gal model rats decreased, indicating that D-galactose can cause the body's PI3K / AKT signaling pathway to be blocked, which will cause apoptosis of hippocampal neurons; the ratio of Bax / Bcl-2 increased significantly. After the intervention of the drug of Zhumian Anshen Fang, the relative expression of P-AKT and P-PI3K increased; the ratio of Bax / Bcl-2 decreased significantly. This shows that Zhumian Anshen Fang can indeed improve the apoptosis of hippocampal neurons, perhaps by activating the PI3K / AKT signaling pathway.

[0167] Example 3 Network pharmacology experiment

[0168] Network pharmacology is a cutting-edge discipline that can more systematically and comprehensively study the pharmacological mechanisms of traditional Chinese medicine and explain the complex network relationship between "drug-target-disease". Traditional Chinese medicine has the advantages of multiple targets and multiple pathways. In the face of AD with complex pathogenesis, it demonstrates the unique advantages and unlimited potential of traditional Chinese medicine in treating AD. Therefore, this embodiment uses the method of network pharmacology to screen, predict and analyze the multi-component, multi-target and multi-pathway mechanisms of the sleep-aiding and tranquilizing prescription for the prevention and treatment of AD. The details are as follows:

[0169] 1. Methods

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

[0171] (1) Screening of ZAF active ingredients: Log in to the Herb website and input “suan jujube seed”, “ginseng”, “polygonatum”, etc. to collect the names, aliases and other information of the active ingredients of 10 herbs; log in to the STRING11.0 database and obtain the CAS numbers of the active ingredients that are not marked with MOL IDs based on the collected names, aliases, molecular formulas and other information of the active ingredients; log in to the TCMSP database and convert the herb IDs of the active ingredients into MOL IDs based on the obtained CAS numbers, and collect the OB values, DL values ​​and other data information of the active ingredients. Use OB values ​​≥ 30 and DL values ​​≥ 0.18 as screening conditions to remove the active ingredients without targets, and screen out the names of the active ingredients and their targets. Log in to PubChem, input the molecular structure of the active ingredient in the sleep-aiding and tranquilizing prescription without MOL ID and CAS number, obtain the relevant information of the active ingredient, and then record the Isomeric SMILES of the active ingredient obtained by screening according to Lipinski's five principles (molecular weight less than 500Da; the number of hydrogen bond donors does not exceed 5; the number of hydrogen bond acceptors does not exceed 10; the logarithm of the lipid-water partition coefficient (logP) of the compound does not exceed 5; the number of rotatable bonds in the compound does not exceed 10).

[0172] (2) Correction of the official name of the target protein of the active ingredient of ZAF: The isosomeric SMILES collected in (1) were searched and the pharmacokinetic information of the relevant ingredients was obtained through SwissADME. The SMILES were screened based on the criteria of GI absorption (High) and at least three Yess. The Swiss Target prediction was used for target prediction. The targets of each active ingredient were integrated and the target protein names were corrected to their official names.

[0173] 2. Screening of ZAF targets for AD treatment

[0174] Log in to GeneCards, OMIM, and DisGeNET respectively, enter "Alzheimer's disease" as a keyword to search for all targets for treating AD, export the search results, merge the results obtained from each database search and delete duplicate data, and intersect the final results with the targets corresponding to the active ingredients of ZAF to obtain the relevant target data of ZAF for treating AD. Organize the target data of ZAF for treating AD, log in to the Weishengxin Cloud Platform, and draw a Venn diagram to plot the data results. Display the STRING11.0 database.

[0175] 3. Construction of protein-protein interaction (PPI) network

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

[0177] 3.9.1 software was used to modify and beautify the PPI map, and the centiscape 2.2 plug-in was used to perform topological analysis on the PPI map. Finally, the color difference of the nodes was displayed according to the degree value, and the strength of the color tone reflected the size of the combine score between the nodes. Potential core targets were screened according to the size of the degree value.

[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 of ZAF active ingredients for treating AD collected in step 2. Complete GO and KEGG pathway enrichment analysis for all potential targets and export the enrichment results. Delete invalid data and sort the target protein count in descending order. Plot the results using the bioinformatics online mapping platform.

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

[0181] The data of ZAF drugs, active ingredients and ZAF anti-AD therapeutic targets were sorted, and the sorted data were entered using Cytoscape 3.9.1 software to construct a "drug-ingredient-target" network relationship diagram. The CytoHubba plug-in tool was imported to analyze the degree values ​​of each node in the network diagram and the relationship between each node, and the main active ingredients that play a role in the prevention and treatment of AD were identified based on the degree value results.

[0182] 2. Results

[0183] 1. Results of screening of active ingredients and targets of ZAF against AD

[0184] Finally, 1087 active ingredients of the sleep-inducing and tranquilizing formula were collected through Herb, and 60 active ingredients were finally obtained through screening through relevant information. The gene target information of the obtained active ingredients was collected, and a total of 985 gene targets of the sleep-inducing and tranquilizing formula were obtained; 14956 potential targets of AD disease were collected through GeneCards and DisGeNET, and 1605 potential disease targets that met the conditions after median screening. After the intersection of ZAF active ingredient targets and AD potential targets, a total of 328 potential targets related to ZAF anti-AD were obtained (see Fig.11 )

[0185] 2. PPI network construction

[0186] The targets of the sleep-control and tranquilizing prescription for treating AD were input into the STRING11.0 database. After PPI network analysis, the network interaction relationship data was saved as a tsv format file and imported into the Cytoscape3.9.1 software. The resulting PPI network diagram was drawn as shown in Fig.12 As shown on the left, the nodes in the figure represent target proteins, and each edge represents its interaction relationship. The PPI network interaction diagram shows that there are 328 interacting proteins and 7926 edges. According to centiscape 2.2, topological analysis was performed, and three parameters, namely, betweenness, closeness, and node connectivity (degree) values, were selected and screened according to the median of these three parameters. Key potential targets that simultaneously meet the three conditions of betweenness ≥ 328.63, closeness ≥ 0.0016, and degree ≥ 48.32 were obtained. The top five core targets ranked by degree are AKT1, GAPDH, ALB, BCL2, and CASP3 ( Fig.13 ).

[0187] 3. Gene Ontology (GO) functional enrichment analysis

[0188] The 328 targets of the sleep-aiding and tranquilizing prescription for the treatment of AD were entered into the DAVID database, and the data were obtained by GO functional analysis and sorted according to the number of proteins involved. A total of 1179 biological processes, 168 cellular components, and 271 molecular effects were obtained. Among them, the biological processes mainly involve signal transduction, protein phosphorylation, positive regulation of RNA polymerase II promoter transcription, negative regulation of cell apoptosis, etc.; the cellular components mainly involve plasma membrane, cytoplasm, cytosol, etc.; the molecular functions involve protein binding, same protein binding, ATP binding, protein serine / threonine / tyrosine kinase activity, etc. The top 10 related target gene counts in the GO functional analysis were imported into the microbial information platform for visual analysis (see Fig.14), BP is represented by bamboo green, CC is represented by pumpkin color, and MF is represented by blue-purple.

[0189] 4. KEGG pathway enrichment analysis

[0190] The 328 anti-AD targets of the sleep-aiding and tranquilizing prescription were input into the DAVID database, and after enrichment analysis and sorting by the number of proteins involved, a total of 195 pathways were obtained. Based on the number (count) of target genes enriched in the signaling pathway and their significance, this example screened the top 20 pathways related to AD. Next, this example used KEGG Pathway to draw a bubble chart through the microbial information platform. The figure shows that the larger the bubble, the more genes are enriched in the pathway, which means that the importance of the pathway is higher (see Fig.15 Among these signaling pathways, those closely related to AD include the AD pathway, PI3K / Akt pathway, and neurodegeneration pathway.

[0191] 5. Construction of a network diagram of ZAF's drugs, ingredients, and potential target proteins

[0192] Based on the aforementioned screening data, this example visualizes the information of the drug, its active ingredients, and the target of the sleep-aiding and tranquilizing prescription on AD. Using Cytoscape 3.9.1 software, this example constructs a drug-ingredient-potential target network diagram, which shows the interactive relationship between the drug, active ingredients, and target protein pathways related to AD in ZAF (see Fig.16 ). Use the CytoHubba plugin to Fig.16 The main active ingredients of ZAF were analyzed and it was found that Beta-sitosterol, Kaempferol, Quercetin, Naringenin, Baicalein, Beta-carotene, Wogonin, Nobiletin, DFV, and Diosgenin scored at the top, and they can be considered as the key ingredients of ZAF in the prevention and treatment of AD.

[0193] The above results show that the sleep-aiding and calming prescription has the advantages of multiple targets and multiple pathways. In the face of AD with a 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] Sleep-aiding and tranquilizing prescription: according to the dosage of the prescription, weigh 10g of ginseng, 15g of polygonatum, 15g of mulberry fruit, 10g of poria, 10g of lotus seed, 15g of spiny jujube seed, 10g of lily, 10g of longan pulp, and 15g of Huai wheat, mix these 9 herbs evenly, prepare the sleep-aiding and tranquilizing prescription freeze-dried powder according to the method described in Example 1, and conduct AD treatment test according to the method described in Example 2. The results show that the sleep-aiding and tranquilizing prescription has the effect of treating Alzheimer's disease and improving aging-related learning and memory disorders.

[0196] Example 5

[0197] Sleep-aiding and tranquilizing prescription: according to the dosage of the prescription, weigh 10g of ginseng, 15g of polygonatum, 15g of mulberry fruit, 15g of poria, 15g of lotus seed, 15g of spiny jujube seed, 15g of lily, 15g of longan pulp, 15g of Huai wheat, and 10g of citron, mix these 10 herbs evenly, prepare the sleep-aiding and tranquilizing prescription freeze-dried powder according to the method described in Example 1, and conduct AD treatment test according to the method described in Example 2. The results show that the sleep-aiding and tranquilizing prescription has the effect of treating Alzheimer's disease and improving aging-related learning and memory disorders.

[0198] Example 6

[0199] Sleep-aiding and tranquilizing prescription: according to the dosage of the prescription, weigh 10g of ginseng, 10g of polygonatum, 10g of mulberry fruit, 15g of poria, 15g of lotus seed, 30g of spiny jujube seed, 15g of lily, 15g of longan pulp, 30g of Huai wheat, 6g of tangerine peel, and 5g of gardenia, mix these 11 herbs evenly, prepare the sleep-aiding and tranquilizing prescription freeze-dried powder according to the method described in Example 1, and conduct AD treatment test according to the method described in Example 2. The results show that the sleep-aiding and tranquilizing prescription has the effect of treating Alzheimer's disease and improving aging-related learning and memory disorders.

[0200] Example 7

[0201] Sleep-aiding and tranquilizing prescription: according to the dosage of the prescription, weigh 5g of ginseng, 15g of polygonatum, 15g of mulberry fruit, 15g of poria, 15g of lotus seed, 20g of spiny jujube seed, 20g of lily, 10g of longan pulp, 15g of Huai wheat, 10g of alpinia oxyphylla, and 10g of malt, mix these 11 herbs evenly, prepare the sleep-aiding and tranquilizing prescription freeze-dried powder according to the method described in Example 1, and conduct AD treatment test according to the method described in Example 2. The results show that the sleep-aiding and tranquilizing prescription has the effect of treating Alzheimer's disease and improving aging-related learning and memory disorders.

Claims

1. Use of a sleep-aiding and tranquilizing composition in the preparation of a product for preventing and / or treating Alzheimer's disease, characterized in that: The sleep-aiding and tranquilizing composition comprises the following raw material components in parts by weight: 5-10 parts of ginseng, 10-15 parts of polygonatum, 10-15 parts of mulberry fruit, 10-15 parts of poria, 10-15 parts of lotus seed, 15-30 parts of spiny jujube kernel, 10-20 parts of lily, 10-15 parts of longan pulp, and 15-30 parts of Huai wheat.

2. Use of a sleep-aiding and tranquilizing composition in the preparation of a product for improving aging-related learning and memory disorders, characterized in that: The sleep-aiding and tranquilizing composition comprises the following raw material components in parts by weight: 5-10 parts of ginseng, 10-15 parts of polygonatum, 10-15 parts of mulberry fruit, 10-15 parts of poria, 10-15 parts of lotus seed, 15-30 parts of spiny jujube kernel, 10-20 parts of lily, 10-15 parts of longan pulp, and 15-30 parts of Huai wheat.

3. The use according to claim 1 or 2, characterized in that: The sleep-aiding and calming composition further comprises the following raw material components in parts by mass: 4 to 8 parts of dried tangerine peel.

4. The use according to claim 1 or 2, characterized in that: The sleep-aiding and tranquilizing composition further comprises the following raw material components in parts by mass: 8 to 12 parts of bergamot.

5. The use according to claim 1 or 2, characterized in that: The sleep-aiding and calming composition further comprises the following raw material components in parts by weight: 4 to 8 parts of dried tangerine peel and 3 to 7 parts of gardenia.

6. The use according to claim 1 or 2, characterized in that: The sleep-aiding and tranquilizing composition further comprises the following raw material components in parts by weight: 8 to 12 parts of Alpinia oxyphylla kernels and 8 to 12 parts of malt.

7. The use according to claim 1 or 2, characterized in that: The product is a medicine or a health product.

8. The use according to claim 1 or 2, characterized in that: The preparation method of the sleep-aiding and tranquilizing composition comprises the following steps: uniformly mixing the raw material components according to mass fractions, soaking in water, decocting for several times, combining the filtrate, concentrating, and freeze-drying to obtain the freeze-dried powder of the sleep-aiding and tranquilizing composition.

9. The use according to claim 8, characterized in that: The concentration is carried out by rotary evaporation.

10. The use according to claim 7, characterized in that: The product also includes excipients acceptable in pharmaceutical or health products.

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