Compound granules for resisting Alzheimer's disease and Parkinson's disease and preparation method thereof

Abstract

The invention relates to the technical field of medicines, in particular to a composition for resisting Alzheimer's disease and Parkinson's disease and a preparation method thereof. The composition disclosed by the invention has obvious capability of inhibiting AChE and MAO-B, can obviously improve the memory of mice, and has obvious effects of resisting Alzheimer's disease and Parkinson's disease.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a compound granule for resisting Alzheimer's disease and Parkinson's disease and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) and Parkinson's disease (PD) are neurodegenerative diseases with insidious onset. After the age of 60, the incidence and severity of the disease gradually increase with age. At present, it is one of the third leading causes of death in the elderly after cardiovascular disease and tumors. The number of patients with Alzheimer's and Parkinson's disease worldwide is expected to increase to more than 150 million by 2050. According to statistics from the Chinese Center for Disease Control and Prevention, the number of patients is expected to exceed 25 million in 2040. Alzheimer's disease and Parkinson's disease seriously threaten human health and also bring serious economic burdens to families. It is necessary to increase investment to develop effective drugs and preventive measures. At present, clinical treatment is mainly based on synthetic drugs, but the onset of degenerative diseases is relatively complicated, and synthetic drugs also have certain adverse reactions. Summary of the invention

[0003] In one aspect, the present invention provides a composition for combating Alzheimer's disease and Parkinson's disease, the composition comprising chitosan oligosaccharide, myricetin and astaxanthin.

[0004] As a preferred technical solution, chitosan oligosaccharide in the composition is the monarch drug, which has the effects of inhibiting cholinesterase and Aβ-induced hippocampal neuron apoptosis, scavenging free radicals, reducing oxidative stress damage, protecting the intestinal barrier, and improving AD and PD status; myricetin is the ministerial drug, and the hydroxyl group in its structure can bind to ChE, MAO-B and Aβ core structure, thereby inhibiting ChE and MAO-B, inhibiting the production of Aβ and reducing its toxic effects, so as to achieve the effect of preventing and treating AD and PD; astaxanthin is the guiding drug, which helps the monarch drug and the ministerial drug to inhibit the effects of ChE and MAO-B and reduce the level of Aβ, resist oxidative stress, and regulate intestinal microorganisms.

[0005] As a preferred technical solution, the chitosan oligosaccharide: myricetin: astaxanthin = 10:10:1 to 10:10:9 (weight ratio).

[0006] As a further preferred technical solution, the chitosan oligosaccharide: myricetin: astaxanthin = 10:10:3 (weight ratio).

[0007] On the other hand, the present invention provides a method for preparing a composition for treating Alzheimer's disease and Parkinson's disease, characterized in that it comprises the following steps:

[0008] (1) weighing chitosan, myricetin, and astaxanthin in proportion, and mixing them to form a mixture;

[0009] (2) The above mixture is stirred and ground, and then ultrasonically mixed with an ultrasonic device to prepare a composite material.

[0010] As a preferred technical solution, the chitosan is prepared by the following method: preparing a chitosan solution by an alkali method, adding chitosanase to dissolve it, and separating it by preparative high performance liquid chromatography.

[0011] As a preferred technical solution, the myricetin is prepared by the following method: taking bayberry, peeling the kernel, drying, crushing, extracting with an organic solvent, drying with anhydrous sodium sulfate, and separating by preparative high performance liquid chromatography.

[0012] As a preferred technical solution, the astaxanthin is prepared by the following method: the shrimp skin of Antarctic krill is dried and crushed, extracted with an organic solvent, dried with anhydrous sodium sulfate, and separated by preparative high performance liquid chromatography.

[0013] In still another aspect, the present invention provides use of the composition in preparing a medicament for preventing / treating Alzheimer's disease.

[0014] In another aspect, the present invention provides use of the composition in preparing a drug for preventing / treating Parkinson's disease.

[0015] In another aspect, the present invention provides use of the composition in preparing a drug for preventing / treating Alzheimer's disease and Parkinson's disease.

[0016] The invention obtains "chitooligosaccharide + astaxanthin + myricetin" with the function of regulating human body by extracting and separating the natural product "crab shell + shrimp skin or seaweed + bayberry kernel" to inhibit AChE, BuChE and MAO-B, and provides a composition for preventing and treating Alzheimer's disease and Parkinson's disease, so as to improve or enhance people's health level and quality of life.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) Using Zhejiang Province's (marine resources) specialty food and medicine - crab shells, shrimp skins or seaweed as the main raw materials, combined with bayberry kernels, we developed a "chitooligosaccharide + astaxanthin + myricetin" granule powder with the function of preventing and treating Alzheimer's disease and Parkinson's disease. The product has an inhibitory effect on acetylcholinesterase, butyrylcholinesterase, and monoamine oxidase B, and shows anti-Alzheimer's disease and Parkinson's disease activity, which is the first reported.

[0019] (2) The present invention designs and prepares a multi-component small molecule easily absorbed composition, which makes up for the nutritional deficiencies of a single plant or animal in preventing or treating nutrition, and can enhance or improve people's health level and quality of life.

[0020] (3) The present invention makes full use of marine waste and plant waste to prepare a new type of "chitosan oligosaccharide + astaxanthin + myricetin" composition, which greatly reduces environmental pollution and diversifies nutritional categories, and will have unlimited prospects.

[0021] The composition of the present invention has the advantages of clear chemical composition, clear pharmacological action, small dosage, quick onset of effect, easy control of product quality, and product appearance and taste that are easily accepted by consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the docking result of chitosan oligosaccharide and AChE.

[0023] Figure 2 This is the docking result of myricetin and AChE.

[0024] Figure 3 This is the docking result of astaxanthin and AChE.

[0025] Figure 4 This is the docking result of chitosan oligosaccharide and MAO-B.

[0026] Figure 5 This is the docking result of myricetin and MAO-B.

[0027] Figure 6 This is the docking result of astaxanthin and MAO-B. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the accompanying drawings.

[0029] Example 1 Preparation of compound granules and activity determination

[0030] Preparation of chitosan oligosaccharides (preparation of chitosan oligosaccharides by degradation of chitosan): ① Preparation of chitosan by alkaline method, crab shells (purchased from Zhoushan aquatic product market) were dried and crushed into powder. Weigh 10g of powder and add 6% NaOH aqueous solution (W / V) in a round-bottom flask and mix at a ratio of 1:20, and extract at 90℃ for 2h (deproteinization). The extract was cooled and centrifuged, and the filtrate was collected and washed with water until the filtrate was neutral. Then 6% NaOH (W / V) was added to the filtrate to make it alkaline, and it was extracted at 110℃ for 6h (deacetylation), and the extract was cooled, centrifuged and washed with water until the pH was 7.0. The extract was adjusted to acidity by adding 10% acetic acid (W / V), and extracted twice at 90℃, each time for 2h. After the extract was cooled and centrifuged, the filtrate was combined, the pH was adjusted to 9.0 with 1mol / L NaOH, and it was allowed to stand overnight and centrifuged. The precipitate was washed with water until it was neutral, and then washed twice with 75% ethanol, and freeze-dried in a vacuum to obtain crude chitosan. ② Enzymatic hydrolysis of chitosan: Add chitosanase to 10% chitosan solution (W / V), mix well and react at 50°C for 10 hours. After the hydrolysis is completed, inactivate the enzyme in a boiling water bath, centrifuge, collect the precipitated chitosan, wash with water to pH 7, concentrate and dry the supernatant collected by centrifugation, and use preparative high performance liquid chromatography to separate and purify pure (using standard chromatography for comparison) chitosan oligosaccharides.

[0031] Preparation of astaxanthin: Antarctic krill (purchased from Zhoushan aquatic product market), take the shrimp skin and dry it, and then crush the dried shrimp shell powder. Accurately weigh 10g of uniform shrimp shell powder, add different types of anhydrous ethanol according to a certain material-liquid ratio (1:20, W / V), and extract at a constant temperature of 50°C for 3h. After the extract is cooled, the solvent is evaporated, a certain amount of water is added to mix, and extracted with dichloromethane, the extracts are combined, dried with anhydrous sodium sulfate, dichloromethane is removed, and preparative high performance liquid chromatography is used to separate and purify to obtain pure astaxanthin.

[0032] Preparation of myricetin: Take fresh bayberry (purchased from Zhoushan Farmer's Market), peel off the kernel, place it in a 40°C oven overnight to evaporate the water, grind the bayberry kernel into powder with a grinder, and pass it through a 40-mesh sieve. Weigh 10g of bayberry kernel powder, put it into a round-bottom flask, add 80% ethanol solid-liquid ratio (1:20, W / V), and use ultrasonic assisted extraction. The extraction time is 2h, the extract is filtered and concentrated to obtain a crude solid. Then take the crude product and add water to mix, extract with ethyl acetate, combine the extracts, dry with anhydrous sodium sulfate, remove ethyl acetate, and use preparative high performance liquid chromatography to separate and purify to obtain a pure product. Standard products: Myricetin and astaxanthin were purchased from Maclean Company, and chitosan oligosaccharide was purchased from Tokyo Chemical Industry Co., Ltd.

[0033] Preparation of compound granules with different components:

[0034] 1. Chitosan oligosaccharides: myricetin: astaxanthin

[0035] (1) Weigh 5 portions of chitosan oligosaccharide and myricetin using an electronic analytical balance, each with a mass of 1 g, mix them, and place them in a centrifuge tube. Weigh 0.1 g, 0.3 g, 0.5 g, 0.7 g, and 0.9 g of astaxanthin, and add them to the centrifuge tube in order to prepare a mixture of chitosan oligosaccharide: myricetin: astaxanthin of 10:10:1, 10:10:3, 10:10:5, 10:10:7, and 10:10:9.

[0036] (2) stirring and grinding the above mixture, and then ultrasonically mixing the mixture to prepare composite granules.

[0037] 2. Astaxanthin: Chitosan Oligosaccharide: Myricetin

[0038] (1) Weigh 5 parts of astaxanthin and chitosan oligosaccharide using an electronic analytical balance, each weighing 1 g, mix them and put them in a centrifuge tube. Weigh 0.1 g, 0.3 g, 0.5 g, 0.7 g and 0.9 g of myricetin and add them to the centrifuge tube in that order to make a mixture of astaxanthin: chitosan oligosaccharide: myricetin in the ratio of 10:10:1, 10:10:3, 10:10:5, 10:10:7 and 10:10:9.

[0039] (2) The mixture is stirred and ground, and then ultrasonically mixed with an ultrasonic device to prepare composite granules.

[0040] 3. Astaxanthin: Myricetin: Chitooligosaccharide

[0041] (1) Using an electronic analytical balance, 5 parts of astaxanthin and myricetin were weighed, each with a mass of 1 g, and mixed. The mixture was placed in a centrifuge tube. 0.1 g, 0.3 g, 0.5 g, 0.7 g, and 0.9 g of chitosan oligosaccharide were weighed and added to the centrifuge tube in order to prepare a mixture of astaxanthin: myricetin: chitosan oligosaccharide of 10:10:1, 10:10:3, 10:10:5, 10:10:7, and 10:10:9.

[0042] (2) The mixture is stirred and ground, and then ultrasonically mixed with an ultrasonic device to prepare composite granules.

[0043] Table 1

[0044]

[0045] Example 2 Inhibitory activity of composite granules of different components on cholinesterase

[0046] Preparation of main reagents

[0047] (1) Preparation of 0.1 mol / L phosphate buffer solution (PBS):

[0048] ①2.84gNa2HPO4 plus 100mL distilled water, take 94.7mL of this part;

[0049] ② Add 25 mL of distilled water to 0.6 g of NaH2PO4, and take 5.3 mL of this part;

[0050] ③ Add the two parts together to make a 0.2 mol / L PBS solution (100 mL);

[0051] ④ Dilute the solution by half to obtain 0.1 mol / LPH=8.0 phosphate buffer.

[0052] (2) Preparation of 4% SDS: Accurately weigh 2 g SDS (sodium dodecyl sulfate), dissolve it completely in 50 mL of distilled water, and place it at room temperature for later use;

[0053] (3) Preparation and concentration dilution of the sample to be tested:

[0054] ① Mother solution: add 1 mL DMSO to 2 mg of compound;

[0055] ②100U / mL concentration solution: Take 10μL of the stock solution and add it to 990μL PBS for dilution.

[0056] (4) 0.1U / mL acetylcholinesterase (AChE) solution:

[0057] ① 1U / mL stock solution: weigh 5mg of acetylcholinesterase and add 1mL of distilled water to dissolve;

[0058] ②0.1U / mL solution: Take 100μL of the stock solution and add it to 900μL of PBS.

[0059] (5) 0.1U / mL butyrylcholinesterase (BuChE) solution:

[0060] ① 2U / mL stock solution: weigh 0.25mg of butyrylcholinesterase and add 0.5ml of distilled water to dissolve;

[0061] ②0.1U / mL solution: Take 50μL of the stock solution and add it to 950μL of PBS.

[0062] (6) 1 mM iodine thioacetylcholine (ATCI) solution: Weigh 0.0105 g of iodine thioacetylcholine powder, add 5 mL of PBS, stir thoroughly to dissolve, and store at 4°C in the dark.

[0063] (7) 1 mM iodothiobutyrylcholine (BTCI) solution: Weigh 0.012 g of iodothiobutyrylcholine powder, add 5 mL of PBS, stir thoroughly to dissolve, and store at 4°C in the dark.

[0064] (8) 100 U / mL Donepezil: 0.01 mol / L stock solution: Accurately weigh 0.00416 g of Donepezil and add 1 mL of DMSO to dissolve. ② Pipette 10 μL of the stock solution and add 990 μL of PBS to dilute the concentration to 100 U / mL.

[0065] (9) 100 U / mL tacolin: 0.01 mol / L stock solution: Accurately weigh 0.00235 g of tacolin and add 1 mL of DMSO to dissolve. Pipette 10 μL of the stock solution and add 990 μL of PBS to dilute the concentration to 100 U / mL.

[0066] (10) Color developer 5,5-dimethylbis-2-nitrobenzoic acid (DTNB): weigh 0.02 g of DTNB, add 5 mL of PBS buffer, and store in a refrigerator at 4°C until use.

[0067] Experimental methods

[0068] (1) Inhibition rate of acetylcholinesterase

[0069] Table 2 Experimental method for acetylcholinesterase inhibition rate

[0070]

[0071] (2) Inhibition rate of butyrylcholinesterase

[0072] Table 3 Experimental method for butyrylcholinesterase inhibition rate

[0073]

[0074] Inhibition rate (%) = [(blank control group - blank background) - (experimental sample or positive control group - sample background)] / (blank control - blank background) × 100%

[0075] Experimental Results

[0076] The inhibitory effects of the compound granules prepared by "chitosan oligosaccharide + myricetin + astaxanthin" on AChE and BuChE activities are shown in Table 4. As can be seen from Table 4, the compound granules prepared by "chitosan oligosaccharide + myricetin + astaxanthin" can significantly inhibit AChE, while the inhibition on BuChE is relatively weak. Studies have shown that chitosan oligosaccharide: myricetin: astaxanthin = 10:10:3 shows the strongest inhibitory effect on AChE. At the same time, chitosan oligosaccharide in this compound granule is the main drug, which can scavenging free radicals, reduce oxidative stress damage, protect intestinal barrier, and improve AD and PD status induced by cholinesterase and Aβ. Myricetin is the ministerial drug, and the hydroxyl group in its structure can bind to ChE, MAO-B and Aβ core structure, thereby inhibiting ChE, MAO-B, inhibiting the production of Aβ and reducing its toxicity, so as to achieve the effect of preventing and treating AD and PD. Astaxanthin is the assistant drug, helping the main drug and ministerial drug to inhibit the effects of ChE and MAO-B and reduce the level of Aβ, fight oxidative stress, and regulate intestinal microorganisms. The optimal dose is chitosan oligosaccharide: myricetin: astaxanthin = 10:10:3.

[0077] Table 4 Experimental results of the compound granules inhibiting AChE and BuChE

[0078] Astaxanthin: Chitosan Oligosaccharide: Myricetin 10:10:1 10:10:3 10:10:5 10:10:7 10:10:9 Acetylcholinesterase inhibition rate 13％ 39％ 38％ 46％ 52％ Butyrylcholinesterase inhibition rate 19％ 7％ 15％ 19％ 20％ Chitosan oligosaccharide: Myricetin: Astaxanthin 10:10:1 10:10:3 10:10:5 10:10:7 10:10:9 Acetylcholinesterase inhibition rate 69％ 72％ 61％ 55％ 57％ Butyrylcholinesterase inhibition rate 22％ 45％ 19％ 38％ 15％ Myricetin: Astaxanthin: Chitooligosaccharide 10:10:1 10:10:3 10:10:5 10:10:7 10:10:9 Acetylcholinesterase inhibition rate 56％ 54％ 48％ 48％ 36％ Butyrylcholinesterase inhibition rate 47％ 13％ 19％ 24％ 3％

[0079] Example 3 Molecular docking results

[0080] Steps and methods: PyRx 0.8 is a free virtual screening software that integrates AutoDock and Vina programs and can perform computational drug discovery on the Windows platform. The protein crystal structure of human AChE comes from the Protein Crystal Database (PDB ID: 4BDS) with a resolution of The pocket is the tacrine small molecule ligand center. The initial structure of the protein crystal is processed through the Yinfu cloud computing platform. The optimized protein crystal structure is first re-docking with its co-crystallized ligand and then molecularly simulated docking with the parent structure containing chitosan oligosaccharides, myricetin, and astaxanthin to evaluate the inhibitory effects of the three substances at the molecular level.

[0081] The docking results of chitosan oligosaccharide, myricetin, astaxanthin and AChE are shown in Figure 1-3The results showed that the hydroxyl groups in the structure of chitosan oligosaccharide mainly combined with HIS-447, SER-125 and ASP-74 in the active center of AChE by hydrogen bonding, the O atom formed a hydrogen bonding interaction with TYR-1249 in the active center of AChE, the hydrogen of -NH2 formed a hydrogen bonding interaction with ASN-87 in the active center of AChE, and the docking binding energy between chitosan oligosaccharide and AChE was -7.5 kcal / mol; the hydroxyl groups in the structure of myricetin mainly combined with TYR-72 and PHE-295 in the active center of AChE by hydrogen bonding, the oxygen atom on the carbonyl group formed a hydrogen bonding interaction with SER-293 and TYR-341 in the active center of AChE, and the docking binding energy between myricetin and AChE was -11.9 kcal / mol; the hydroxyl groups in the structure of astaxanthin formed a hydrogen bonding interaction with ASN-87 in the active center of AChE, and the docking binding energy between astaxanthin and AChE was -9.3 kcal / mol.

[0082] Example 4: Monoamine oxidase inhibitory activity of the composite granules

[0083] Extraction of monoamine oxidase:

[0084] Solution preparation

[0085] (1) PBS solution (PH = 7.6)

[0086] ①0.6gNaH2PO4 dissolved in 25mL distilled water;

[0087] ②2.84gNaHPO4 dissolved in 100mL distilled water;

[0088] (2) Mix 5.3 mL of ① and 94.7 mL of ②;

[0089] (3) 0.3 M sucrose solution: 0.268 g of sucrose solid was dissolved in 100 mL of PBS buffer.

[0090] How to make rat liver

[0091] Cut rat liver into small pieces and rinse the liver surface with pH 7.6 PBS buffer. After washing, take 4g liver, add 20mL 0.3 M sucrose buffer to fully homogenize (use a homogenizer to repeat more than ten times until the bottom is clear to prevent the appearance of mucus blocks later), and then add them to 5mL centrifuge tubes. After balancing, perform low-temperature differential centrifugation and centrifuge at 1000×g for 10min. Aspirate the supernatant, balance the supernatant, centrifuge at 1200×g for 15min, aspirate the supernatant, balance the supernatant, centrifuge at 10000×g for 30min, discard the supernatant, add 4mL phosphate buffer to the precipitate and mix well. The obtained SD rat liver mitochondrial concentrate is the mother solution of monoamine oxidase for the experiment, which is stored in a -80℃ refrigerator.

[0092] Solution preparation

[0093] (1) 10% DMSO solution: Take 1 ml of DMSO solution and add 9 ml of deionized water.

[0094] (2) Pargyline Solution

[0095] ① Mother solution: 0.00195 g of paroxysmal and 1 ml of DMSO are added to prepare the mother solution.

[0096] ②100uM concentration solution: take 10ul of the mother solution and add 990ul (10%) DMSO.

[0097] ③ 10uM concentration solution: Take 100ul of the above solution (concentration is 100uM) and add 900ul (10%) DMSO.

[0098] (3) Clorgyline solution

[0099] ① Mother solution: 0.00308 g of clorgyline plus 1 ml of DMSO is prepared into the mother solution.

[0100] ②100uM concentration solution: take 10ul of the mother solution and add 990ul (10%) DMSO.

[0101] ③10uM concentration solution: Take 100ul of solution (concentration is 100uM) and add 900ul (10%) DMSO.

[0102] (4) PBS solution

[0103] ①2.84g Na2HPO4 plus 100ml distilled water, take 94.7ml of this part;

[0104] ② Add 25ml of distilled water to 0.6g NaH2PO4, and take 5.3ml of this part.

[0105] ③ Add the two parts together to make a 0.2 mol / L PBS solution (100 ml).

[0106] (5) Substrate (tyramine): 2.5 mM concentration solution: 0.00343 g tyramine plus 10 ml PBS solution.

[0107] (6) Color developing solution

[0108] ①4U / mL horseradish peroxidase: 1mg horseradish plus 50ml PBS solution;

[0109] ②0.5mM 4-aminoantipyrine: 2.03mg 4-aminoantipyrine plus 10ml PBS solution;

[0110] ③1mM vanillic acid: 1.68mg vanillic acid plus 10ml PBS solution;

[0111] ④ Mix the three in a ratio of 1:1:1 to prepare a color developing solution.

[0112] Preparation of test compounds

[0113] Weigh 2 mg of each test compound and add 1 ml of DMSO to prepare a sample solution.

[0114] Table 5 MAO-B inhibition rate detection method

[0115]

[0116] The experimental results are shown in Table 6. The results show that the compound granules can significantly inhibit MAO-B and show a certain anti-Parkinson's disease effect, among which the dosage ratio of chitosan oligosaccharide: myricetin: astaxanthin = 10:10:3 has the best activity.

[0117] Table 6 Experimental results of compound granules inhibiting MAO-B

[0118] Chitosan oligosaccharide: Myricetin: Astaxanthin 10:10:1 10:10:3 10:10:5 10:10:7 10:10:9 MAO-B inhibition rate 76％ 92％ 76％ 72％ 13％ Myricetin: Astaxanthin: Chitooligosaccharide 10:10:1 10:10:3 10:10:5 10:10:7 10:10:9 MAO-B inhibition rate 70％ 87％ 35％ 52％ 30％ Astaxanthin: Chitosan Oligosaccharide: Myricetin 10:10:1 10:10:3 10:10:5 10:10:7 10:10:9 MAO-B inhibition rate 4％ 26％ 27％ 52％ 72％

[0119] Example 5 Docking of chitosan oligosaccharides, myricetin, and astaxanthin with MAO-B

[0120] Steps and methods: PyRx 0.8 is a free virtual screening software that integrates AutoDock and Vina programs and can perform computational drug discovery on the Windows platform. The protein crystal structure of human MAO-B comes from the Protein Crystal Database (PDB ID: 2V5Z) with a resolution of The initial structure of the MAO-B protein crystal was processed through the Yinfu cloud computing platform. The pocket was the safinamide small molecule ligand center. The optimized protein crystal structure was first re-docking with its co-crystallized ligand and then molecularly simulated docking with the parent structures of chitosan oligosaccharide, myricetin, and astaxanthin to evaluate the inhibitory effect of the synthetic compounds at the molecular level.

[0121] The results of docking chitosan oligosaccharides, myricetin, and astaxanthin with MAO-B showed that: Figure 4-6The hydroxyl groups in the structure of chitosan oligosaccharide mainly hydrogen bond with GLN-206 and ILE-198 in the active center of MAO-B, and the hydrogen of -NH2 forms hydrogen bond interactions with TYR-435, GLY-434 and CYS-172 in the active center of MAO-B. The docking binding energy between chitosan oligosaccharide and MAO-B is -6.7 kcal / mol. The hydroxyl groups in the structure of myricetin mainly hydrogen bond with SER-59, TYR-60 and GLY-434 in the active center of MAO-B, and the oxygen atom on the carbonyl group forms hydrogen bond interactions with TYR-398 in the active center of MAO-B. The docking binding energy between myricetin and MAO-B is -9.8 kcal / mol. The hydroxyl groups in the structure of astaxanthin form hydrogen bond interactions with LEU-56 in the active center of MAO-B, and also bind to amino acid residues TYR-435, TYR-398 and TRP-388. The docking binding energy of astaxanthin is -6.7 kcal / mol. Molecular docking showed that the compound could stably bind to the target.

[0122] Example 6 In vivo experiment

[0123] Dark avoidance test to detect the latency and number of errors of each group of mice

[0124] The preliminary preparation of compound Chinese medicine granules was carried out with reference to the Chinese Pharmacopoeia, and the prepared Chinese medicine granules were subjected to the mouse water maze test to evaluate the pharmaceutical activity. After oral gavage for 2 weeks, on the day of the experiment, in the memory test after medication, compared with the blank group, the latency of mice in the model group was significantly shortened (p<0.01), and the number of errors was significantly increased (p<0.01); compared with the model group, the latency of mice in the 100 and 300 mg / kg groups was significantly prolonged (p<0.01), and the number of errors was significantly reduced (p<0.05, p<0.01); the number of errors of mice in the 50 mg / kg group was more than that of the 100 and 300 mg / kg groups (p<0.05), and the number of errors of mice in the 100 mg / kg grapefruit peel granule group was more than that of the 300 mg / kg group (p<0.05) (Table 7).

[0125] Table 7 Latency and number of errors of mice in each group in the dark avoidance experiment

[0126]

[0127] * p<0.01νs blank group; # p<0.05, ## p<0.01νs model group

[0128] Morris water maze test

[0129] Compared with the blank group, the time for mice in each group to find the platform, the number of times they crossed the platform and the time they stayed in the quadrant showed that the time for mice in the model group to find the platform was significantly increased (p<0.01), the number of times they crossed the original platform was significantly reduced (p<0.01), and the time they stayed in the target quadrant was shortened (p<0.01); compared with the model group, the time for mice in the 100 and 300 mg / kg groups to find the platform was significantly shortened (p<0.05), and the number of times they crossed the platform and the time they stayed in the quadrant were significantly increased (p<0.05); the time for mice in the 50 mg / kg group to find the platform was longer than that in the 100 and 300 mg / kg groups (p<0.01), and the time for mice in the 100 mg / kg group to find the platform was longer than that in the 300 mg / kg group (p<0.05); the number of times mice in the 100 mg / kg group crossed the platform and the time they stayed in the target quadrant were less than those in the 300 mg / kg group (p<0.05, p<0.01), see Table 8.

[0130] Table 8 Water maze experiment observation of the time for each group of mice to find the platform, the number of times they crossed the platform, and the time they stayed in the target quadrant

[0131]

[0132] * p<0.01νs blank group; # p<0.05, ## p<0.01νs model group

[0133] The results showed that compared with the model group, all doses of granules showed significant learning and memory abilities in both the dark avoidance test and the water maze test, and the effect gradually increased with the increase of the dose, suggesting that the effect of granules in improving memory may have a dose-effect relationship.

[0134] The in vivo and in vitro experiments and molecular docking of the present invention show that the compound granules (chitosan oligosaccharide: myricetin: astaxanthin = 10:10:3) are the best doses, which can significantly inhibit AChE and MAO-B, improve the memory of mice, and show the effects of resisting Alzheimer's disease and Parkinson's disease.

[0135] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A composition for treating Alzheimer's disease and Parkinson's disease, characterized in that: The composition comprises chitosan oligosaccharide, myricetin and astaxanthin.

2. The composition for treating Alzheimer's disease and Parkinson's disease according to claim 1, characterized in that: In the composition, chitosan oligosaccharide is the monarch drug, which has the effects of inhibiting the apoptosis of hippocampal neurons induced by cholinesterase and Aβ, scavenging free radicals, reducing oxidative stress damage, protecting the intestinal barrier, and improving the AD and PD states; myricetin is the ministerial drug, and the hydroxyl group in its structure can be combined with ChE, MAO-B and Aβ core structure, thereby inhibiting ChE and MAO-B, inhibiting the production of Aβ and reducing its toxicity, so as to achieve the effect of preventing and treating AD and PD; astaxanthin is the assistant drug, which helps the monarch drug and the ministerial drug to inhibit the effects of ChE and MAO-B and reduce the level of Aβ, resist oxidative stress, and regulate intestinal microorganisms.

3. A composition for combating Alzheimer's disease and Parkinson's disease according to claim 1, characterized in that: The chitosan oligosaccharide: myricetin: astaxanthin = 10:10:1-10:10:9, preferably chitosan oligosaccharide: myricetin: astaxanthin = 10:10:

3.

4. A method for preparing a composition for treating Alzheimer's disease and Parkinson's disease according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) weighing chitosan, myricetin, and astaxanthin in proportion, and mixing them to form a mixture; (2) The above mixture is stirred and ground, and then ultrasonically mixed with an ultrasonic device to prepare a composite material.

5. The composition for treating Alzheimer's disease and Parkinson's disease according to any one of claims 1 to 3, characterized in that: The chitosan is prepared by the following method: preparing a chitosan solution by an alkali method, adding chitosanase to dissolve the chitosan, and separating the chitosan by preparative high performance liquid chromatography.

6. The composition for treating Alzheimer's disease and Parkinson's disease according to any one of claims 1 to 3, characterized in that: The myricetin is prepared by the following method: taking bayberry, peeling the kernel, drying, crushing, extracting with an organic solvent, drying with anhydrous sodium sulfate, and separating by preparative high performance liquid chromatography.

7. The composition for treating Alzheimer's disease and Parkinson's disease according to any one of claims 1 to 3, characterized in that: The astaxanthin is prepared by the following method: the shrimp skin of Antarctic krill is dried and crushed, then extracted with an organic solvent, dried with anhydrous sodium sulfate, and separated by preparative high performance liquid chromatography.

8. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for preventing / treating Alzheimer's disease.

9. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for preventing / treating Parkinson's disease.

10. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for preventing / treating Alzheimer's disease and Parkinson's disease.

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