Application of Cyperus rotundus extract A in the preparation of drugs for the prevention, improvement or treatment of Alzheimer's disease

Abstract

This invention relates to the pharmaceutical use of Cyperus rotundus extract A in the prevention, improvement, or treatment of Alzheimer's disease, belonging to the pharmaceutical field. Pharmacological experiments have shown that Cyperus rotundus extract A has a good therapeutic effect on Alzheimer's disease, and therefore it can be used to prepare drugs for the prevention, improvement, or treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of Cyperus rotundus extract A in the preparation of drugs for the prevention, improvement or treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD), commonly known as senile dementia, primarily affects people over 65 years of age. This disease not only causes immense physical and psychological suffering for patients but also places a heavy burden on their families and society. With the accelerating aging of the world's population, Alzheimer's disease has become the "number one chronic killer" threatening the health and lives of people in their later years.

[0003] The main clinical manifestations of Alzheimer's disease are memory loss and cognitive impairment. The main pathological symptoms include the formation of insoluble β-amyloid plaques (Aβ), also known as dense senile plaques (SPs), and filamentous neurofibrillary tangles (NFTs) formed by abnormal phosphorylation of Tau protein in the brain.

[0004] Donepezil is a specific central acetylcholinesterase inhibitor (AChEI) that specifically inhibits the breakdown of acetylcholine in the brain, increases acetylcholine levels in brain tissue, and improves dementia symptoms, thus becoming the first-line drug for treating mild to moderate Alzheimer's disease. However, when using higher doses to treat severe dementia, adverse reactions such as gastrointestinal reactions, arrhythmias, mental confusion, neurological damage, liver dysfunction, and hypokalemia may occur, especially with overdose and combination therapy.

[0005] Globally, there remains a persistent and urgent need for new drugs that are effective, have few side effects, and are well-tolerated for the prevention, improvement, or treatment of Alzheimer's disease. Summary of the Invention

[0006] The present invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the present invention provides a drug for preventing, improving or treating Alzheimer's disease.

[0007] According to one aspect of the invention, the use of cyperus oleracea extract A or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable ester thereof or a pharmaceutically acceptable glycoside thereof in the preparation of a medicament for the prevention, improvement or treatment of Alzheimer's disease is provided.

[0008] Preferably, the use of cyperus rotundus extract A as the sole active ingredient in the preparation of the drug.

[0009] Preferably, the use of the bamboo rhizome extract A together with other active ingredients in the preparation of the drug.

[0010] Preferably, the other active ingredients include donepezil.

[0011] Preferably, the drug is selected from one or more oral or non-oral formulations.

[0012] Preferably, the oral preparation is one or more of the following: capsules, tablets, oral liquids, granules, pills, powders, elixirs, or ointments.

[0013] Preferably, the non-oral preparation is one or more of the following: injection, cream, patch, ointment or spray.

[0014] Preferably, the injection is administered via one or more of the following methods: subcutaneous administration, intramuscular administration, or intravenous administration.

[0015] Preferably, the application is to mammals.

[0016] According to another aspect of the invention, a medicament for preventing, improving or treating Alzheimer's disease is provided, comprising: cyperus oleracea extract A or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable ester thereof or a pharmaceutically acceptable glycoside thereof.

[0017] Compared with the prior art, this invention has the following beneficial effects: This invention is the first to discover that Cyperus rotundus extract A can be used to prevent, improve, or treat Alzheimer's disease. Therefore, it can be used to prepare drugs for the prevention, improvement, or treatment of Alzheimer's disease. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0019] Figure 1 and Figure 1 (Continued) The experimental data graph of cyperus rotundus A against β-amyloidosis is shown;

[0020] Figure 2 and Figure 2 (Continued) The experimental data on the inhibition of β-amyloid β-protein (Aβ) fibril formation by cyperus rotundus A are shown in the figure.

[0021] Figure 3 , Figure 3 (Continued 1) and Figure 3(Continued 2) shows the experimental data graph of the effect of cyperus rotundus A on the cognitive function of 3×Tg-AD mice;

[0022] Figure 4 and Figure 4 (Continued) The experimental data of the effects of cyperus rotundus extract A on the cerebral cortex and hippocampus of 3×Tg-AD mice are shown in the figure.

[0023] Figure 5 The experimental data on the effects of cyperus rotundus extract A on amyloid precursor protein (APP) and Aβ oligomer levels are shown in the figure; and

[0024] Figure 6 and Figure 6 (Continued) Experimental data and effects of cyperus rotundus extract A on the levels of glial fibrillary acidic protein (GFAP), anti-apoptotic protein Bcl-2, and pro-apoptotic protein Bax are shown in the figure. Detailed Implementation

[0025] The following examples are provided to enable those skilled in the art to better understand the present invention. It should be noted that the following examples do not constitute a limitation on the scope of protection claimed by the present invention, but are merely illustrative examples. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained by known existing methods. It should be noted that the figures and figures (continued) can be considered as a single overall diagram.

[0026] Raddeeanin A (RDA) is a triterpenoid saponin isolated from Cyperus rotundus (also known as anemone, or two-pointed cyperus). The earliest record of its use in treating breast cancer is found in the ancient medical text *Yifang Gekuo*. Modern pharmacological studies have confirmed that RDA possesses significant antitumor, anti-inflammatory, antipyretic, analgesic, anticonvulsant, and sedative effects. Current experimental studies show that RDA primarily exhibits inhibitory effects on tumors; its pharmacological effects in the treatment of Alzheimer's disease have not yet been reported. In this invention, we demonstrated the neuroprotective effect of RDA on β-amyloid-related proteins.

[0027] More specifically, we demonstrated the effects of cyperus rotundus extract A on amyloid precursor protein (APP), β-secretase (BACE1) activity, and β-amyloid protein (Aβ), as well as its effects on the behavior and pathology of the 3xTg-AD Alzheimer's disease model mice. This invention employed various biological assays on β-amyloid protein (Aβ), such as Western blot analysis, β-secretase activity assays, and dot matrix assays, confirming the effect of cyperus rotundus extract A in attenuating β-amyloidosis in nerve cells. Animal experiments confirmed that cyperus rotundus extract A improved learning and memory impairments and neuropathological defects in the hippocampus of 3xTg-AD model mice.

[0028] In conclusion, Cyperus rotundus extract A can play an important role in the development of prevention, improvement and treatment methods for Alzheimer's disease.

[0029] Example 1

[0030] To evaluate the effect of cyperus oleracea extract A on β-amyloidosis, molecular docking was first performed using Glide 9.1. The chemical structure of cyperus oleracea extract A and the calculated docking structure of amyloid precursor protein (APP) are shown below. Figure 1 As shown in A and 1B. PCR and immunoblotting results confirmed that, compared with the control treatment ( Figure 1 Compared to F, 1G, and 1H, treatment with cyperus rotundus extract A for 24 h significantly reduced the gene and protein levels of amyloid precursor protein (APP) in U87 cells. Figure 1 In I, the activity of β-secretase (BACE1) decreased in a dose-dependent manner after treatment with cyperus rotundus A.

[0031] β-amyloid protein (Aβ) is a hydrolysis product of β precursor protein (APP). APP is processed via an amyloidogenic pathway, meaning that APP is sequentially cleaved by β-secretase (BACE1) and γ-secretase to produce Aβ, which is prone to self-aggregation and is toxic.

[0032] β-amyloid protein (1-42) (Aβ42) is neurotoxic and is a major component of amyloid plaques in the brains of Alzheimer's disease patients.

[0033] U87 cells were treated with β-amyloid protein (1-42) (Aβ42) (30 μM) and cyperus tinctoria extract A for 24 h. Dot analysis showed that cyperus tinctoria extract A reduced the levels of Aβ oligomers and total Aβ ( ). Figure 1 The level of J).

[0034] To confirm the effective dose of cytotoxicity of cypermethrin A in U87 cells, crystal violet staining and LDH determination were used. Figure 1 As shown in C, 1D, and 1E, 2–6 μM of cyperus rotundus A showed no significant cytotoxicity in cells.

[0035] Example 2

[0036] Numerous studies have shown that the aggregation of Aβ monomers can form neurotoxic amyloid fibers, which directly damage neurons. To assess the ability of cyperus oleracea extract A to inhibit Aβ fibrosis, ThT fluorescence detection was performed.

[0037] β-amyloid protein (1-42) (Aβ42) was incubated with cyperus rotundus extract A for 1, 2, 3, 4, and 5 days, respectively. The wavelength was 482 nm. Figure 2 A low fluorescent benzothiazole dye (ThT) signal was detected at A), indicating that cyperus oleracea extract A has the effect of inhibiting Aβ fiber formation.

[0038] To further confirm the direct binding affinity of cyperus oleracea extract A to the Aβ peptide, biomembrane interferometry (BLI) was performed. This was achieved by monitoring the binding tendency of cyperus oleracea extract A to the Aβ peptide immobilized on the surface of the biosensor tip. Upon binding of cyperus oleracea extract A to the Aβ peptide, the optical thickness of the biosensor tip increased, which was reflected by the shift value Δλ in the interference spectrum. These interactions were quantified as follows: Figure 2 The binding or dissociation rates are shown in the kinetic binding sensor plots for increasing concentrations of B and 2C. The binding / dissociation curves for cyperin A show a dose-dependent increase in the optical thickness (nm) of the sensor layer, indicating that cyperin A binds directly to the Aβ peptide.

[0039] Further analysis using Forte BIO software yielded a kinetic constant (Kd) of cyperus rotundus A of 27.63 μM. Figure 2 D). Our research group previously reported a novel application of UHPLC-QTOF-MS / MS in identifying β-amyloid (1-42) (Aβ42) fibrosis inhibitors, but... Figure 2 E and 2F showed that after incubation with 30 μM β-amyloid protein (1-42) (Aβ42) alone, the peak area of ​​the extracted ion chromatography (EIC) of cyperus rotundus A was significantly reduced.

[0040] Example 3

[0041] The Morris water maze test is an indicator of working memory and spatial memory in mice. Mice participating in the water maze test must have normal, undamaged eyes, normal limb motor function, and no wounds. The water maze used for testing mice has a diameter of 1.5m. Before testing AD model mice, an appropriate amount of edible titanium dioxide is added to the water to improve the contrast between the animals and the background. The platform diameter is 10cm, and the platform plane is 2cm underwater. Different colored and shaped markers are attached to the side walls of the water maze to help the mice remember the relative positions of the platforms. Indoor lighting avoids direct light shining onto the water surface. A camera is positioned above and in the center of the water maze. The camera's focus and resolution are adjusted to identify the mouse's movement. The camera is connected to a computer, and the camera's tracking system records and analyzes the mouse's movement trajectory.

[0042] The mouse water maze experiment consists of two phases: a training phase and a detection phase. In the training phase, mice undergo five consecutive days of training to memorize the platform locations using cues from surrounding landmarks. During these five days, mice are placed into the water from the quadrant opposite the platform each day. When placing the mouse against the side wall of the pool, ensuring its back faces the wall, the software is activated and records the time for 90 seconds. If the mouse climbs onto the platform and stays there for 5 seconds, it is considered to have successfully found the platform, and the timer stops. If the mouse fails to find the platform after 90 seconds, an experimenter guides the mouse to locate it in the water and stay there for 1 minute. The detection phase is conducted on the sixth day, starting at least 24 hours after the previous training session. The platform is removed from the water, and the mouse is placed into the water from the quadrant opposite the platform. The timer is 90 seconds, then the mouse is retrieved, its fur is dried, and it is returned to its cage.

[0043] A quiet environment must be maintained during the experiment. The latency period for mice during the training experiment is the time from when they enter the water until they climb onto the platform. During the exploration experiment, the number of times a mouse moves back and forth between the original platform location and the current platform location is recorded as the "platform crossing count."

[0044] Four- to six-month-old 3×Tg-AD mice were administered bamboo rhizome extract A daily by gavage, and its effects on cognitive function were evaluated. Figure 3 As shown in Figure B, cyperus rotundus extract A had no significant effect on mouse body weight throughout the experimental period. At 4 months of age, 3×Tg-AD mice began to show significant spatial learning and memory impairment compared to WT mice. To investigate the spatial memory and learning ability of 3×Tg-AD mice, the Morris water maze (MWM) test was performed at weeks 5 and 9 after administration of cyperus rotundus extract A. Compared with the model group, donepezil (positive control) treatment reduced the escape latency in AD mice, and cyperus rotundus extract A also significantly reduced the escape latency in a dose-dependent manner. During training, five groups of mice ( Figure 3There was no significant difference in swimming speed and total swimming distance in the E group, indicating that cyperus rotundus extract A did not affect the kinetic behavior of mice. After plateau removal on day 6 of the MWM test, compared with the donepezil and cyperus rotundus extract A treatment groups ( Figure 3 Compared to F), the escape latency time was prolonged in the model group. Furthermore, treatment with cyperus rotundus extract A and donepezil significantly increased the 5-day training period ( Figure 3 The number of times G and 3H cross the a3 quadrant where the platform is located. Similarly, in the MWM test ( Figure 3 On day 6 of I), there was no significant difference in swimming speed among the mice in each treatment group, demonstrating that Cyperus rotundus extract A improved the learning and memory deficits in 3×Tg-AD mice.

[0045] Cyperus rotundus extract A can function as the sole active ingredient or in combination with other active ingredients. It can be administered orally or in non-oral formulations. Oral formulations can be one or more of the following: capsules, tablets, oral liquids, granules, pills, powders, elixirs, or ointments. Non-oral formulations can be one or more of the following: injections, creams, patches, ointments, or sprays. Injections can be administered subcutaneously, intramuscularly, or intravenously.

[0046] Example 4

[0047] A portion of Alzheimer's patients have a genetic predisposition, so by transferring the DNA sequence of genes related to the disease into mice, a congenital Alzheimer's model can be established. Currently, there are single-transgenic, double-transgenic, and triple-transgenic animals.

[0048] Single transgenic animals mainly involve introducing the human PS1 gene into mice. This gene controls the production of progerin, which promotes aging. Double transgenic animals, such as APP / PS1 transgenic mice

[55] , are currently recognized by scholars at home and abroad as Alzheimer's model animals, and are the most widely used and mature. The AP gene is a human precursor protein gene that can promote the production of APP, which in turn promotes the deposition of Aβ. Combined with the effect of progerin, this transgenic animal already has mild symptoms of Alzheimer's disease at 6 to 7 months of age.

[0049] In the past two years, three transgenic animals have been established, which are transgenic animals with the addition of genes that promote tau protein phosphorylation, such as the 3xTg transgenic model. Because the pathogenesis of Alzheimer's disease is complex, the 3xTg transgenic model can more comprehensively simulate Alzheimer's disease. In this experiment, to study the pathological defects in AD mice, the effects of cyperus rotundus extract A on the cerebral cortex and hippocampus of 3×Tg-AD mice were assessed using hematoxylin and eosin (H&E) staining and Nissl staining.

[0050] H&E staining includes: dewaxing paraffin sections: xylene I for 30 min; xylene II for 30 min (at this point, the tissue is transparent and cannot be observed); anhydrous ethanol I for 3 min; anhydrous ethanol II for 3 min; 95% ethanol I for 3 min; 95% ethanol II for 3 min; 80% ethanol for 2 min; 70% ethanol for 2 min; rinsing with tap water; rinsing once with purified water. After dewaxing and rehydration, the sections are immersed in hematoxylin staining solution; rinsed with tap water; immersed in differentiation solution for 30 s; quickly transferred to tap water; immersed in blueing solution for at least 2 min, at which point the sections will show a faint blue-purple color; rinsed with purified water; stained in (water-soluble) eosin staining solution for 2 min; after staining, quickly transferred to anhydrous ethanol I to wash away excess stain; then transferred to anhydrous ethanol II for further washing and dehydration for 3 min; the sections are cleared in xylene for 3 min; the sections are removed and mounted with neutral resin. After mounting, the slides should be placed on a flat surface and left to stand for at least 2 days before being transferred to the slide box for later use.

[0051] Nissl staining involves: centrifuging 5 mL of Nissl stain solution beforehand and collecting the supernatant to avoid precipitate affecting the results. Using a pipette, add a few drops of Nissl stain solution to completely cover the brain slice. Incubate the brain slice at 37°C for 5 minutes, discard the stain solution, rinse with double-distilled water for a few seconds, then dehydrate, clear, and mount the slice as with H&E staining. Note that when rinsing with double-distilled water, do not rinse the brain slice directly; instead, rinse the area around the slice to remove excess stain. After mounting, store the slice away from light to prevent fading.

[0052] H&E staining results showed that neurons in the WT group were intact, regular, and morphologically normal. Neuronal nuclei in the cerebral cortex and hippocampal CA3, CA1, and DG regions were clearly stained. In contrast, neurons in the model group showed shrinkage, loosening, disordered arrangement, and altered contours and morphology. Furthermore, neuronal nuclei in the model group were fragmented or missing. Notably, these pathological defects were observed after treatment with Cyperus rotundus extract A (…). Figure 4 A) Relief is subsequently achieved. Under pathological conditions, Nissl bodies, subcellular structures found in nerve cell bodies and dendrites undergo severe alterations or loss within the cell. If the neuron successfully repairs the damage, Nissl bodies will gradually reappear and restore their characteristic intracellular distribution. Figure 4 B-4F showed that Nissl bodies were significantly lost in the model group. Administration of cyperus rotundus extract A or donepezil could reduce the loss of Nissl bodies in the cortex and the CA3, CA1, and DG regions of the hippocampus.

[0053] Example 5

[0054] APP is highly expressed in the central nervous system (CNS). APP is sequentially cleaved by β-secretase and γ-secretase to generate Aβ. In Alzheimer's disease (AD), Aβ is a major component of senile plaques, and Aβ oligomers are a primary cause of neuronal damage due to their high toxicity. To investigate whether cyperus rotundus extract A could alleviate the levels of APP and Aβ oligomers, immunoblotting and dot blot analysis were performed on whole-brain and hippocampal lysates from mice. Figure 5 A-5F analysis showed that cyperus rotundus extract A reduced APP, Aβ oligomer, and total Aβ levels in the whole brain and hippocampus. In conclusion, cyperus rotundus extract A may exert a neuroprotective effect in the AD transgenic mouse model by reducing APP and Aβ levels.

[0055] The immunoblotting method for detecting protein content includes the following steps:

[0056] 1. Mounting the plates: Align the two glass plates and clamp them together. Hold them vertically on the frame and wait for the adhesive to be applied. (The bottom edges of the two glass plates must be parallel to prevent adhesive leakage).

[0057] 2. Prepare the SDS-PAGE separating gel (if using a 1.5mm glass plate, one gel requires 7.5ml, and two gels require 15ml). Before adding TEMED, mix the other components thoroughly (mix gently to avoid air bubbles). After adding TEMED, shake well immediately and pour the gel immediately.

[0058] 3. When pouring the glue, use a 1ml pipette to slowly pour the glue along the edge of the glass plate, avoiding air bubbles. The glue level should rise slightly above the protruding edge of the clamp. Then, fill the top part of the glue with 70% ethanol for liquid sealing. (Add the ethanol slowly to avoid deforming the glue).

[0059] 4. After about 20 minutes, a clear refractive line will appear between the ethanol and the gel, indicating that the gel has solidified. Pour off the ethanol from the top of the gel, carefully absorb the remaining ethanol with filter paper strips, and allow it to air dry.

[0060] 5. After about 20 minutes of concentrated gel solidification, hold the comb by both sides with your hands, keeping it vertical and horizontal, and gently pull the comb out. (To preserve the gel, keep it moist and store it in a refrigerator at 4°C for short-term preservation.)

[0061] 6. Protein Sample Loading: Remove the protein samples from the -80℃ freezer beforehand and heat them in a 95℃ metal bath for 5 minutes. Mix thoroughly and set aside. If there is sediment at the bottom, centrifuge, collect the supernatant, and remove the sediment. Clamp the gel securely, ensuring no leakage, and place it in the electrophoresis tank (according to the correct red and black markings). Add an appropriate amount of 1× running buffer and check for leakage. Use a pipette to carefully load the sample into the wells of the gel, being careful to avoid overflow. Leave a well for the protein marker; use its bands to determine the molecular weight of the target protein.

[0062] 7. Set a constant voltage of 80V during electrophoresis. When the protein enters the separating gel, adjust the voltage to 120V until the electrophoresis is complete.

[0063] 8. Wet transfer is used. A constant current of 300mA is set during transfer. A low temperature must be maintained during transfer, so the transfer process should be conducted on ice. The transfer buffer also needs to be prepared in advance and cooled at 4°C. High temperatures can easily deform the gel, resulting in uneven bands. The electroporation time depends on the molecular weight of the target protein; the smaller the molecular weight, the shorter the required electroporation time, and vice versa.

[0064] 9. After the transfer is complete, place the membrane in a 5% BSA (bovine serum albumin) blocking solution and block at room temperature for 1 hour.

[0065] 10. Place the membrane in the antibody containing the target protein or internal reference protein, and incubate overnight on a shaker at 4°C.

[0066] 11. First, bring the membrane to room temperature, rinse three times with 1×TBST for 10 minutes each time, add the secondary antibody and incubate at room temperature for 1 hour, then rinse three times with 1×TBST for 10 minutes each time. Shake the membrane on a shaker during rinsing.

[0067] 12. Finally, develop using chemiluminescence.

[0068] Example 6

[0069] Astrocytes are the most abundant cell type in the central nervous system, playing a crucial role in maintaining normal central nervous system physiological functions, such as providing nutrients to neurons, regulating blood-brain barrier permeability, modifying synaptic signals, and maintaining the neuronal microenvironment. In Alzheimer's disease (AD), adenosine beta (Aβ) activates astrocytes. These reactive astrocytes produce pro-inflammatory cytokines, leading to Aβ accumulation due to decreased Aβ uptake and clearance rates. Astrocytes in the central nervous system contain glial fibrillary acidic protein (GFAP), a primary intermediate filament protein constituting the astrocyte cytoskeleton and serving as a marker of astrocyte activity. GFAP expression is significantly increased in activated astrocytes compared to quiescent astrocytes. Upregulation of GFAP in astrocytes is also a typical marker of AD pathology.

[0070] To investigate the effects of cyperus rotundus extract A on astrocytes, this invention utilizes immunohistochemical (IHC) staining on the DG region (…). Figure 6 The number of GFAP-positive cells in *Cyperus rotundus* A was visualized and quantified. Results showed that treatment with *Cyperus rotundus* A and donepezil reduced the number of GFAP-positive cells. Immunoblot analysis confirmed that *Cyperus rotundus* A and donepezil significantly reduced GFAP (…). Figure 6 The levels of B-6C (a type of cyperus oleracea extract) suggest that cyperus oleracea extract A inhibits the activation of astrocytes in 3×Tg-AD mice. To investigate the effect of cyperus oleracea extract A on neuronal apoptosis, this invention detected the expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2. Figure 6 D-6H showed that cyperus rotundus extract A and donepezil downregulated the level of the pro-apoptotic protein Bax in mouse brain tissue and upregulated the level of the anti-apoptotic protein Bcl-2, suggesting that they have anti-apoptotic effects in 3×Tg-AD mice.

[0071] Immunofluorescence staining (ICC) includes the following steps: Dewaxing paraffin sections: xylene for 30 min (at this point the tissue is transparent and cannot be observed); 100% ethanol for 5 min; 95% ethanol for 5 min; 85% ethanol for 5 min; 70% ethanol for 5 min; washing with tap water; rinsing once with purified water. After dewaxing and rehydration, antigen retrieval is performed: cross-linked antigenic epitopes in the tissue are opened in a high-temperature citrate buffer solution, exposing normal antigenic epitopes. The tissue sections are placed in an incubation box and heated in a water bath at 95°C for 30 min. After retrieval, the sections are cooled to room temperature before further processing.

[0072] Remove the slides and immerse them in 3% hydrogen peroxide solution for 10 min, then wash once in Tris buffer. Add 5% BSA to the tissue and incubate at room temperature for 1 h to block non-specific binding sites. Remove the sheep serum from the tissue and add antibody solution, incubating overnight at 4°C. Recover the primary antibody and wash three times in TBST. Add secondary antibody to the tissue and incubate at room temperature in the dark for 1 h, then wash the slides with TBST. Air dry until semi-dry and mount.

[0073] In summary, identifying effective natural components for β-amyloidosis may be a promising research direction for the prevention and treatment of Alzheimer's disease (AD). The results of this invention confirm that Cyperus rotundus extract A, as a potential novel traditional Chinese medicine preparation for treating AD, reduced β-amyloid protein deposition and improved learning and memory deficits in a 3×Tg-AD mouse model, and revealed multiple neuroprotective effects through pathological studies. These findings provide a scientific basis for the potential clinical use of Cyperus rotundus extract A in the prevention or treatment of AD.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of cyperus oleracea extract A or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention, improvement or treatment of β-amyloidosis-driven Alzheimer's disease.

2. The application according to claim 1, characterized in that, The application of the bamboo rhizome cyperus extract A as the sole active ingredient in the preparation of the drug.

3. The application according to claim 1, characterized in that, The use of the bamboo rhizome cyperus extract A together with other active ingredients in the preparation of the drug.

4. The application according to claim 3, characterized in that, The other active ingredients include donepezil.

5. The application according to claim 3, characterized in that, The drug is selected from oral preparations.

6. The application according to claim 5, characterized in that, The oral preparation is one or more of the following: capsules, tablets, oral liquids, granules, pills, powders, elixirs, or ointments.

7. The application according to any one of claims 1 to 6, characterized in that, The target audience for this application is mammals.

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