A pharmaceutical compound composition for preventing or treating Alzheimer's disease and use thereof

Abstract

The present application relates to a kind of drug compound composition for preventing or treating alzheimer disease and its application, the drug compound composition includes the following weight ratio of components: Ganoderma lucidum polysaccharide 1-10 parts, wolfberry polysaccharide 1-10 parts, turmeric extract 1-10 parts.It is found that the drug compound composition can significantly improve the learning ability of APP / PS1 mouse, reduce the level of A beta in the brain of mouse, improve the intestinal microorganism environment of mouse.Therefore the drug group can be applied in prevention or treatment of alzheimer disease.

Description

[0001] This application claims the benefit of Chinese Patent Application No. 2023100594545, filed January 18, 2023, in the China National Intellectual Property Office, the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0002] The present application relates to the technical field of traditional Chinese medicine, in particular to a compound composition for preventing or treating Alzheimer's disease and application thereof. BACKGROUND

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease with insidious onset, characterized by memory impairment, aphasia, apraxia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavior changes, and other comprehensive dementia. With the aging of the population, dementia has become a common disease among the elderly and is the main cause of disability and death in the elderly.

[0004] Ganoderma lucidum is a fungus with both medicinal and edible properties, which has the effects of strengthening the body and delaying aging. Modern pharmacological studies have shown that it also has the pharmacological activities of lowering blood sugar, anti-tumor, anti-inflammatory, and lowering blood pressure. Its components mainly include triterpenes, polysaccharides, sterols, fatty acids, etc., and the first two are generally considered to be the main active substances of Ganoderma lucidum. Shichao Huang et al. (Huang S, Mao J, Ding K, et al. Polysaccharides from Ganoderma lucidum Promote Cognitive Function and Neural Progenitor Proliferation in Mouse Model of Alzheimer's Disease. Stem Cell Reports. 2017 Jan 10; 8(1): 84-94.) found that Ganoderma lucidum polysaccharides can activate FGFR1 (fibroblast growth factor receptor 1) and downstream ERK (excellular signal-regulated kinase) and AKT pathways; by promoting neural progenitor cell proliferation, thereby enhancing neurogenesis in transgenic AD mice and reducing cognitive impairment.

[0005] Lycium barbarum L. is a fruit of Solanaceae Lycium barbarum L. It is a medicine and food homologous medicinal material, and has the effects of nourishing liver and kidney, and benefiting essence and eyesight. Modern pharmacological studies have shown that it has immunomodulatory, antioxidant, antitumor, and neuroprotective activities. The main components of Lycium barbarum L. include polysaccharides, flavonoids, polyphenols betaine, etc. Lycium barbarum L. polysaccharides are a group of water-soluble glycoconjugates with a molecular weight of 10-230 kDa, accounting for 5%-8% of the dry weight of Lycium barbarum L. and are the main functional active substances in Lycium barbarum L. It has been proved that it can play a significant role in immunomodulation, antioxidant, and anti-aging. Yue Zhou et al. (Zhou Y, Duan Y, Huang S, et al. Polysaccharides from Lycium barbarum ameliorate amyloid pathology and cognitive functions in APP / PS1 transgenic mice. Int J Biol Macromol. 2020 Feb 1; 144: 1004-1012.) found that Lycium barbarum L. polysaccharides can reduce the level of amyloid-β (Aβ) in APP / PS1 transgenic mice and improve cognitive function.

[0006] Curcuma longa L. is a dried rhizome of Zingiberaceae Curcuma longa L. It has the effects of activating blood and qi, and relieving pain. Curcuma longa L. mainly contains volatile oil components and curcumin components, and the curcumin components are the main active components of Curcuma longa L. Modern pharmacological studies have shown that it has anti-inflammatory, antioxidant, antitumor, and antiviral functions. And it has low toxicity and small adverse reactions. Zhang L et al. (Zhang L, Fiala M, Cashman J, et al. Curcuminoids enhance amyloid-beta uptake by macrophages of Alzheimer's disease patients. J Alzheimers Dis. 2006 Sep; 10(1): 1-7.) found that the decrease of macrophage activity and the insufficient clearance of Aβ widely exist in Alzheimer's disease, which promotes the accumulation of Aβ, and the macrophages pretreated with curcumin can make 50% of the macrophages uptake Aβ, and delay the progression of AD.

[0007] Therefore, the present application is proposed. SUMMARY

[0008] The application adopts APP / PS1 mice as a model to evaluate the anti-Alzheimer's disease pharmacological activity of the composition of Ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract, and it is found that the composition can significantly improve the learning ability of APP / PS1 mice, reduce the level of Aβ in the brain of the mice, and improve the intestinal microbial environment of the mice. Therefore, the drug combination can be applied in the prevention or treatment of Alzheimer's disease.

[0009] One of the purposes of the application is to provide a drug combination composition for preventing or treating Alzheimer's disease.

[0010] The second purpose of the application is to provide an application of a drug combination composition for preventing or treating Alzheimer's disease.

[0011] In order to achieve the above-mentioned purposes of the application, the following technical solutions are adopted:

[0012] The application provides a drug combination composition for preventing or treating Alzheimer's disease, which comprises the following components in a weight ratio: 1-10 parts of Ganoderma lucidum polysaccharide, 1-10 parts of medlar polysaccharide, and 1-10 parts of turmeric extract.

[0013] Preferably, the composition comprises the following components in a weight ratio: 2 parts of Ganoderma lucidum polysaccharide, 2 parts of medlar polysaccharide, and 1 part of turmeric extract.

[0014] The above-mentioned components can be obtained by commercial channels or self-preparation.

[0015] The weight ratio of Ganoderma lucidum polysaccharide is typically but not limitedly, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts.

[0016] The weight ratio of medlar polysaccharide is typically but not limitedly, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts.

[0017] The weight ratio of turmeric extract is typically but not limitedly, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts.

[0018] In one embodiment, the weight ratio of Ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract is 8:1:1.

[0019] In another embodiment, the weight ratio of Ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract is 2:2:1.

[0020] In another embodiment, the weight ratio of Ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract is 1:8:1.

[0021] In another embodiment, the weight ratio of Ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract is 9:9:2.

[0022] In another embodiment, the weight ratio of Ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract is 6:3:1.

[0023] In another embodiment, the weight ratio of Ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract is 3:6:1.

[0024] Ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract all have certain effects on reducing the level of Aβ at the cellular level, and the combination of the three has a better effect on reducing the level of Aβ in cells than single use or combination of two.

[0025] The "comprising" in the present application means that other components can be included in addition to the components described, and in addition, the "comprising" in the present application can be replaced by the closed "consisting of" or "consisting of" and the like.

[0026] In some embodiments, Ganoderma lucidum polysaccharide and medlar polysaccharide can be obtained by water extraction and alcohol precipitation of Ganoderma lucidum and medlar fruit, respectively, and the precipitate is redissolved in water, filtered and dried to prepare.

[0027] In some embodiments, turmeric extract can be prepared by ethyl acetate extraction and isopropyl alcohol crystallization.

[0028] In specific examples, Ganoderma lucidum polysaccharide is prepared by a preparation method comprising the following steps:

[0029] The Ganoderma lucidum medicinal material is first degreased with 90% ethanol, decocted twice with water, and then the extract is concentrated to a specific gravity of 1.10-1.20. 95% ethanol is added to a final alcohol concentration of 80%, and the alcohol precipitate is redissolved in water. The solution is filtered through a 0.4 μm microfiltration membrane and a 10 kDa ultrafiltration membrane in sequence, and the ultrafiltration membrane retentate is collected, concentrated, dried and pulverized to obtain the Ganoderma lucidum polysaccharide. The molecular weight distribution of the prepared Ganoderma lucidum polysaccharide is 10 kDa-200 kDa.

[0030] The medlar fruit medicinal material is first degreased with 90% ethanol, decocted twice with water, and then the extract is concentrated to a specific gravity of 1.10-1.20. 95% ethanol is added to a final alcohol concentration of 80%, and the alcohol precipitate is redissolved in water. The solution is filtered through a 0.5 μm microfiltration membrane and a 30 kDa ultrafiltration membrane in sequence, and the ultrafiltration membrane retentate is collected, concentrated, dried and pulverized to obtain the medlar polysaccharide. The molecular weight distribution of the prepared medlar polysaccharide is 30 kDa-900 kDa.

[0031] In specific examples, turmeric extract is prepared by a preparation method comprising the following steps:

[0032] The turmeric medicinal material is pulverized, heated and extracted with ethyl acetate, the extract is concentrated, isopropyl alcohol is added for crystallization, and the filter cake is collected and dried under reduced pressure to obtain the turmeric extract.

[0033] In some embodiments, the pharmaceutical compound composition of the present application further comprises a pharmaceutically acceptable excipient.

[0034] The excipient can be a pharmaceutical excipient known in the art, such as a filler (e.g. starch, glucose, anhydrous lactose and lactose beads), a wetting agent (e.g. water, ethanol), a binder (e.g. microcrystalline cellulose, starch paste), a disintegrant (e.g. cross-linked PVP, cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), a lubricant (e.g. magnesium stearate, talc), a sweetener (e.g. sucrose), an excipient, etc.

[0035] The dosage form of the pharmaceutical compound composition is not particularly limited, and can be a capsule, a tablet, a granule, a powder, a pill, etc.

[0036] Another aspect of the present application provides a method for preparing a pharmaceutical compound composition for preventing or treating Alzheimer's disease, comprising the following steps:

[0037] (1) Preparing Ganoderma lucidum polysaccharide: Ganoderma lucidum medicinal materials are first degreased with 80-90 vol% ethanol, water decocted (preferably, water decoction is performed for 1.5-2.5 hours each time), the extract is concentrated, 90-95 vol% ethanol is added for alcohol precipitation, the precipitate is redissolved with water, and is successively filtered through a microfiltration membrane and an ultrafiltration membrane, the ultrafiltration membrane retentate is collected, and is concentrated and dried to obtain; preferably, the molecular weight distribution range of the prepared Ganoderma lucidum polysaccharide is 10 kDa-200 kDa;

[0038] (2) Preparing Lycium barbarum polysaccharide: Lycium barbarum medicinal materials are first degreased with 80-90 vol% ethanol, water decocted (preferably, water decoction is performed for 1.5-2.5 hours each time), the extract is concentrated, 90-95 vol% ethanol is added for alcohol precipitation, the precipitate is redissolved with water, and is successively filtered through a microfiltration membrane and an ultrafiltration membrane, the ultrafiltration membrane retentate is collected, and is concentrated and dried to obtain; preferably, the molecular weight distribution range of the prepared Lycium barbarum polysaccharide is 30 kDa-900 kDa;

[0039] (3) Preparing curcuma extract: curcuma medicinal materials are crushed, ethyl acetate is added for extraction (preferably, the extraction time is 0.5-1.5 hours, and the extraction temperature is 60-70℃), the extract is concentrated, isopropyl alcohol is added for crystallization (preferably, 3-4 times the amount of isopropyl alcohol is added, stirring is performed at 45-55℃ for 30 minutes, and filtration is performed after standing for 2-4 hours), the filter cake is collected, and is dried under reduced pressure to obtain;

[0040] (4) Mixing Ganoderma lucidum polysaccharide, Lycium barbarum polysaccharide and curcuma extract.

[0041] The curcuma extract in the above step (3) can also be purchased from Sabinsa Corporation, USA, Curcumin C3 Complex;

[0042] The pharmaceutical compound composition of the present application is prepared by extracting ganoderma polysaccharide, medlar polysaccharide and curcuma extract respectively.

[0043] In another aspect of the present application, the pharmaceutical compound composition of the present application is used for preventing or treating Alzheimer's disease.

[0044] The pharmaceutical compound composition of the present application can be administered to mammals, including but not limited to, human, mouse, horse, pig, dog, cow, sheep, etc.

[0045] The pharmaceutical compound composition of the present application can significantly improve the memory and cognitive ability of mice, reduce the Aβ plaque in the brain of mice, and improve the intestinal microbial imbalance related to the disease.

[0046] Beneficial effects

[0047] The present application first screens the effect of different proportions of drug combinations on the Aβ level of human neuroblastoma SK-N-SH cells, and optimizes the proportion of ganoderma polysaccharide, medlar polysaccharide and curcuma extract to 8:1:1, 4:4:2 and 1:8:1. Experiments on APP / PS1 mice (a pathological model of Alzheimer's disease) with the three drug proportions show that in the behavior detection "gold standard" - water maze experiment, it is found that the combination of ganoderma polysaccharide, medlar polysaccharide and curcuma extract can significantly improve the memory and cognitive ability of APP / PS1 model mice; in the histological detection "gold standard" - brain slice Aβ plaque staining, it is found that the combination of ganoderma polysaccharide, medlar polysaccharide and curcuma extract can significantly reduce the Aβ plaque in the brain of mice; in the behavior pharmacodynamic detection experiment - novelty exploration, it is further determined that the combination of ganoderma polysaccharide, medlar polysaccharide and curcuma extract can significantly improve the memory and cognitive ability of APP / PS1 model mice; in the new anti-AD pharmacological mechanism - intestinal microbial effect detection, the results show that the combination of ganoderma polysaccharide, medlar polysaccharide and curcuma extract can improve the intestinal microbial imbalance related to the disease of APP / PS1 model mice. The drug combination of the present application can relieve the cognitive dysfunction of APP / PS1 model mice, and is a very potential candidate drug for anti-AD.

[0048] The present application has been described in detail in the foregoing, but the above-mentioned embodiments are only illustrative in nature and are not intended to limit the present application. In addition, the present application is not limited by any theory described in the foregoing prior art or summary of the invention or in the following examples.

[0049] Unless specifically stated otherwise as apparent from the above disclosure, throughout the application file, ranges of values are to be understood to encompass any and all subranges of the same order of magnitude unless otherwise indicated. Unless otherwise stated, throughout the application file, the numerical values of the parameters (e.g., amounts or conditions) in the examples provided in the detailed description section are to be understood to be approximations rather than being strictly bound by the conditions set forth. All parameters including endpoint points stated above are meant to be approximations since some variations can occur whenever any industrial processes or other processes are used. Unless otherwise stated, all parameters, including endpoint points, are approximations and are understood to be open-ended ranges. At the very least, each numerical parameter should at least be construed in light of the number of significant digits and by applying ordinary rounding techniques. Furthermore, any numerical value can contain certain errors associated with the measuring process inherent in any such process. These errors should be considered to be inherent in and can be accommodated by the numerical values that have been reported for the parameters. Unless otherwise stated, the term "about" as applied to one or more values of the parameters herein is meant to encompass minor variations (plus or minus ten percent) from the stated values. For example, "about" can include variations less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The body weight of each group of mice in Example 2 of the present application is shown.

[0051] Figure 2 The time to reach the platform of mice in the training period of the water maze in Example 2 of the present application is shown.

[0052] Figure 3 The time to first cross the platform and the number of times to cross the platform in the probe trial of Example 2 of the present application are shown.

[0053] Figure 4 The swimming speed and distance of mice in the probe trial of Example 2 of the present application are shown.

[0054] Figure 5 The time spent in the quadrant where the platform was located and the surrounding quadrants of mice in Example 2 of the present application are shown.

[0055] Figure 6 The effect of the drug on the new object exploration behavior of APP / PS1 mice in Example 3 of the present application is shown.

[0056] Figure 7 The results of the area of Aβ plaque and the total number of plaques in the hippocampus and cortex in Example 4 of the present application are shown.

[0057] Figure 8 The level of Desulfovibrio in the intestine of mice in Example 5 of the present application is shown.

[0058] Figure 9Figure 5 shows the level of cyanobacteria in the gut of mice of Example 5. DETAILED DESCRIPTION

[0059] The application will be further described in conjunction with the following examples. It should be noted that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the application.

[0060] Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are conventional raw materials, reagents, methods in the art.

[0061] The materials, reagents and instruments used in the following examples are as follows:

[0062] 1. Experimental materials and equipment

[0063] Human Aβ enzyme-linked immunosorbent assay kit (Ekoase Biotechnology Co., Ltd.); Morris water maze (General Electric Company); Morris water maze video analysis system (Shanghai Jiliang Software Technology Co., Ltd.); SK-N-SH cells purchased from ATCC; APP / PS1 transgenic mice purchased from Jackson Laboratory; C57BL / 6 mice purchased from Shanghai Animal Experimental Center.

[0064] 2. Experimental raw materials

[0065] Ganoderma lucidum medicinal materials were purchased from Zhejiang Keda Biotechnology Co., Ltd., batch number: KDL18A01-191123. Ganoderma lucidum polysaccharide, self-made sample, batch number LZ-20200910-01, preparation method: Ganoderma lucidum medicinal materials were first degreased with 90% ethanol, then water decocted twice (in particular, two hours each time), the extract was concentrated to a specific gravity of 1.10-1.20, 95% ethanol was added to a final alcohol concentration of 80%, the alcohol-sedimented precipitate was redissolved with water, and then passed through 0.4 μm microfiltration membrane and 10 kDa ultrafiltration membrane in sequence, the ultrafiltration membrane retentate was collected, concentrated, dried, and pulverized to obtain the product. The molecular weight distribution range of the prepared Ganoderma lucidum polysaccharide sample was 10 kDa-200 kDa.

[0066] Medicinal materials of Fructus Lycii were purchased from Ningxia Wofu Baierui Lycium Industry Co., Ltd., batch number: 200202. Lycium barbarum polysaccharide, self-made sample, batch number GQ-20200910-01, preparation method: Fructus Lycii medicinal materials were first degreased with 90% ethanol, then water decocted twice (in particular, two hours each time), the extract was concentrated to a specific gravity of 1.10-1.20, 95% ethanol was added to a final alcohol concentration of 80%, the alcohol-sedimented precipitate was redissolved with water, and then passed through 0.5 μm microfiltration membrane and 30 kDa ultrafiltration membrane in sequence, the ultrafiltration membrane retentate was collected, concentrated, dried, and pulverized to obtain the product. The molecular weight distribution range of the prepared Fructus Lycii polysaccharide sample was 30 kDa-900 kDa.

[0067] Turmeric medicinal materials were purchased from Shanghai Huayu Pharmaceutical Co., Ltd., batch number: T2019052101. Turmeric extract, self-made sample, batch number JH-20200910-01, preparation method: after the turmeric medicinal materials were crushed, ethyl acetate was added for heating extraction (in particular, 10 times the amount of ethyl acetate can be added, 65°C extraction twice, 1 hour each time), the extract was concentrated, isopropyl alcohol was added for crystallization (in particular, 3 times the amount of isopropyl alcohol can be added, 45-55°C stirring for 30 minutes, standing for 2 hours and then filtering, refining twice), filtering, collecting the filter cake and drying under reduced pressure to obtain the product.

[0068] 3. Preparation and administration of animal test drugs

[0069] According to different proportions of ganoderma lucidum polysaccharide, medlar polysaccharide and turmeric extract, they were mixed and dissolved in 0.5% CMC-Na aqueous solution, and the drug concentration was 18 mg / mL. Shake before each gavage to make the drug evenly dispersed. Administered once a day, the dose was 300 mg / kg, orally gavaged, and administered continuously for 3 months.

[0070] 4. Test animals and feeding conditions

[0071] 4.1. Species: APP / PS1 mice, C57BL / 6 mice; SPF level; body weight 28-35 g.

[0072] All animal experiments in this report were carried out in accordance with the National Institutes of Health (NIH) Laboratory Animal Care and Use Guidelines. The animal experiment plan was approved by the Biological Research Ethics Committee of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The pain and discomfort of the animals were minimized as much as possible.

[0073] 4.2. Feeding management

[0074] 4.2.1. Environmental adaptation: environmental adaptation for 1 day before the test.

[0075] 4.2.2. Experimental feeding conditions: the feeding density was 1 per cage; the cage space displacement frequency was 1 time / week.

[0076] 4.2.3. Feeding environmental conditions

[0077] Feeding environmental condition standards: GB14925-2010 of the People's Republic of China;

[0078] Feeding environment control system: automatic control of fresh air central air conditioning system;

[0079] Temperature: 20-26°C (daily temperature difference ≤4°C);

[0080] Humidity: relative humidity 30-70%;

[0081] Light: artificial illumination 12h light-dark cycle (light on at 07:30, light off at 19:30, if necessary for the experiment, illumination during the dark period is possible);

[0082] Air changes: minimum air changes index 15 / h for barrier system, 8 / h for general animal room, 20 / h for isolator, 100% fresh air.

[0083] Bedding changed at least once a week, along with the cages, and at any time in case of abnormal conditions. The disinfectant water bottles and stoppers were changed daily, and the cages were disinfected every two weeks. All the changed cages were sterilized by autoclaving.

[0084] 4.2.4. Feed

[0085] Feeding method: ad libitum (except when the experiment requires otherwise);

[0086] Nutritional composition testing: provided by the feed supplier for each batch;

[0087] Routine nutritional composition indicators: crude protein, crude fat, crude fiber, crude ash, moisture, calcium, and phosphorus.

[0088] 4.2.5. Drinking water

[0089] Type: drinking water for experimental animals (tap water autoclaved);

[0090] Water supply method: drinking water bottles filled, ad libitum.

[0091] 5. Statistical methods

[0092] The data are expressed as Mean ± SEM, and the significant analysis uses two-way ANOVA and one-way ANOVA of Graph Pad software to test whether there is a difference in the sample population, if there is a difference, then the Bonferroni test is used to test the difference between each group and the model group, and p < 0.05 is considered statistically significant. In addition, if the mice in the experiment behave abnormally, such as circling, floating, etc., they should be removed. The behavior of the mice is calculated using Graph Pad's Identify outliers Q = 0.5%, if the outliers are removed, the mouse is removed. The calculation of the intestinal flora microorganism outliers of the mice: data outside the range of mean plus or minus three standard deviations.

[0093] Example 1 Drug ratio in vitro screening experiment

[0094] I. Cell culture and drug administration

[0095] SK-N-SH cells were cultured in MEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 0.1 mg / mL streptomycin. The cells were incubated with drugs at a total drug concentration of 500 μg / mL for 24 h.

[0096] II. Detection of Aβ levels

[0097] The Aβ levels in the culture supernatants of the control and drug-treated groups were quantitatively analyzed according to the instructions of the human Aβ ELISA kit. The Aβ levels in the drug-treated groups were compared with those in the control group.

[0098] III. Experimental results

[0099] The SK-N-SH cells were incubated with drug combinations at different ratios. The results are shown in Table 1.

[0100] Table 1. Extracellular Aβ levels at different drug ratios

[0101]

[0102] The results showed that each ratio could reduce the extracellular Aβ levels. The three-drug combinations (Experiments 1-6) were superior to the two-extract combinations (Experiments 7-10) and the single extracts (Experiments 11-12).

[0103] The optimal ratio of Ganoderma lucidum polysaccharides, Lycium barbarum polysaccharides and turmeric extract was 4:4:2.

[0104] Example 2. Mouse water maze test

[0105] I. Grouping of mice in the water maze

[0106] Table 2. Grouping of mice in the water maze

[0107]

[0108] II. Behavior of mice in the water maze

[0109] The APP / PS1 mice were grouped and administered at the age of 4-5 months, and the administration lasted for three months, followed by behavior testing.

[0110] Preparation of mice before the experiment: The mice that needed to be tested in the water maze were transferred to the behavior room in advance, one cage per mouse, and new mouse cages (SPF level) were replaced. They were randomly renumbered 1-N and placed in disorder. The experimenter and the mice were double-blinded to the grouping information. After the single-caged mice were adapted to the water maze room for at least one day, the experiment began. The experimenter observed the room temperature, which was maintained at about 23°C using an air conditioner.

[0111] III. Preparation of materials before the water maze experiment:

[0112] 1. Bucket. Diameter 1.2 meter. White paper is used on the bottom and inner wall of the bucket.

[0113] 2. Platform. Diameter 11 cm, height 19 cm.

[0114] The surface of the platform is made of organic glass, and the surface is made of horizontal and vertical deep lines to prevent slipping.

[0115] 3. Computer (software EthoVision XT).

[0116] 4. Camera.

[0117] 5. Light. Non-natural light source, the light cannot be directly above the bucket to prevent the water surface from appearing inverted and affecting the collection of the trajectory. The light needs to be slightly dark, and a white cloth can be used to cover the light.

[0118] 6. Visible markers (Cues). Currently, four black and white patterns are used: square, square, triangle, circle, and cross. They are located on the inner wall of the bucket.

[0119] The size of each Cue: square (25 x 25 cm, black and white each 3 cm wide); triangle (25 cm equilateral; from outside to inside: black-white-black); circle (diameter 25 cm; from outside to inside: white-black-white-black); cross (all black, 25 cm long and wide).

[0120] 7. Plastic particles (transparent or white) floating on the water surface. (Make the water opaque, and the mouse cannot see the platform).

[0121] 8. Paper, strainer, thermometer, gloves, mask, etc.

[0122] Four, daily water maze process:

[0123] 1. Fill the water. Before filling the water on the first day, first determine the position of the outer bucket and the relative position of the inner bucket, and make a mark. Mark the four directions of the inner bucket (direction: northwest, southeast, paste paper strip N, W, E, S). Adjust the position of the bucket in the software to make the WE line and the NW line horizontal and vertical. The above position should not change during the 7-day experiment process. Fill the tap water below the platform surface, then adjust the water temperature to 21.5°C ± 0.5°C according to the existing water temperature (by heating water or adding ice, etc.), and the room temperature is 22°C ± 0.5°C. Add plastic particles to make the water level reach 1 cm above the platform.

[0124] 2. Preparation before placing the mouse: wait for the water surface to be still, make sure the surrounding environment is normal (no unexpected debris in the field of view, etc.). Make sure the experimenter wears the experimental clothes (wear the same clothes every day). Record the room temperature and water temperature. Invert the water bottle on the mouse cage. Two experimenters are responsible for operating the mouse (Experimenter A) and operating the computer (Experimenter B).

[0125] 3、Training: 6 days of training, each mouse is trained four times a day (from four different directions, the order of the directions for each mouse is determined by a random number, to ensure that each mouse is trained in four different directions each day and not repeated). The experimenter A uses a long-handled scoop to transfer the mouse to the water surface, so that the mouse faces the barrel wall and enters the water, while the scoop is quickly retracted, and the experimenter A immediately leaves the mouse's field of view. The experimenter B clicks the start key of the computer at the moment the mouse enters the water.

[0126] 4、For each training trial, the mouse has a maximum of 60s to find the platform. If the mouse finds the platform, the mouse is allowed to stay on the platform for 30s, and then the mouse is taken back (using a scoop, gently and non-stimulating). If the mouse has not found the platform by the end of 60s, the experimenter guides it onto the platform. (When guiding, the tail is pulled with the hand, so that the mouse faces the platform and climbs on, avoiding being dragged onto the platform with the back. ) Then it is allowed to stay on the platform for 30s before the mouse is taken back. The mouse is gently wiped with toilet paper in an empty cage and then put back in the cage. Then the next one is prepared.

[0127] 5、The experimenter B records the time the mouse gets on the platform in the experiment notebook, and tells the experimenter A by voice: "to" means 60s is up, the mouse has not found the platform, and guidance is needed; "good" means the mouse has found the platform and stayed on it for 30s, and it can be taken back. Before each mouse starts the experiment (when it is prepared to be put on the scoop), the experimenter B needs to tell the experimenter A the current direction: e.g. "north", to ensure that the direction is not put wrong. In addition, there should be no other loud voices.

[0128] 6、In principle, the number of mice in each direction is controlled within 25-30 per day, and the experimental time for each direction is maintained at about 30-50 minutes. If there are too many mice and the time per round is too long, the difficulty of learning and memory will increase, which will affect the effect of mouse learning and memory. If the number of samples in the experiment is more than the amount of one experiment, the mice in each group must be evenly distributed in each experiment, especially the number of samples in the two control groups should be evenly distributed, so that the parallelism of each experiment can be measured.

[0129] 7、On the 4th and 7th days, there is a probe trial, i.e. the platform is removed in advance, and the mouse is searched for 60s in the barrel without the platform, and then it is taken out. On the 4th day, after the 4 directions are completed, the probe trial is performed. Note: When the mouse is taken out after 60s, the experimenter still needs to guide the mouse to the original position (nearby) of the platform and take it out (try to minimize the misunderstanding brought by the probe trail).

[0130] 8、After the experiment is completed on the same day, a water bottle is given to each mouse (to drink water).

[0131] V. Water maze test results

[0132] 1. Body weight record

[0133] The body weight of each group of mice was as shown in Table 1, and the food intake of mice during the administration period was normal, and the body weight growth of mice in each administration group was normal, indicating that the drug would not produce toxicity to mice. Figure 1 Figure 1 It can be seen that the food intake of mice during the administration period was normal, and the body weight growth of mice in each administration group was normal, indicating that the drug would not produce toxicity to mice.

[0134] 2. Water maze test results

[0135] The time for mice to reach the platform during the water maze training period was as shown in Table 2. Figure 2

[0136] During the six-day training process, the time for mice to reach the platform in the three administration groups was significantly different from that in the model group (***p<0.001), indicating that the three different administration groups improved the learning ability of APP / PS1 mice.

[0137] The first time for mice to cross the platform and the number of times for mice to cross the platform in the platform removal test were as shown in Table 3. The swimming speed and distance of mice in the platform removal test were as shown in Table 4. The time for mice to cross the platform and the surrounding quadrants was as shown in Table 5. Figure 3 Figure 4 Figure 5 In the platform removal test, compared with the model group, the time for mice to reach the platform in the three administration groups was significantly reduced (*p<0.05; **p<0.01; ***p<0.001), and the number of times for mice to cross the platform was significantly increased. The swimming speed and distance of each administration group were not reduced, and the swimming speed and time of mice in administration group 3 were slightly increased (*p<0.05; ***p<0.001), indicating that the motor ability of mice was not impaired. Compared with the model group, the search time for mice in administration group 1 and administration group 2 in the platform quadrant (northwest quadrant) was significantly increased (***p<0.001), indicating that the drug combination could improve the memory of APP / PS1 mice.

[0138]

[0139] Example 3 Novelty exploration test of mice

[0140] I. Novelty exploration grouping

[0141] Table 3 Grouping of novelty exploration test

[0142]

[0143] II. Novel object recognition

[0144] 1. Square frosted opaque box (45 cm x 45 cm x 45 cm) ​​​​​

[0145] 2. Computer (software EthoVision XT).

[0146] 3. Camera.

[0147] 4. Light. Non-natural light source, the light needs to be slightly dark, you can use white cloth cover lamp.

[0148] Three, new object recognition experiment process:

[0149] Put the APP / PS1 mice facing the middle of the box wall and against the object, take out after 10 minutes of free exploration on the first day. Wipe the object and the box with 75% alcohol, and put it in the second mouse to be tested after natural drying. Change one of the same objects to a new object on the second day, and explore freely for 10 minutes as on the first day. Preference calculation method: time of new object B / (total time of old object A + new object B).

[0150] Four, novelty exploration test results

[0151] The results of the preference time of each group are shown in Figure 6 .

[0152] From Figure 6 The results of the novelty exploration experiment show that the preference degree of the model group for the new object is significantly lower than that of the control group, and the preference of the three drug groups for the new object is significantly higher than that of the model group (*p<0.05; **p<0.01; ***p<0.001), indicating that the drug combination can improve the cognitive ability of mice.

[0153] Example 4: APP / PS1 mouse Aβ plaque staining test

[0154] I. Brain slice Aβ plaque staining grouping:

[0155] Table 4: Brain slice Aβ plaque staining grouping

[0156]

[0157] II. Materials and methods

[0158] 1. Sample preparation:

[0159] After the completion of the mouse behavior test, the mouse brain sample was obtained after 1xPBS perfusion, the left brain was fixed in 4% PFA, and after overnight replacement with 30% sucrose water, it was dehydrated for three to five days, then placed in an embedding box, embedded with OCT, and frozen in a-80℃ refrigerator. After the right brain was taken out, the hippocampus and cortex were placed in liquid nitrogen and then stored in a-80℃ refrigerator.

[0160] The left half brain was taken out from -80°C refrigerator and placed in -20°C ice cutter for 45 min before frozen sectioning. The brain slices were taken every 30 μm from hippocampus and 6 groups were collected in parallel. 2000 μL tissue protective solution (sucrose 150 g, 0.2 M PB 250 mL, glycol 150 mL) was placed in 24-well plate.

[0161] 2. Test method:

[0162] 1) The sample was taken out from the tissue protective solution and placed in 1 x PBS for 5 min on a shaker.

[0163] 2) Block buffer (3% BSA + 0.2% Triton X-100 + 50 mL 1 x PBS) was added for 1 h at room temperature.

[0164] 3) 1 x PBS was added for 5 min on a shaker.

[0165] 4) Primary antibody (1:1000) was added at 200 μL per well, and the primary antibody anti-rabbit GFAP, anti-mouse 6E10 was diluted with antibody diluent (1% BSA + 0.2% Triton X-100 + 50 mL 1 x PBS) at 4°C overnight.

[0166] 5) After overnight, the primary antibody was aspirated and recovered. 1 x PBS was added to the well for 5 min on a shaker.

[0167] 6) Secondary antibody donkey anti-rabbit Cy3, donkey anti-mouse Alex488 was added at 1:1000, and the antibody was diluted with antibody diluent, protected from light, for 1 h.

[0168] 7) 1 x PBS was added for 5 min on a shaker.

[0169] 8) DAPI (1 x PBS dilution) was added at 1:5000 for 10 min.

[0170] 9) 1 x PBS was added for 5 min on a shaker.

[0171] 10) The sample was mounted on a glass slide, and the mounting was performed with anti-fluorescence quenching mounting medium.

[0172] 11) Carl Zeiss fluorescence microscope was used for observation.

[0173] III. Test results

[0174] The Aβ plaque area and total plaque number in hippocampus and cortex area were as shown in Table 1. Figure 7 Table 1

[0175] Depend on Figure 7 As can be seen, after staining with 6E10 antibody, the area of ​​Aβ plaques in the cortex and hippocampus of mice was significantly reduced in all three treatment groups compared with the model group (*p<0.05; **p<0.01; ***p<0.001). The total number of Aβ plaques in the mouse brain was also significantly reduced. Furthermore, treatment group 2 showed slightly better results than treatment groups 1 and 3.

[0176] Example 5: Detection of desulfurized bacteria and cyanobacteria in the intestinal microbiota of mice.

[0177] I. Experimental Grouping

[0178] 1. Grouping of desulfurized bacteria spp. in mouse gut microbiota

[0179] Table 5 Grouping of desulfurization bacteria in mouse gut microbiota

[0180]

[0181] 2. Grouping of Cyanobacteria Species Detection in Mouse Intestinal Microbiota

[0182] Table 6 Grouping of mouse gut microbiota (cyanobacteria)

[0183]

[0184] II. Intestinal Microbial Materials and Methods

[0185] 1. Stool sample collection

[0186] 2. DNA extraction and PCR amplification

[0187] according to Total DNA was extracted from the microbial community using the soil DNAkit (Omega Bio-tek, Norcross, GA, US) according to the instructions. DNA extraction quality was assessed using 1% agarose gel electrophoresis, and DNA concentration and purity were determined using NanoDrop 2000. PCR amplification of the 16S rRNA gene V3-V4 variable region was performed using 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The amplification program was as follows: 95℃ pre-denaturation for 3 min, 27 cycles (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s), followed by a stable extension at 72℃ for 10 min, and finally storage at 4℃ (PCR instrument: ABI). 9700). The PCR reaction system was as follows: 5x TransStart FastPfu buffer 4 μL, 2.5 mmol dNTPs 2 μL, upstream primer (5 μM) 0.8 μL, downstream primer (5 μM) 0.8 μL, TransStart FastPfu DNA polymerase 0.4 μL, template DNA 10 ng, and the rest was supplemented to 20 μL. Each sample was repeated 3 times.

[0188] 3. Illumina Miseq sequencing

[0189] The PCR products of the same sample were mixed and recovered by 2% agarose gel, and the recovered products were purified by AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), detected by 2% agarose gel electrophoresis, and quantified by Quantus Fluorometer (Promega, USA). TM The NEXTFLEX Rapid DNA-Seq Kit was used for library construction:

[0190] 1) Linker ligation

[0191] 2) Use magnetic beads to remove linker self-ligation fragments

[0192] 3) Use PCR amplification to enrich library templates

[0193] 4) Recover the PCR product by magnetic beads to obtain the final library. The Miseq PE300 / NovaSeq PE250 platform of Illumina Company was used for sequencing.

[0194] 4. Data processing

[0195] The raw sequencing sequences were quality controlled by fastp (https: / / github.com / OpenGene / fastp, version 0.20.0) software, and spliced by FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.7) software.

[0196] III. Experimental results

[0197] The mouse intestinal Desulfovibrio level was as shown in Figure 8 .

[0198] The Figure 8It can be seen that the levels of Desulfobacterota in the control group were significantly lower than those in the model group, and the levels in the treatment group 2 were also significantly lower than those in the model group (***p<0.001). After three months of treatment, the levels of Desulfobacterota in the APP / PS1 mice in the treatment group 2 had decreased to levels approaching those of the control group.

[0199] IV. Cyanobacterial Level

[0200] mouse gut cyanobacterial levels such as Figure 9 As shown.

[0201] Depend on Figure 9 It can be seen that Cyanobacteria levels were significantly lower in mice in treatment groups 2 and 3 compared to the APP / PS1 model group (**p<0.01; ***p<0.001). Cyanobacteria or cyanobacteria in the gut microbiota may produce the neurotoxin β-n-methylamino-l-alanine (BMAA), which is considered to be associated with AD.

[0202] Oral administration of Ganoderma lucidum polysaccharides, Lycium barbarum polysaccharides, and Curcuma longa extract reduced the time APP / PS1 mice took to reach the platform for the first time in the water maze test, indicating that the drug combination improved the learning ability of APP / PS1 mice. In the platform removal test, compared with the model group, the three drug-treated groups increased the number of platform shuttles, shortened the time to reach the platform, and increased the search and exploration time in the quadrant where the platform was located, indicating that the drug combination can improve the memory ability of APP / PS1 mice. In the novelty exploration test, the proportion of mice exploring new objects increased in the three drug-treated groups, suggesting that it improved the cognitive function of APP / PS1 mice. In the mouse brain slice immunofluorescence staining experiment, the three drug-treated groups significantly reduced the area and total number of Aβ plaques in the hippocampus and cortical areas of APP / PS1 mice. In addition, in the analysis of some bacteria related to the course of AD in the mouse gut microbiota, it was found that the gut microbiota of the drug-treated groups also had a corresponding reduction in Cyanobacteria and Desulfurobacteria compared with the APP / PS1 model group. Therefore, the combination of Ganoderma lucidum polysaccharide, Lycium barbarum polysaccharide and turmeric extract can alleviate cognitive dysfunction in APP / PS1 model mice and is a promising candidate drug for anti-AD.

[0203] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and substance defined by the claims of the present invention; and such modifications or substitutions are still within the scope defined by the claims of the present invention.

Claims

1. A pharmaceutical combination composition for preventing or treating Alzheimer's disease, which is composed of the following components in the following weight ratio: 1-10 parts of Ganoderma lucidum polysaccharide, 1-10 parts of Lycium barbarum polysaccharide, and 1-10 parts of Curcuma longa extract, The pharmaceutical compound composition is prepared by a preparation method comprising the following steps: (1) Ganoderma lucidum polysaccharide is prepared as follows: Ganoderma lucidum is first defatted with 80-90 vol% ethanol, then water decocted, and the extract is concentrated; 90-95 vol% ethanol is added for alcohol precipitation, the precipitate is dissolved in water, and then passed through a microfiltration membrane and an ultrafiltration membrane in sequence; the ultrafiltration membrane cut-off liquid is collected, concentrated, dried, and pulverized to obtain the Ganoderma lucidum polysaccharide; (2) Lycium barbarum polysaccharide is prepared as follows: Lycium barbarum is first defatted with 80-90 vol% ethanol, then water decocted, and the extract is concentrated; 90-95 vol% ethanol is added for alcohol precipitation, the precipitate is dissolved in water, and then passed through a microfiltration membrane and an ultrafiltration membrane in sequence; the ultrafiltration membrane cut-off liquid is collected, concentrated, dried, and pulverized to obtain the Lycium barbarum polysaccharide; (3) Curcuma longa extract is prepared as follows: Curcuma longa is pulverized, then extracted with ethyl acetate, and the extract is concentrated; isopropyl alcohol is added for isopropyl alcohol crystallization, filtration is performed, the filter cake is collected, and then dried under reduced pressure to obtain the Curcuma longa extract; (4) Ganoderma lucidum polysaccharide, Lycium barbarum polysaccharide, and Curcuma longa extract are mixed.

2. The pharmaceutical composition of claim 1, wherein The pharmaceutical compound composition comprises the following components in the following proportions by weight: 2 parts of Ganoderma lucidum polysaccharide, 2 parts of Lycium barbarum polysaccharide, and 1 part of Curcuma longa extract.

3. The pharmaceutical composition of claim 1, wherein The molecular weight distribution range of the Ganoderma lucidum polysaccharide is 10 kDa-200 kDa.

4. The pharmaceutical composition of claim 1, wherein The molecular weight distribution range of the Lycium barbarum polysaccharide is 30 kDa-900 kDa.

5. The pharmaceutical composition of claim 1, wherein, In step (1), Ganoderma lucidum is first defatted with 90% ethanol, then water decocted twice, the extract is concentrated to a specific gravity of 1.10-1.20, 95% ethanol is added to a final alcohol concentration of 80%, the precipitate is dissolved in water, and then passed through a 0.4 μm microfiltration membrane and a 10 kDa ultrafiltration membrane in sequence; the ultrafiltration membrane cut-off liquid is collected, concentrated, dried, and pulverized to obtain the Ganoderma lucidum polysaccharide.

6. The pharmaceutical composition of claim 1, wherein, In step (2), Lycium barbarum is first defatted with 90% ethanol, then water decocted twice, the extract is concentrated to a specific gravity of 1.10-1.20, 95% ethanol is added to a final alcohol concentration of 80%, the precipitate is dissolved in water, and then passed through a 0.5 μm microfiltration membrane and a 30 kDa ultrafiltration membrane in sequence; the ultrafiltration membrane cut-off liquid is collected, concentrated, dried, and pulverized to obtain the Lycium barbarum polysaccharide.

7. The pharmaceutical compound composition of claim 1, wherein: In step (1), water decoction is performed for 1.5-2.5 hours each time; and / or In step (2), water decoction is performed for 1.5-2.5 hours each time; and / or In step (3), the extraction time is 0.5-1.5 hours, and the extraction temperature is 60-70 °C; and / or In step (3), 3-4 times the amount of isopropyl alcohol is added for isopropyl alcohol crystallization, stirring is performed at 45-55 °C for 30 minutes, and then filtration is performed after standing for 2-4 hours.

8. A composition comprising: The pharmaceutical compound composition for preventing or treating Alzheimer's disease according to any one of claims 1-7; and a pharmaceutically acceptable adjuvant.

9. The composition of claim 8, wherein, The adjuvant comprises one or more selected from a filler, a wetting agent, a binder, a disintegrant, a lubricant, a sweetener, and an excipient.

10. The composition according to claim 8 or 9, characterized in that, The dosage form of the composition is selected from any one of a capsule, a tablet, a granule, a powder, and a pill.

11. A method for preparing a pharmaceutical combination composition for preventing or treating Alzheimer's disease, characterized by, The method comprises the following steps: (1) Ganoderma lucidum polysaccharide: Ganoderma lucidum medicinal materials are first defatted with 80-90 vol% ethanol, water decocted, the extract is concentrated, 90-95 vol% ethanol is added for alcohol precipitation, the precipitate is redissolved with water, and then passed through a microfiltration membrane and an ultrafiltration membrane successively, the ultrafiltration membrane cut-off liquid is collected, concentrated, dried and pulverized to obtain; (2) Wolfberry polysaccharide: Wolfberry medicinal materials are first defatted with 80-90 vol% ethanol, water decocted, the extract is concentrated, 90-95 vol% ethanol is added for alcohol precipitation, the precipitate is redissolved with water, and then passed through a microfiltration membrane and an ultrafiltration membrane successively, the ultrafiltration membrane cut-off liquid is collected, concentrated, dried and pulverized to obtain; (3) Curcuma longa extract: Curcuma longa medicinal materials are pulverized, extracted with ethyl acetate, the extract is concentrated, isopropyl alcohol is added for crystallization, filtered, the filter cake is collected, and dried under reduced pressure to obtain; (4) Ganoderma lucidum polysaccharide 1-10 parts, wolfberry polysaccharide 1-10 parts and curcuma longa extract 1-10 parts are mixed.

12. The method of claim 11, wherein, In step (4), 2 parts of Ganoderma lucidum polysaccharide, 2 parts of wolfberry polysaccharide and 1 part of curcuma longa extract are mixed.

13. The method of claim 11 or 12, wherein, In step (1), the Ganoderma lucidum medicinal materials are first defatted with 90% ethanol, water decocted twice, the extract is concentrated to a specific gravity of 1.10-1.20, 95% ethanol is added to a final alcohol concentration of 80%, the alcohol precipitate is redissolved with water, and then passed through a 0.4 μm microfiltration membrane and a 10 kDa ultrafiltration membrane successively, the ultrafiltration membrane cut-off liquid is collected, concentrated, dried and pulverized to obtain.

14. The method of claim 11 or 12, wherein, In step (2), the Wolfberry medicinal materials are first defatted with 90% ethanol, water decocted twice, the extract is concentrated to a specific gravity of 1.10-1.20, 95% ethanol is added to a final alcohol concentration of 80%, the alcohol precipitate is redissolved with water, and then passed through a 0.5 μm microfiltration membrane and a 30 kDa ultrafiltration membrane successively, the ultrafiltration membrane cut-off liquid is collected, concentrated, dried and pulverized to obtain.

15. The method of claim 11, wherein: In step (1), water decoction is performed for 1.5-2.5 hours each time; and / or In step (2), water decoction is performed for 1.5-2.5 hours each time; and / or In step (3), the extraction time is 0.5-1.5 hours, and the extraction temperature is 60-70°C; and / or In step (3), 3-4 times the amount of isopropyl alcohol is added for isopropyl alcohol crystallization, stirred at 45-55°C for 30 minutes, and then filtered after standing for 2-4 hours.

16. The method of claim 11 or 12, wherein: The molecular weight distribution range of the prepared Ganoderma lucidum polysaccharide is 10 kDa-200 kDa; and / or The molecular weight distribution range of the prepared Wolfberry polysaccharide is 30 kDa-900 kDa.

17. Use of the pharmaceutical compound composition of any one of claims 1-7 or the composition of any one of claims 8-10 in the preparation of a drug for preventing or treating Alzheimer's disease.

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