A pharmaceutical composition for preventing and treating AD and its application

Abstract

The present invention discloses a pharmaceutical composition for preventing and treating AD, comprising 50 to 100 parts by weight of salidroside, 60 to 80 parts by weight of protocatechuic acid, 70 to 80 parts by weight of wedelol, 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. The present invention also discloses the use of a pharmaceutical composition for preventing and treating AD as a central nervous system protective agent. The present invention illustrates through behavioral, morphological and immunological experiments that intraperitoneal injection of D-gal can successfully replicate a mouse learning and memory disorder model, and that both the pharmaceutical composition and the positive drug can improve the mouse's ability to explore new things, working memory ability and spatial exploration ability, improve the structural morphology of hippocampal neurons, increase the number of hippocampal neurons and the number of Nissl bodies, thereby exerting a neuroprotective effect on D-gal model mice, providing an experimental and theoretical basis for the clinical application of the pharmaceutical composition in preventing and treating AD and the 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 show that due to the increasing 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 a certain extent hinders social development [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, research on traditional AD pathogenesis such as neuroinflammation and oxidative stress has not obtained effective therapeutic targets and strategies, and the development of AD therapeutic drugs has been hindered. Memantine is the only approved treatment for moderate to severe AD as an adjunct to cholinesterase inhibitors. While it may help treat behavioral disturbances, it only temporarily improves symptoms, carries numerous side effects, and cannot reverse established pathology. Currently, there is no drug that completely cures the disease, and the few available that improve clinical symptoms cannot fully cure it. Therefore, there is an urgent need for a pharmaceutical combination that can address the complex pathogenesis of AD and address it from multiple perspectives and targets.

[0003] Based on the theory of "tonifying the kidney and producing marrow," the laboratory conducted in-depth experimental research on the use of Erzhi Pills in the prevention and treatment of Alzheimer's disease. The results showed that Erzhi Pills can improve learning and memory impairments in D-gal model mice, and that the components absorbed into the bloodstream and brain are mostly phytoestrogens. It is preliminarily speculated that phytoestrogens may be the key component in Erzhi Pills' ability to improve learning and memory impairments in D-gal model mice. The Erzhi Pill extract contains monomeric components such as salidroside, wedelolactone, luteolin, oleanolic acid, and ligustrin. This patent aims to explore the synergistic effects of these drug combinations in order to provide new insights and approaches for the treatment of neurodegenerative diseases such as AD. Summary of the Invention

[0004] 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 wedelolactone, 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 acceptable 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 anti-AD drugs to delay the development of AD diseases.

[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-enhancing effects.

[0012] Protocatechuic acid, chemically named 3,4-dihydroxybenzoic acid, has a molecular formula of C8H8O4 and a molecular weight of 168.15 g / mol. It is a white crystal that is soluble in water, methanol, and ethanol. Protocatechuic acid can reduce the expression levels of AchE, ROS, caspase-3, Aβ, and MAP mRNA, while increasing BDNF mRNA expression, exerting a neuroprotective effect. Salidroside can significantly enhance learning and memory in AD rats, reduce damage to hippocampal neurons, regulate central nervous system inflammation, repair pathological damage to hippocampal neurons, and improve memory in AD mice.

[0013] Echinacoside, chemically named (3S,4S)-3-(4-hydroxyphenyl)-4-(2-hydroxypropyl)hexanoic acid 1-O-β-D-glucopyranoside, has a molecular formula of C24H38O13 and a molecular weight of 522.56 g / mol. It is a colorless crystal soluble in water, ethanol, and methanol. Echinacoside exhibits anti-infective, antioxidant, immune-enhancing, and antidepressant effects. Echinacoside has a protective effect on neurons, effectively inhibiting the activation of calpain I and downregulating inflammatory cytokines. It improves motor learning in rats, prevents the formation of neurofibrillary tangles, and protects neurons from damage.

[0014] Apigenin, chemically known as 4',5,7-trihydroxyflavone, has a molecular formula of C15H10O5 and a molecular weight of 270.24 g / mol. It is a yellow crystal with poor water solubility. Apigenin has antioxidant, anticancer, anti-inflammatory, and sedative properties.

[0015] Luteolin, chemically named 5,7,3',4'-tetrahydroxyflavone, has a molecular formula of C15H10O6, a molecular weight of 286.24 g / mol, and is a yellow crystal that is easily soluble in alcohol and ether. Luteolin has antioxidant, anti-inflammatory, anti-cancer, and neuroprotective properties. Luteolin protects nerve cells by improving neuroinflammation and enhancing autophagy, anti-oxidation, anti-apoptosis, and other pathways, thereby improving cognitive impairment in AD model animals. Luteolin has a certain regulatory effect on glycogen metabolism, affecting CREB phosphorylation, regulating the brain insulin / IGF-1 signaling pathway, enhancing synaptic plasticity, and reducing Aβ production. The latest studies have shown that luteolin can also serve as a new inhibitor of the β-amyloid precursor protein cleaving enzyme 1 receptor, reducing Aβ production and exerting a neuroprotective effect.

[0016] Oleanolic acid, chemically named 3β-hydroxy-olean-12-en-28-oicacid, has a molecular formula of C30H48O3 and a molecular weight of 456.71 g / mol. It occurs as colorless crystals or off-white powder with low polarity and poor solubility. Luteolin has antioxidant, anti-inflammatory, anti-cancer, and neuroprotective properties. Oleanolic acid may achieve neuroprotective effects by inhibiting the expression of proteins involved in the MAPK / ERK pathway, including Ras, Raf-1, and MEK1 / 2, in the hippocampus.

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

[0018] There are currently no 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 pharmaceutical composition can successfully replicate an AD animal model by intraperitoneal injection of D-gal through general state, behavioral and morphological experiments, and that the pharmaceutical composition can improve the mice's ability to explore new things, working memory ability and spatial exploration ability, improve the structural morphology of hippocampal neurons, increase the number of hippocampal neurons and the number of Nissl bodies, thereby exerting a neuroprotective mechanism in D-gal model mice, providing an experimental and theoretical basis for the clinical application of the pharmaceutical 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: ** indicates P < 0.01 compared with the Control group; ## indicates P < 0.01 compared with the Model group.

[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 represent the control group, model group, positive group, and EZW group, respectively. ** indicates P < 0.01 compared with the control group; ## indicates P < 0.01 compared with the model group.

[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 represent the control group, model group, positive group, and EZW group, respectively. ** indicates P < 0.01 compared with the control group; ## indicates P < 0.01 compared with the model group.

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

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

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

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

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

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

[0032] Note: A to D represent the control group, model group, positive group, and EZW group, respectively. ** indicates P < 0.01 compared with the control group; ## indicates P < 0.01 compared with the model group. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended 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 wedelolide, 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 acceptable 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] Sixty clean-grade, Kunming strain mice, 3 months old, male, weighing 25 ± 5 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. (license number: SCXK(Beijing)2019-0010). They were housed in an environment with a temperature of 21 ± 1°C and a relative humidity of 60 ± 10%, with free access to water and food. The animal experiments were approved by the Experimental Animal Ethics Committee of the Yunnan Branch of the 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] Experimental groups and treatments are shown in Table 2. Sixty three-month-old male Kunming mice were randomly divided into control, model, positive, and EZW groups. After one week of acclimation, modeling began on day 8. The blank group received a subcutaneous injection of normal saline, while the other groups received continuous intraperitoneal injections of D-gal. Drug administration was performed every two days during modeling, and behavioral studies began on day 57. Simultaneously with modeling, drug administration was performed: the control and model groups received pure water by gavage; the positive and EZW groups received the positive drug and drug combination, respectively, once daily.

[0049] In this experiment, D-gal was injected intraperitoneally to replicate the AD model and simulate the neuropathological and behavioral changes of Alzheimer's disease.

[0050] D-gal solution: Normal saline was used as the solvent and D-gal was prepared to a concentration of 25 μg.mL -1 Liquid, 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 16.25 μg.mL -1 Liquid medicine, dosage is 0.13mg.kg -1 .d -1 The positive group was gavaged with 0.2 mL of the solution.

[0052] Pharmaceutical composition solution: Take 34g of the pharmaceutical composition of salidroside: protocatechuic acid: wedelilide: apigenin: luteolin: oleanolic acid = 5:6:7:5:3:1, place it in a 100mL volumetric flask, and dissolve it in pure water to the mark to obtain a concentration of 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 experiments

[0056] 2.2.1 Morris water maze test

[0057] The Morris water maze includes three experimental parts: adaptation, 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 were placed in the water facing the pool wall and adaptively swam for 90 seconds. The mice were taken out, their fur was blown dry, and then they were placed back in the cage.

[0059] Positioning navigation experiment: The water maze was divided into four equal quadrants, and a transparent platform was placed 2 cm underwater in the third quadrant. A small amount of ink was added to the water to make the background black, so that the computer could identify the mouse. The mouse entered 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 was recorded within 90 seconds. The mouse that found the platform stayed on the platform for 30 seconds. The mouse that did not find the platform was manually placed on the platform for 30 seconds to enhance its memory. The time it took the mouse to find the platform was recorded as the escape latency of the mouse. The experimental period lasted for 4 days, and the changes in the escape latency were recorded.

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

[0061] 2.2.2 Y-maze experiment

[0062] The Y-maze consists of three arms of equal length and a central area. The arms are visually marked with different geometric shapes. Each arm has a removable partition in the center of the maze. During the experiment, the three arms were randomly assigned to the following positions: the novel arm, the starting arm, and the other arm. The novel arm was blocked by a partition during the training phase and opened during the test phase.

[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 throughout the 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 and moves freely in the starting arm and other arms for 10 minutes. After the training, the mouse is returned to the breeding cage.

[0064] One hour later, the second phase of the experiment was conducted. This phase was the testing phase. All three arms were open, and mice were placed in the starting arm and allowed to move freely among the three arms for 5 minutes. The time the mice spent in each arm and the number of times they crossed over were observed over the 5-minute period. The number of times the mice entered the novel arm and the percentage of their total time spent there were analyzed to assess their novelty exploratory behavior. The spontaneous alternation rate (spontaneous alternation rate = number of non-repeated entries into the three arms / (total number - 2) × 100%) was analyzed to assess the mice's spontaneous alternation behavior.

[0065] 2.3 Animal sampling and application

[0066] After behavioral testing, all mice were fasted for 24 hours, anesthetized, and perfused with saline until the liver turned white. Six mice were selected from each group. After perfusion of the heart with saline, they were perfused with 4% paraformaldehyde. After muscle contraction and tail lift were observed, they were decapitated and the brains were removed on an ice tray. After perfusion of saline with saline, the remaining mice in each group had their whole brains or hippocampi removed and quenched in liquid nitrogen. After removal, they were stored at -80°C for subsequent Western Blot analysis.

[0067] After perfusion with 4% paraformaldehyde, the brain tissue was fixed in 4% paraformaldehyde for 24 h, trimmed and flattened, 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, and paraffin I and paraffin II for 90 min each. The tissue was then embedded and sliced ​​to a thickness of about 4-6 μm. After flattening, the slices were removed and dried in a 37°C oven overnight for subsequent HE and Nissaurus staining.

[0068] 2.4 Pathomorphological 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: Hematoxylin staining for 10 min, rinse with running water, hydrochloric acid differentiation solution for 30 s, rinse with running water, eosin alcohol counterstaining for 1 min, rinse with running water.

[0072] (3) Dehydration and clearing: 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 clearing for 5 minutes.

[0073] (4) Observation under a microscope: Seal the slide with neutral gum. After the gum dries, observe it 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 clearing: Dehydrate with 95% ethanol I and II for 2 minutes each, and clear with xylene I and II for 5 minutes each.

[0078] (4) Observation under a microscope: Seal the slide with neutral gum. After the gum dries, observe it 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 thoroughly ground in a homogenizer. Ultrasonication on ice was performed for 10 min each, followed by pipetting once, and repeated three times. The tube was then centrifuged at 4°C, 12,000 rpm for 40 min. The supernatant was collected and the volume was recorded.

[0082] 2.5.2 BCA method for protein concentration determination and preparation of the protein to be tested

[0083] Protein concentration was determined according to the BCA kit instructions. Three rows of replicate wells were set for both the standard curve and the samples. The standard curve was constructed based on the measured OD values. The sample OD values ​​were substituted into the standard curve equation to determine the sample protein concentration. Using a 10 μL loading volume and an optimal loading concentration of 3.33 μg / μL, the required volumes of diluted lysate and loading buffer were calculated according to the protein loading buffer instructions. Finally, the diluted protein solution was denatured by boiling for 5 minutes.

[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 kit instructions, mix thoroughly, and quickly pour the lower glue layer into the glue plate to a height of about 1.5 cm from the upper edge.

[0086] Seal the surface of the gel with isopropyl alcohol and let it stand at room temperature for 30 minutes until the separation gel solidifies. Discard the upper layer of isopropyl alcohol on the gel surface, 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, 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, and load the sample. Run the gel at 80V for the stacking gel and 100V for the separating gel. Adjust the time based on the protein band position.

[0088] 2.5.4 Wet transfer

[0089] Prepare the PVDF membrane and thick transfer filter paper in advance. Soak the PVDF membrane in methanol for 5 minutes (to activate), deionized water for 2 minutes (to remove the methanol), and then soak in transfer buffer for 30 minutes (to equilibrate). After electrophoresis, cut the gel and soak it in transfer buffer for 10 minutes. Soak the thick transfer filter paper in transfer buffer for 10 minutes. Simultaneously, inject transfer buffer into the transfer electrophoresis apparatus to thoroughly moisten the transfer chuck, 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 the negative electrode to the 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 blocking solution and blocked for 2 h at room temperature on a horizontal shaker.

[0093] 2.5.6 Antibody incubation

[0094] Remove the blocked PVDF membrane and add the appropriate amount of target antibody (concentration according to the instructions), incubating overnight at 4°C. The next day, rinse the PVDF membrane three times with TBST, 10 minutes apart. Then, add the secondary antibody and incubate on a shaker at room temperature for 2 hours. Rinse the PVDF membrane three times with TBST, 10 minutes apart. Finally, perform ECL imaging. Antibody dilution ratios are shown in Table 3.

[0095] Table 3 Antibody dilution ratio

[0096]

[0097] 2.6 Results Analysis

[0098] Relative mRNA expression was calculated based on the Ct values ​​obtained using QuantStudio 5 software. 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 expressed as mean ± standard deviation. Data with normal distribution and homogeneity of variance were analyzed using one-way ANOVA. Pairwise comparisons of means between groups were performed using the LSD test, with SNK correction for statistical significance. Data with heterogeneity of variance were compared between groups using the Tamhane's ST2 test. P < 0.05 indicated a significant difference.

[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 in the figure, 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, ## Indicates 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] Table 5 Morris water maze space exploration ability of each group of mice (x±S, n=15)

[0110]

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

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

[0115]

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

[0117] The results of the spontaneous alternation experiment are shown in Table 7. Figure 4 The total number of arm entries was not statistically different among the groups (P>0.05). Compared with the Control group, the spontaneous alternation rate of mice in the Model group was significantly decreased (P<0.01); compared with the Model group, the spontaneous alternation rate of mice in the Positive and EZW groups 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: ** indicates P < 0.01 compared with the Control group; ## indicates P < 0.01 compared with the Model group.

[0121] 3.2 Pathomorphological 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 hippocampal CA1 region of the Control group mice had clear outlines and compact structures, and the intracellular Nissl bodies were abundant, patchy, and larger in size; compared with the Control group, the neurons in the hippocampal CA1 region of the Model group mice had fuzzy outlines and loose structures, and the intracellular Nissl bodies were irregular in shape and significantly fewer in number (P<0.01); compared with the Model group, the neurons in the hippocampal CA1 region of the Positive group and the EZW group mice had clearer outlines and more compact structures, and the intracellular Nissl bodies were more regular in shape and significantly increased in number (P<0.01).

[0126] Table 8 Changes in the number of Nissl bodies in the hippocampal CA1 region 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: ** indicates P < 0.01 compared with the Control group; ## indicates P < 0.01 compared with the Model group;

[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 EZW group was significantly decreased (P<0.01).

[0133] Table 9 p-Tau protein expression 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: ** indicates P < 0.01 compared with the Control group; ## indicates P < 0.01 compared with the Model group.

[0136] Example 2

[0137] The only difference between this embodiment and Example 1 is that: a pharmaceutical composition for preventing and treating AD, comprising 60 parts by weight of salidroside, 70 parts by weight of protocatechuic acid, 70 parts by weight of wedelolactone, 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 Example 1 is that: a pharmaceutical composition for preventing and treating AD, comprising 70 parts by weight of salidroside, 60 parts by weight of protocatechuic acid, 60 parts by weight of wedelolactone, 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 Example 1 is that: a pharmaceutical composition for preventing and treating AD, comprising 50 parts by weight of salidroside, 80 parts by weight of protocatechuic acid, 70 parts by weight of wedelolactone, 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 Example 1 is that: a pharmaceutical composition for preventing and treating AD, comprising 100 parts by weight of salidroside, 60 parts by weight of protocatechuic acid, 70 parts by weight of wedelolactone, 50 parts by weight of apigenin, 30 parts by weight of luteolin and 10 parts by weight of oleanolic acid.

[0144] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

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

2. The 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 wedelolactone, 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 is prepared into one or more of oral liquid, powder, tablet, capsule and pill.

4. The pharmaceutical composition for preventing and treating AD according to claim 3, characterized in that: The pharmaceutical composition includes auxiliary agents within the pharmaceutically acceptable 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 in preparing a central nervous system protective agent.

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