Pharmaceutical composition for preventing and treating AD (Alzheimer's disease) and application thereof

Abstract

The invention discloses a pharmaceutical composition for preventing and treating AD (Alzheimer's disease). The pharmaceutical composition is prepared from the following components in parts by weight: 50 to 100 parts of salidroside, 60 to 80 parts of protocatechuic acid, 70 to 80 parts of wedelolactone, 50 to 80 parts of apigenin, 30 to 60 parts of luteolin and 10 to 30 parts of oleanolic acid. The invention further discloses application of the pharmaceutical composition for preventing and treating AD as a central nervous protective agent. The behavioral, morphological and immunological experiments show that a mouse learning and memory disorder model can be successfully copied by intraperitoneal injection of D-gal, and the pharmaceutical composition and the positive drug can improve the structural morphology of neurons in a hippocampus region by improving the exploration ability, the working memory ability and the space exploration ability of a mouse on new things, so that the learning and memory disorder of the mouse can be successfully copied. The number of neurons in the hippocampus and the number of nissl's somes are increased, so that the neuroprotective effect on D-gal model mice is achieved, and experimental and theoretical basis is provided for clinical application of the pharmaceutical composition for preventing and treating AD and research and development of new anti-AD traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing and treating AD and application thereof, belonging to the technical field of pharmaceuticals. Background Art

[0002] Alzheimer's disease (AD) is an age-related neurodegenerative disease. Patients will show symptoms such as progressive memory impairment and cognitive dysfunction, which have a huge impact on social life and seriously affect the physical health and quality of life of the elderly. Epidemiological studies have shown that due to the aging of the population, the number of AD patients worldwide may increase to 139 million by the middle of this century [2]. AD has a long course of disease and high treatment and nursing costs, which imposes a heavy mental and economic burden on patients and their families, and to some extent hinders the development of society [3]. AD has a slow or hidden onset, a slow course of disease, and a complex pathogenesis. The current mainstream theories include the Aβ cascade hypothesis, the cholinergic damage hypothesis, the Tau protein hyperphosphorylation hypothesis, the glutamate receptor hypothesis, the inflammatory response and free radical damage hypothesis, etc. At present, studies on traditional AD pathogenesis such as neuroinflammation and oxidative stress have not obtained effective therapeutic targets and strategies, and the development of AD therapeutic drugs has been hindered. Memantine is approved as the only treatment for moderate to severe AD. It is an adjunct to cholinesterase inhibitors and may help treat behavioral disorders, but it only temporarily improves symptoms and has many side effects. It cannot reverse the pathological state that has already formed. So far, there is no drug that can completely cure the disease, and the few drugs that improve the clinical symptoms of AD cannot completely treat AD. It can be seen that there is an urgent need for a drug composition that can treat AD from a multi-angle and multi-target treatment concept based on the complex pathogenesis of the disease.

[0003] Based on the theory of "tonifying the kidney and producing marrow", the laboratory conducted in-depth experimental research on the prevention and treatment of Alzheimer's disease with Erzhi Pills. It was found that Erzhi Pills can improve the learning and memory disorders of D-gal model mice, and most of the components absorbed into the blood and brain are phytoestrogens. It is preliminarily speculated that phytoestrogens may be the key components of Erzhi Pills to improve the learning and memory disorders of D-gal model mice. The extract of Erzhi Pills contains monomer components such as salidroside, wedelolactone, luteolin, oleanolic acid, and privet glycoside. This patent aims to explore the joint effects of drug combinations in order to provide new ideas and methods for the treatment of neurodegenerative diseases such as AD. Summary of the invention

[0004] In order to solve the technical problems mentioned in the background technology, the present invention provides a pharmaceutical composition for preventing and treating AD and its application.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A pharmaceutical composition for preventing and treating AD comprises 50-100 parts by weight of salidroside, 60-80 parts by weight of protocatechuic acid, 70-80 parts by weight of wedelilide, 50-80 parts by weight of apigenin, 30-60 parts by weight of luteolin and 10-30 parts by weight of oleanolic acid.

[0007] The pharmaceutical composition can be prepared into one or more of oral liquid, powder, tablet, capsule and pill.

[0008] The pharmaceutical composition includes auxiliary agents within the pharmaceutically permitted range, and the auxiliary agents are one or more of wetting agents, antibacterial agents, emulsifiers, antioxidants, solubilizers, thickeners, suspending agents, excipients, sweeteners or stabilizers.

[0009] A pharmaceutical composition for preventing and treating AD is used as a central nervous system protective agent.

[0010] The pharmaceutical composition is used in combination with an anti-AD drug to delay the development of AD disease.

[0011] Salidroside (Salidroside or Rhodioloside), chemical name: 4-(β-D-glucopyranosyloxy)phenyl(2E)-3-(4-hydroxyphenyl)prop-2-enoate, molecular formula is C16H18O8, molecular weight is 342.31g / mol, white or light yellow crystalline powder, good water solubility. Salidroside has antioxidant, anti-fatigue, anti-depression and immune enhancement effects.

[0012] Protocatechuic acid, chemical name: 3,4-dihydroxybenzoic acid, molecular formula: C8H8O4, molecular weight: 168.15g / mol, white crystals, easily soluble in water, methanol, ethanol. Protocatechuic acid can reduce the expression levels of AchE, ROS, caspase-3, Aβ and MAP mRNA, increase the expression of BDNF mRNA, and play a neuroprotective role. Salidroside can significantly enhance the learning and memory ability of AD rats, reduce the damage of hippocampal neurons, regulate the central inflammatory state, repair the pathological damage of hippocampal neurons, and improve the memory of AD mice.

[0013] Echinacoside, chemical name: (3S,4S)-3-(4-hydroxyphenyl)-4-(2-hydroxypropyl)hexanoic acid1-O-β-D-glucopyranoside, molecular formula: C24H38O13, molecular weight: 522.56g / mol, colorless crystals, soluble in: water, ethanol, methanol. Echinacoside has anti-infection, antioxidant, immune enhancement, anti-depression and other effects. Echinacoside has a protective effect on neurons. Echinacoside can effectively inhibit the activation of calpain I, downregulate inflammatory cytokines, improve the motor learning ability of rats, prevent the formation of neurofibrillary tangles, and protect neurons from damage.

[0014] Apigenin, chemical name: 4',5,7-trihydroxyflavone, molecular formula: C15H10O5, molecular weight: 270.24g / mol, yellow crystal, poor water solubility. Apigenin has antioxidant, anti-cancer, anti-inflammatory, sedative and other effects.

[0015] Luteolin, chemical name: 5,7,3',4'-tetrahydroxyflavone, molecular formula is C15H10O6, molecular weight is 286.24g / mol, yellow crystals, easily soluble in alcohol and ether. Luteolin has antioxidant, anti-inflammatory, anti-cancer, neuroprotective and other effects. Luteolin protects nerve cells by improving neuroinflammation and enhancing autophagy, anti-oxidation, anti-apoptosis and other pathways, and plays a role in improving cognitive impairment in AD model animals. Luteolin has a certain regulatory effect on glycogen metabolism, can affect CREB phosphorylation, regulate the brain insulin / IGF-1 signaling pathway, enhance synaptic plasticity, and reduce the production of Aβ. The latest research shows that luteolin can also be used as a new inhibitor of the β-amyloid precursor protein cleavage enzyme 1 receptor, reduce the production of Aβ, and play a neuroprotective role.

[0016] Oleanolic acid, chemical name: 3β-hydroxy-olean-12-en-28-oicacid, molecular formula C30H48O3, molecular weight 456.71g / mol, colorless crystal or off-white powder, low polarity and poor solubility. Luteolin has antioxidant, anti-inflammatory, anti-cancer, and neuroprotective effects. Oleanolic acid can achieve neuroprotective effects by inhibiting the expression of Ras, Raf-1, MEK1 / 2 and other related proteins in the MAPK / ERK pathway in the hippocampus tissue.

[0017] Salidroside, protocatechuic acid, wedelilide, apigenin, luteolin and oleanolic acid are all commercially available.

[0018] There are no relevant reports on the application of salidroside, protocatechuic acid, wedelilide, apigenin, luteolin and oleanolic acid in the treatment of AD.

[0019] The present invention illustrates that the drug composition can successfully replicate the AD animal model by intraperitoneal injection of D-gal through general state, behavioral and morphological experiments, and the drug composition can improve the exploration ability of mice to new things, working memory ability and spatial exploration ability, improve the structural morphology of neurons in the hippocampus, increase the number of neurons and Nissl bodies in the hippocampus, thereby exerting a neuroprotective mechanism on D-gal model mice, providing an experimental and theoretical basis for the clinical application of the drug composition in preventing and treating AD and the research and development of new anti-AD traditional Chinese medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Morris water maze escape latency of each group of mice provided in the examples of this application

[0021] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01.

[0022] Figure 2 Comparison of spatial exploration ability of Morris water maze in each group of mice provided in the examples of this application

[0023] Note: A to D are Control group, Model group, Positive group, and EZW group. Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01.

[0024] Figure 3 The activity of the novel arm of each group of mice provided in the examples of this application

[0025] Note: A to D are Control group, Model group, Positive group, and EZW group. Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01.

[0026] Figure 4 The spontaneous alternation of each group of mice provided in the examples of this application

[0027] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01.

[0028] Figure 5Pathological morphology of neurons in the CA1 region of the hippocampus of each group of mice provided in the examples of the present application Note: A to D are: Control group, Model group, Positive group, and EZW group, respectively.

[0029] Figure 6 Nissl staining results of the CA1 region of the hippocampus of each group of mice provided in the examples of this application

[0030] Note: A~D are: Control group, Model group, Positive group, EZW group.

[0031] Figure 7 The p-Tau protein expression levels in the hippocampus of each group of mice provided in the examples of this application

[0032] Note: A to D are Control group, Model group, Positive group, and EZW group. Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0034] Example 1

[0035] A pharmaceutical composition for preventing and treating AD, comprising 50 parts by weight of salidroside, 60 parts by weight of protocatechuic acid, 70 parts by weight of wedelilide, 50 parts by weight of apigenin, 30 parts by weight of luteolin and 10 parts by weight of oleanolic acid

[0036] The pharmaceutical composition can be prepared into one or more of oral liquid, powder, tablet, capsule and pill.

[0037] The pharmaceutical composition includes auxiliary agents within the pharmaceutically permitted range, and the auxiliary agents are one or more of wetting agents, antibacterial agents, emulsifiers, antioxidants, solubilizers, thickeners, suspending agents, excipients, sweeteners or stabilizers.

[0038] A pharmaceutical composition for preventing and treating AD is used as a central nervous system protective agent.

[0039] 1 Materials

[0040] 1.1 Experimental animals

[0041] Clean grade, Kunming mice, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., 60 mice, 3 months old, male, weighing 25±5g, license number: SCXK (Beijing) 2019-0010. The breeding environment temperature was 21±1℃, the relative humidity was 60±10%, and water and food were available freely. The animal experiment was approved by the Experimental Animal Ethics Committee of Yunnan Branch of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, approval number: 202206007.

[0042] 1.2 Reagent configuration

[0043] The reagents required for the experiment are prepared as shown in Table 1

[0044] Tab.1Main Reagent Configuration

[0045]

[0046] 2 Methods

[0047] 2.1 Experimental groups and drug administration

[0048] The experimental groups and treatment methods are shown in Table 2. 60 3-month-old male Kunming mice were randomly divided into the Control group, Model group, Positive group and EZW group. After one week of adaptation, modeling began on the 8th day. The blank group was subcutaneously injected with normal saline, and the other groups were continuously intraperitoneally injected with D-gal. The modeling was administered once every two days, and behavioral studies began on the 57th day. Drugs were administered at the same time as modeling. The Control group and the Model group were gavaged with pure water; the Positive group and the EZW group were given positive drugs and drug combinations, respectively, once a day.

[0049] In this experiment, the AD model was established by intraperitoneal injection of D-gal to simulate the neuropathological physiological changes and behavioral changes of Alzheimer's disease.

[0050] D-gal solution: physiological saline was used as the solvent, and D-gal was prepared to a concentration of 25 μg.mL -1 Liquid medicine, modeling dose is 125μg.kg -1 d -1 The control group was intraperitoneally injected with 0.2 mL of normal saline, and the other groups were intraperitoneally injected with 0.2 mL of D-gal solution.

[0051] Positive drug solution: Pure water is used as the solvent, and the positive drug is prepared to a concentration of 16.25μg.mL -1 Liquid medicine, dosage is 0.13mg.kg -1 .d -1 The Positive group was intragastrically administered with 0.2 mL of the solution.

[0052] Drug composition solution: Take 34g of the drug composition of salidroside: protocatechuic acid: wedelilide: apigenin: luteolin: oleanolic acid = 5:6:7:5:3:1, put it in a 100mL volumetric bottle, dissolve it in pure water to the mark, and the concentration is 3.4mg.mL -1 The EZW group was gavaged with 0.2 mL of the test solution.

[0053] Table 2 Mold making and Drug Administration Schedule

[0054]

[0055] 2.2 Behavioral Experiment

[0056] 2.2.1 Morris water maze test

[0057] The Morris water maze consists of three experimental parts: adaptability, positioning navigation, and spatial exploration.

[0058] Adaptation experiment: On the first day of the experiment, a certain amount of water was injected into the water maze, and the water temperature was controlled at 25±2℃. The mice entered the water facing the pool wall and swam adaptively for 90 seconds. The mice were taken out, their fur was dried, and then they were put back into the cage.

[0059] Positioning navigation experiment: The water maze is divided into 4 quadrants, and a transparent platform is placed 2 cm underwater in the third quadrant. A little ink is added to the water to make the background black, so that the computer can identify the mouse. The mouse enters the water at the entry point in the first quadrant, facing the pool wall. The time required for the mouse to find the target platform within 90 seconds is recorded. The mouse that finds the platform stays on the platform for 30 seconds. The mouse that does not find the platform is placed on the platform for 30 seconds to enhance its memory. The time it takes for the mouse to find the platform is recorded as the escape latency of the mouse. The experimental period is 4 days, and the changes in the escape latency are recorded.

[0060] Space exploration experiment: 24 hours after the positioning navigation experiment, the platform was removed and the mice were put into the water again at the entry point in the first quadrant. The time they stayed in the target quadrant (third quadrant) and the platform position and the number of times they crossed the effective area (2 times the platform diameter) within 90 seconds were recorded.

[0061] 2.2.2 Y-maze experiment

[0062] The Y-maze consists of three arms of equal length and a central area. Different geometric shapes are attached to the three arms as visual markers. In the center of the maze, each arm has a movable partition. During the experiment, the three arms were randomly set as: novel arm, starting arm, and other arms. The novel arm: blocked by a partition during the training period and opened during the test period;

[0063] Starting arm: the arm where the mouse is when it enters the maze. Other arms: another arm except the novel arm and the starting arm. Among them, the starting arm and other arms are open during the entire experiment, and the animals can enter and exit freely. After the experiment on each mouse, the inner wall of the maze is wiped with 75% alcohol and distilled water respectively to minimize the interference of residual animal odor on the experimental results. The experiment consists of two stages. The first stage is the training stage. The novel arm is closed, and the mouse is placed in the starting arm. It moves freely in the starting arm and other arms for 10 minutes. After the training, the mouse is returned to the breeding cage.

[0064] After 1 hour, the second stage of the experiment was carried out. The second stage was the detection stage. All three arms were open. The mice were placed in the starting arm and moved freely in the three arms for 5 minutes. The time the mice stayed in each arm and the number of shuttles were observed within 5 minutes. The number of times the mice entered the novel arm and the percentage of the total time were analyzed and calculated to evaluate the novelty exploration behavior of the mice. The spontaneous alternation rate was analyzed and calculated. The spontaneous alternation rate = the number of non-repeated entries into the three arms / (total number-2) × 100% was used to evaluate the spontaneous alternation behavior of the mice.

[0065] 2.3 Animal materials and applications

[0066] After the behavioral study, all experimental mice were fasted for 24 hours, anesthetized, and perfused with saline until the liver turned white. Six mice were selected from each group. After the heart was perfused with saline, it was perfused with 4% paraformaldehyde. After muscle contraction and tail lifting were observed, the head was cut off and the brain was peeled off on an ice tray. After the remaining mice in each group were perfused with saline, the whole brain or hippocampus was taken out and quenched in liquid nitrogen, and then stored in a -80℃ refrigerator for subsequent Western Blot experiments.

[0067] The brain tissue after perfusion with 4% paraformaldehyde was fixed in 4% paraformaldehyde for 24 h, trimmed and leveled, and then dehydrated, transparentized, and waxed in 70% ethanol, 80% ethanol, and 90% ethanol for 1 h each, 95% ethanol I and II for 50 min each, 100% ethanol I and II for 30 min each, xylene-anhydrous ethanol (1:1) for 30 min, xylene I and II for 20 min each, paraffin I and paraffin II for 90 min each, then embedded, sliced, with a thickness of about 4-6 μm, flattened, fished out, and dried in a 37°C oven overnight for subsequent HE and Nissl staining.

[0068] 2.4 Pathological morphological observation

[0069] 2.4.1 HE staining to observe hippocampal tissue morphology

[0070] (1) Dewaxing and rehydration: xylene I and II for 5 min each, 100% ethanol for 5 min, 95% ethanol and distilled water for 2 min.

[0071] (2) HE staining: stain with hematoxylin for 10 min, rinse with running water, use hydrochloric acid differentiation solution for 30 s, rinse with running water, counterstain with eosin alcohol for 1 min, rinse with running water.

[0072] (3) Dehydration and transparency: Use 95% ethanol I, 95% ethanol II, 100% ethanol I for 15 seconds each, and 100% ethanol II for 1 minute each. After dehydration, place in xylene for transparency for 5 minutes.

[0073] (4) Observation under a microscope: Seal the slide with neutral gum. After the gum dries, observe under a microscope.

[0074] 2.4.2 Nissl staining of hippocampal neuron morphology

[0075] (1) Dewaxing and rehydration: xylene I, II, and III for 10 min each, 100% ethanol for 5 min, 90% ethanol for 2 min, 70% ethanol for 2 min, and rinse with distilled water for 2 min.

[0076] (2) Nissl staining: Add Nissl staining solution and soak for 10 minutes, then rinse with running water.

[0077] (3) Dehydration and transparency: Dehydrate with 95% ethanol I and II for 2 min each, and transparentize with xylene I and II for 5 min each.

[0078] (4) Observation under a microscope: Seal the slide with neutral gum. After the gum dries, observe under a microscope.

[0079] 2.5 Western blot detection

[0080] 2.5.1 Total protein extraction from tissues

[0081] Protein extraction was performed on ice. An appropriate amount of hippocampus was placed in a centrifuge tube, and RIPA lysis buffer (approximately 10 times the volume of the hippocampus) was added. The tube was fully ground in a homogenizer, and ultrasonicated on ice for 10 min each time. The tube was blown once and repeated for 3 rounds. The tube was then centrifuged at 4°C, 12,000 rpm for 40 min. The supernatant was taken and the volume was recorded.

[0082] 2.5.2 BCA method for protein concentration measurement and preparation of protein to be measured

[0083] The protein concentration was determined according to the instructions of the BCA kit. Three rows of duplicate wells were set for the standard curve and samples. The standard curve was drawn according to the measured OD value. The OD value of the sample was substituted into the standard curve equation to obtain the sample protein concentration. The sample volume was 10 μL and the optimal sample concentration was 3.33 μg / μL. According to the instructions of the protein loading buffer, the required volume of the diluted lysate and loading buffer was calculated. Finally, the diluted protein solution was boiled for 5 minutes for denaturation.

[0084] 2.5.3SDS-PAGE electrophoresis

[0085] Install the glue plate as required, check for leakage with deionized water, prepare the upper and lower glue layers according to the instructions of the kit, mix them thoroughly, and quickly pour the lower glue layer into the glue plate. The pouring height should be about 1.5 cm from the upper edge.

[0086] Seal the surface of the gel with isopropanol and let it stand at room temperature for 30 minutes until the separation gel solidifies. Discard the isopropanol on the upper layer of the gel, absorb the excess liquid with filter paper, pour in the prepared upper layer of gel, slowly insert the comb along one side of the gel plate, and let it stand at room temperature for 30 minutes.

[0087] The polyacrylamide gel is now ready. Place the prepared gel in the electrophoresis tank, add electrophoresis buffer, remove the comb, load the sample, and use the electrophoresis conditions of 80V for the concentrated gel and 100V for the separation gel. The time is adjusted according to the position of the protein bands.

[0088] 2.5.4 Wet transfer

[0089] Prepare the PVDF membrane and transfer thick filter paper in advance. Soak the PVDF membrane in methanol for 5 minutes (activation), deionized water for 2 minutes (methanol removal), and transfer buffer for 30 minutes (equilibrium). After the electrophoresis is completed, cut the gel and soak the gel in transfer buffer for 10 minutes. Soak the transfer thick filter paper in transfer buffer for 10 minutes. At the same time, transfer buffer is also injected into the transfer electrophoresis instrument to fully wet the transfer clip, transfer core, transfer tank and sponge.

[0090] After completion, put the transfer material in the order of sponge---filter paper---separation gel---PVDF membrane---filter paper---sponge from negative electrode to positive electrode, drive out bubbles and wrinkles, put the transfer clip into the transfer core, put the special ice box of the transfer instrument on the side, connect the power supply of the wet transfer instrument, adjust the current to 300mA, and the time to 2h.

[0091] 2.5.5 Closure

[0092] After the transfer, the PVDF membrane was placed in the blocking solution and blocked for 2 h on a horizontal shaker at room temperature.

[0093] 2.5.6 Antibody incubation

[0094] Take out the blocked PVDF membrane, add an appropriate amount of target indicator antibody (concentration is prepared according to the instructions), and incubate at 4°C overnight. The next day, wash the PVDF membrane 3 times with TBST, with an interval of 10 minutes per time. Then, add the secondary antibody, incubate on a shaker at room temperature for 2 hours, and wash the PVDF membrane 3 times with TBST, with an interval of 10 minutes per time. Finally, perform ECL imaging. The dilution ratio of each antibody is shown in Table 3.

[0095] Table 3 Antibody dilution ratio

[0096]

[0097] 2.6 Results Analysis

[0098] According to the Ct value obtained by QuantStudio5 software, the relative expression of mRNA was calculated. The Ct difference comparison formula is: ΔΔCt = intervention group (Ct target gene - Ct internal reference gene) - blank group (Ct target gene - Ct internal reference gene). Relative expression = 2-ΔΔCt.

[0099] 2.7 Data Processing

[0100] The data were processed using Graphpad5.0 and presented as mean ± standard deviation. Indicates. One-way-ANOVA was used for data that were normally distributed and had homogeneous variance. LSD test was used for pairwise comparison of means between groups, and SNK was used for statistical correction. Tamhane'ST2 method was used for inter-group comparison of data with unequal variance, and P<0.05 indicated significant differences.

[0101] 3 Experimental results

[0102] 3.1 Behavioral Experimental Results

[0103] 3.1.1 Morris water maze test results

[0104] The results of the positioning navigation experiment are shown in Table 4. Figure 1 As shown, compared with the Control group, the escape latency of mice in the Model group was significantly increased (P<0.01); compared with the Model group, the escape latency of mice in the Positive group and EZW group was significantly decreased (P<0.01).

[0105] Table 4 Morris water maze escape latency of mice in each group ( n=15)Tab.4Morris water mazeescape latency of mice in each group( n=15)

[0106]

[0107] Note: Compared with the Control group, ** Indicates P < 0.01; compared with the Model group, ## P < 0.01

[0108] The results of the space exploration experiment are shown in Table 5. Figure 2 Compared with the Control group, the number of times the mice in the Model group crossed the platform and the time they stayed in the target quadrant were significantly reduced (P<0.01); compared with the Model group, the number of times the mice in the Positive group and the EZW group crossed the platform and the time they stayed in the target quadrant were significantly increased (P<0.01).

[0109] Tab.5 Morris water maze space exploration ability of each group of mice (x±S, n=15)

[0110]

[0111] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01.

[0112] 3.1.2Y-maze results

[0113] The results of the novel arm exploration experiment are shown in Table 6. Figure 3 Compared with the Control group, the number of times the mice in the Model group entered the novel arm and the percentage of the time they stayed in the novel arm as a percentage of the total time were significantly reduced (P<0.01); compared with the Model group, the number of times the mice in the Positive group and the EZW group entered the novel arm and the percentage of the time they stayed in the novel arm as a percentage of the total time were significantly increased (P<0.01).

[0114] Tab.6 New arm exploration of mice in each group (x±S, n=15)

[0115]

[0116] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01.

[0117] The results of the spontaneous alternation experiment are shown in Table 7. Figure 4 As shown. There was no statistical difference in the total number of arm entries among the mice in each group (P>0.05). Compared with the Control group, the spontaneous alternation rate of mice in the Model group was significantly reduced (P<0.01); compared with the Model group, the spontaneous alternation rate of mice in the Positive group and the EZW group was significantly increased (P<0.01).

[0118] Table 7 Spontaneous alternation of mice in each group ( n=15)Tab.7Spontaneous alternation ofmice in each group n=15)

[0119]

[0120] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01.

[0121] 3.2 Pathological morphology observation

[0122] 3.2.1 HE staining results

[0123] HE staining results are shown in Figure 5 , the cells in the CA1 region of the hippocampus of the mice in the Control group were arranged regularly and tightly, with uniform staining, and the number of pyramidal cells was large; compared with the Control group, the cells in the CA1 region of the hippocampus of the mice in the Model group were arranged sparsely and chaotically, the number of pyramidal cells was significantly reduced, and the cell lines were blurred; compared with the Model group, the cells in the CA1 region of the hippocampus of the mice in the Positive group and the EZW group were arranged neatly and tightly, the number of pyramidal cells was increased, and the cell lines were clearer.

[0124] 3.2.2 Nissau staining results

[0125] Nissl staining results are shown in Figure 6 , Table 8, The neurons in the CA1 region of the hippocampus of the Control group mice had clear outlines, compact structures, abundant intracellular Nissl bodies, which were patchy and large in size; compared with the Control group, the neurons in the CA1 region of the hippocampus of the Model group mice had fuzzy outlines, loose structures, irregular shapes of intracellular Nissl bodies, and significantly fewer in number (P<0.01); compared with the Model group, the neurons in the CA1 region of the hippocampus of the Positive group and the EZW group mice had clearer outlines, tighter structures, regular shapes of intracellular Nissl bodies, and significantly increased in number (P<0.01).

[0126] Table 8 Changes in the number of Nisslerian bodies in the CA1 region of the hippocampus of mice in each group (x±S, n=3)

[0127] Tab.8Changes in the number of Nissl bodies in the hippocampal CA1region of mice in each

[0128] group(±S,n=3)

[0129]

[0130] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01;

[0131] 3.3 Western blot detection of p-Tau protein expression in mouse hippocampus

[0132] The experimental results are as follows Figure 7 As shown in Table 9, compared with the Control group, the expression of p-Tau protein in the hippocampus of the mice in the Model group was significantly increased (P<0.01); compared with the Model group, the expression of p-Tau protein in the hippocampus of the mice in the Positive group and the EZW group was significantly decreased (P<0.01).

[0133] Table 9 Expression of p-Tau protein in the hippocampus of mice in each group ( n=3) Tab.9Expression content ofp-Tau protein in hippocampus of mice in each group( n=3)

[0134]

[0135] Note: Compared with the Control group, ** indicates P < 0.01; compared with the Model group, ## indicates P < 0.01.

[0136] Example 2

[0137] The only difference between this embodiment and embodiment 1 is that: a pharmaceutical composition for preventing and treating AD comprises 60 parts by weight of salidroside, 70 parts by weight of protocatechuic acid, 70 parts by weight of wedeliolide, 50 parts by weight of apigenin, 40 parts by weight of luteolin and 10 parts by weight of oleanolic acid.

[0138] Example 3

[0139] The only difference between this embodiment and embodiment 1 is that: a pharmaceutical composition for preventing and treating AD comprises 70 parts by weight of salidroside, 60 parts by weight of protocatechuic acid, 60 parts by weight of wedelilide, 50 parts by weight of apigenin, 60 parts by weight of luteolin and 30 parts by weight of oleanolic acid.

[0140] Example 4

[0141] The only difference between this embodiment and embodiment 1 is that: a pharmaceutical composition for preventing and treating AD comprises 50 parts by weight of salidroside, 80 parts by weight of protocatechuic acid, 70 parts by weight of wedelilide, 50 parts by weight of apigenin, 30 parts by weight of luteolin and 10 parts by weight of oleanolic acid.

[0142] Example 5

[0143] The only difference between this embodiment and embodiment 1 is that: a pharmaceutical composition for preventing and treating AD comprises 100 parts by weight of salidroside, 60 parts by weight of protocatechuic acid, 70 parts by weight of wedelilacin, 50 parts by weight of apigenin, 30 parts by weight of luteolin and 10 parts by weight of oleanolic acid.

[0144] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing and treating AD, characterized in that: The invention comprises 50 to 100 parts by weight of salidroside, 60 to 80 parts by weight of protocatechuic acid, 70 to 80 parts by weight of wedelolide, 50 to 80 parts by weight of apigenin, 30 to 60 parts by weight of luteolin and 10 to 30 parts by weight of oleanolic acid.

2. A pharmaceutical composition for preventing and treating AD according to claim 1, characterized in that: The invention comprises 80-90 parts by weight of salidroside, 70-80 parts by weight of protocatechuic acid, 75-80 parts by weight of wedelolide, 70-80 parts by weight of apigenin, 50-60 parts by weight of luteolin and 50-30 parts by weight of oleanolic acid.

3. A pharmaceutical composition for preventing and treating AD according to claim 1 or 2, characterized in that: The pharmaceutical composition can be prepared into one or more of oral liquid, powder, tablet, capsule and pill.

4. A pharmaceutical composition for preventing and treating AD according to claim 3, characterized in that: The pharmaceutical composition includes auxiliary agents within the pharmaceutically permitted range, and the auxiliary agents are one or more of wetting agents, antibacterial agents, emulsifiers, antioxidants, solubilizers, thickeners, suspending agents, excipients, sweeteners or stabilizers.

5. Use of the pharmaceutical composition for preventing and treating AD according to claim 1 or 2 as a central nervous system protective agent.

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