Asparagine uniform polysaccharide, its preparation method and application in preparing medicine for treating nervous system diseases

Abstract

The application discloses asparagus uniform polysaccharide, a preparation method thereof and application of the asparagus uniform polysaccharide in preparation of a medicine for treating nervous system diseases. The asparagus uniform polysaccharide is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose and fructose. After asparagus is crushed, hydrochloric acid solution is added for decocting, the decocting liquid is cooled and filtered to obtain asparagus polysaccharide extract; the asparagus polysaccharide extract is adjusted in pH value by using NaOH solution, and then is evaporated and concentrated to obtain a flow extract; the flow extract is subjected to alcohol precipitation to obtain alcohol-precipitated crude polysaccharide; the alcohol-precipitated crude polysaccharide is subjected to protein removal by using a Sevag method to obtain asparagus total polysaccharide; the asparagus total polysaccharide is subjected to separation and purification, and then is concentrated and freeze-dried to obtain the asparagus uniform polysaccharide. The preparation method is suitable for industrial production, provides a basis for quality control and standardized production of ACP, and widens the application range of asparagus polysaccharide. The asparagus uniform polysaccharide has the effect of treating nervous system diseases, and provides a basis for developing a potential new medicine for AD.

Description

I. TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine extraction, and particularly relates to a Radix Asparagi uniform polysaccharide, a preparation method thereof and application of the Radix Asparagi uniform polysaccharide in preparation of a medicine for treating nervous system diseases. II. BACKGROUND

[0002] Radix Asparagi is the dried rhizome of Asparagus cochinchinensis (AC) (Lour.) Merr. of Liliaceae. According to Chinese Pharmacopoeia, Radix Asparagi is sweet, bitter and cold in nature, and is attributed to the lung and kidney channels, has the functions of nourishing yin and moistening dryness, clearing the lung and generating fluid, and is used for treating dry cough, sticky phlegm, dry throat and constipation. Radix Asparagi contains various chemical components, including 19 kinds of amino acids such as asparagine, citrulline, serine and threonine, polysaccharide components such as asparagus polysaccharide A, B, C and D, and other chemical components such as polysaccharide protein, glucose and fructose. Pharmacological research results show that Radix Asparagi has the effects of antioxidant, anti-aging, anti-tumor, bacteriostatic and anti-inflammatory, hypoglycemic and antitussive and expectorant.

[0003] Alzheimer's disease (AD) is an irreversible progressive neurodegenerative disease. The pathogenesis of Alzheimer's disease has not been fully elucidated. The main pathological features of AD include the deposition of β-amyloid (Aβ) to form senile plaques, neurofibrillary tangles, chronic inflammatory response of activated and proliferated glial cells, synaptic dysfunction, and neuronal degeneration and death. Inflammation occurs in the surrounding tissues of Aβ deposition. A number of studies have shown that the deposition of Aβ in brain microvessels promotes the occurrence of neuroinflammation in AD patients. In addition, AD is characterized by the expression of activated microglial cells and astrocytes, release of pro-inflammatory cytokines and chemotactic factors. These adhesion molecules and chemotactic factors are involved in the neuroinflammatory cascade. The presence of inflammatory mediators and the increase in complement cascade expression in the brain of AD patients strongly suggest the role of inflammation in the pathogenesis of AD. This complex network of pathways is mainly induced by adhesion molecules and cell chemotactic mediators.

[0004] In recent years, more and more researches on prevention and treatment of neurodegenerative diseases by using traditional Chinese medicine have been carried out. Polysaccharide is a natural macromolecular polymer, usually composed of more than 10 monosaccharide units connected by glycosidic bond in linear or branched chain, and the molecular weight is tens of thousands or even millions. Polysaccharides of natural origin can significantly affect the immune system. Therefore, they are used as immunomodulators in clinical practice. A number of studies have shown that polysaccharides have various biological activities, including anti-inflammatory, antiviral, antioxidant, anticoagulant, antithrombotic, antitumor, anticomplement and hypoglycemic activities. Studies have shown that the processing technology has a significant impact on the physicochemical, functional and microstructure characteristics of asparagus. Carbohydrates are one of the four basic substances that constitute life, and the polysaccharide components of traditional Chinese medicine have various pharmacological activities, which have become the focus of attention of modern medicine and food functional chemistry in recent years. At present, researchers have reported a variety of methods for extracting plant polysaccharides, but the method for extracting polysaccharides from asparagus is water solution extraction.

[0005] AD treatment is still a medical problem. More and more researches have found that traditional Chinese medicine polysaccharides are concerned in the prevention and treatment of AD due to their immunomodulatory effects. Euonymus polysaccharide, honeysuckle polysaccharide, coptis polysaccharide, schisandra polysaccharide and angelica polysaccharide show good therapeutic effect in the treatment of AD. At present, there are few studies on the preparation and pharmacological activity of asparagus polysaccharide. In the existing preparation process of asparagus polysaccharide, the method is single, the extraction rate of asparagus polysaccharide is low, and the application in actual production is restricted. If a method for preparing asparagus polysaccharide with fixed composition and high yield can be developed, it will greatly promote the industrialization process and application range of asparagus. III. SUMMARY

[0006] The technical problem to be solved by the present application is that based on the current method for extracting polysaccharides from asparagus and the prevention and treatment of neurodegenerative diseases by traditional Chinese medicine, the present application provides an asparagus uniform polysaccharide capable of preventing and treating neurodegenerative diseases and a preparation method and application thereof.

[0007] In order to solve the above problems, the technical scheme adopted by the present application is:

[0008] The present application provides

[0009] The present application provides an asparagus uniform polysaccharide, and the structural formula of the asparagus uniform polysaccharide is as follows:

[0010]

[0011] In addition, a preparation method of asparagus uniform polysaccharide is provided, and the preparation method comprises the following steps:

[0012] a. The raw material asparagus is crushed, and then 15-20 times of hydrochloric acid solution is added for decoction. The obtained decoction liquid is cooled and filtered, and the obtained filtrate is asparagus polysaccharide extract.

[0013] b. adjusting pH of the obtained asparagus polysaccharide extract to 6-8 using NaOH solution, then performing evaporation concentration to obtain a flow extract, and then performing alcohol precipitation on the flow extract to obtain an alcohol-precipitated crude polysaccharide;

[0014] c. removing protein from the obtained alcohol-precipitated crude polysaccharide using a Sevag method to obtain total asparagus polysaccharide;

[0015] d. sequentially performing ion exchange column chromatography and gel column chromatography on the obtained total asparagus polysaccharide to separate and purify the total asparagus polysaccharide, and then performing concentration and freeze-drying to obtain uniform asparagus polysaccharide.

[0016] According to the above method for preparing uniform asparagus polysaccharide, the concentration of the hydrochloric acid solution in step a is 0.05-0.2 mol / L; the temperature during the decoction is controlled to be 80-90℃, and the number of decoction is 2-3 times.

[0017] According to the above method for preparing uniform asparagus polysaccharide, the concentration of the NaOH solution in step b is 0.5-2 mol / L.

[0018] The specific process of the alcohol precipitation is as follows: the obtained flow extract is dispersed and dissolved using distilled water, 95% industrial ethanol is added to the dissolved solution to a concentration of 80-95%, and then the solution is left to stand for 20-30 hours and centrifuged to obtain a precipitate, and the ethanol in the precipitate is evaporated to obtain the alcohol-precipitated crude polysaccharide.

[0019] According to the above method for preparing uniform asparagus polysaccharide, the specific process of the Sevag method for removing protein in step c is as follows: 1-3 times the volume of distilled water is added to the obtained alcohol-precipitated crude polysaccharide, and a chloroform-n-butanol mixed solution in an amount of 1 / 5 of the volume of the solution is added for extraction, and then the solution is shaken on a shaking bed for 15-20 minutes, and then transferred to a separatory funnel and left to stand, and then the upper liquid is taken after standing, and centrifuged for 1-2 minutes to remove the residual protein precipitate; the operation is repeated for 3-5 times to obtain a crude asparagus polysaccharide solution after removing protein, and the obtained crude asparagus polysaccharide solution is sequentially subjected to vacuum concentration and drying to obtain total asparagus polysaccharide.

[0020] According to the above method for preparing uniform asparagus polysaccharide, the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixed solution is 3-5:1.

[0021] According to the above method for preparing uniform asparagus polysaccharide, the filler used in the ion exchange column in step d is DEAE-sepharose FF; and the filler used in the gel column is Superdex-200.

[0022] According to the preparation method of the asparagus cochinchinensis uniform polysaccharide, the elution gradient of the ion exchange column chromatography in step d is 0 mol / L, 0.2 mol / L, 0.4 mol / L and 2.0 mol / L NaCl eluent; the concentration of the NaCl eluent is 0-2 mol / L, and the solution corresponding to the peak of 155-170 min is collected after purification by a Superdex-200 gel column.

[0023] The asparagus cochinchinensis uniform polysaccharide can be used for preparing a drug for treating nervous system diseases.

[0024] According to the application of the asparagus cochinchinensis uniform polysaccharide in the preparation of a drug for treating nervous system diseases, the nervous system disease is Alzheimer's disease.

[0025] The asparagus cochinchinensis uniform polysaccharide is specifically asparagus cochinchinensis polysaccharide (ACP). The asparagus cochinchinensis uniform polysaccharide ACP is obtained from the dry rhizome of Asparagus cochinchinensis (AC) (Lour.) Merr. of the Liliaceae plant, and the production place is Guizhou.

[0026] The asparagus cochinchinensis uniform polysaccharide has the following advantages:

[0027] 1. The technical scheme of the present application uses a sugar purification process to extract a uniform polysaccharide with a new structure from asparagus cochinchinensis. Pharmacological experimental results show that the asparagus cochinchinensis uniform polysaccharide prepared by the present application can regulate the VCAM-1 / MIP-1β / CCR5 signal transduction, thereby providing a basis for developing a potential new drug for Alzheimer's disease.

[0028] 2. The technical scheme of the present application uses an acid extraction method to extract asparagus cochinchinensis polysaccharide, and uses high-performance anion exchange chromatography (HPAEC), gas chromatography-mass spectrometry (GC-MS), gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) to determine its structure. It is found through research that asparagus cochinchinensis polysaccharide can reduce the deposition of Aβ in the brain of a rapid aging model (SAMP8) mouse. Asparagus cochinchinensis polysaccharide significantly reduces the MIP-1β level of SAMP8 mice, down-regulates the expression of VCAM-1 and CCR5, and reduces brain inflammation. The results show that the asparagus cochinchinensis polysaccharide prepared by the present application is a potential drug for treating AD.

[0029] 3、The asparagus cochinchinensis uniform polysaccharide (ACP) extracted by the method is a new uniform polysaccharide compound, and the content of the asparagus cochinchinensis uniform polysaccharide is 1.5-2.0% of the original medicinal material asparagus cochinchinensis; ion chromatogram shows that the asparagus cochinchinensis uniform polysaccharide is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose and fructose according to a molar ratio of 2.854:0.022:0.048:12.185:81.837:0.574:2.481, and the molecular weight is 15580 Da.

[0030] 4、The preparation method of the asparagus cochinchinensis uniform polysaccharide is suitable for industrial production, provides a basis for quality control and standardized production of the ACP, and widens the application range of the asparagus cochinchinensis polysaccharide.

[0031] 5、The asparagus cochinchinensis uniform polysaccharide ACP prepared by the method has a nerve system treatment effect, and provides a basis for developing a potential new drug for AD. IV. DESCRIPTION OF DRAWINGS

[0032] Figure 1 The total ion current chromatogram of the asparagus cochinchinensis uniform polysaccharide ACP polysaccharide bond structure analysis obtained in the embodiment 1 of the present application;

[0033] Figure 2 The hydrogen spectrum of the asparagus cochinchinensis uniform polysaccharide ACP prepared in the embodiment 1 of the present application;

[0034] Figure 3 The carbon spectrum of the asparagus cochinchinensis uniform polysaccharide ACP prepared in the embodiment 1 of the present application;

[0035] Figure 4 The HH-COSY diagram of the asparagus cochinchinensis uniform polysaccharide ACP prepared in the embodiment 1 of the present application;

[0036] Figure 5 The HSQC diagram of the asparagus cochinchinensis uniform polysaccharide ACP prepared in the embodiment 1 of the present application;

[0037] Figure 6 The HMBC diagram of the asparagus cochinchinensis uniform polysaccharide ACP prepared in the embodiment 1 of the present application;

[0038] Figure 7 The NOESY diagram of the asparagus cochinchinensis uniform polysaccharide ACP prepared in the embodiment 1 of the present application;

[0039] Figure 8 The asparagus cochinchinensis uniform polysaccharide ACP treatment can reduce the Aβ deposition in the brain of SAMP8 mice;

[0040] Figure 9 The asparagus cochinchinensis uniform polysaccharide ACP treatment can reduce the expression of VCAM-1, MIP-1β and CCR5 in the brain of SAMP8 mice. V. DETAILED DESCRIPTION

[0041] The present application is further illustrated by the following examples, but the scope of the technical solutions defined by the present application is not limited thereto.

[0042] Example 1:

[0043] The preparation method of the asparagopsis uniform polysaccharide is as follows:

[0044] a. The raw material 5 kg of asparagus was crushed, and then 17 times of 0.1 mol / L hydrochloric acid solution was added for decoction, the decoction temperature was 83℃, the decoction times was 2 times, and the decoction time was 2.5 h each time; the obtained decoction liquid was sequentially cooled and filtered, and the obtained filtrate was asparagus polysaccharide extract liquid;

[0045] b. The obtained asparagus polysaccharide extract liquid was adjusted to pH 7 by using 1 mol / L NaOH solution, and then evaporated and concentrated by using a rotary thin film evaporator to obtain a flow extract; the obtained flow extract was dispersed and dissolved by using 1 L of distilled water, and after dissolution, 95% industrial ethanol was added to 90% alcohol concentration, and then placed for 24 h and centrifuged to obtain a precipitate, and the ethanol was removed to obtain an alcohol precipitation crude polysaccharide;

[0046] c. 1 times volume of distilled water was added to the obtained alcohol precipitation crude polysaccharide, and according to the Sevag method, 1 / 5 solution amount of chloroform-n-butanol mixed liquid was added for extraction (the mixed volume ratio of chloroform-n-butanol mixed liquid was 4:1), and then placed in a shaking bed for 20 min, and then transferred to a separatory funnel for standing, and after standing, the upper liquid was taken and centrifuged for 1 min to remove the residual protein precipitate; the operation was repeated for 5 times to obtain the asparagus crude polysaccharide solution after removing the protein, and the obtained asparagus crude polysaccharide solution was sequentially concentrated under reduced pressure and dried to obtain 1.02 kg of asparagus total polysaccharide;

[0047] d. DEAE-Sepharose FF column was used, and connected with flow fraction collector and peristaltic pump; the asparagus total polysaccharide was eluted by using distilled water, 0.2 mol / L, 0.4 mol / L and 2 mol / L NaCl eluent at a flow rate of 50 mL / h, 10 mL test tube was used to collect the eluent label, phenol-sulfuric acid method was used to measure the absorbance value at 490 nm, and a scatter plot was drawn, and the eluent of 0-20 Tube was collected; the obtained elution was sequentially concentrated, dialyzed by using 3500 Da dialysis bag, and freeze-dried to obtain elution site Fr.A, and the mass was 224 g;

[0048] Take Fr. A polysaccharide component 100 mg, dissolved with 3 mL distilled water, centrifuge (12000 rpm) for 10 min, the supernatant was further separated and purified by Sephadex-200 column, the combined solution was concentrated by rotary evaporator, freeze-dried, and the obtained component was aspartate homogeneous polysaccharide, named ACP, with a mass of 40 mg.

[0049] Example 1 Preparation of aspartate homogeneous polysaccharide ACP polysaccharide bond structure analysis:

[0050] (1) Agilent Technologies Inc. (CA, UAS) 7890A-5977B gas chromatograph-mass spectrometer, automatic sampler model G4567A. The chromatographic system uses Agilent gas chromatographic system (Agilent 7890A; Agilent Technologies, USA), and the chromatographic column is BPX70 (30 m x 0.25 mm x 0.25 μm, SGE, Australia). The injection volume is 1 μl, the split ratio is 10:1, and the carrier gas is high-purity helium; the initial temperature of the column oven is 140℃, which is kept for 2.0 min, and then increased to 230℃ at a rate of 3℃ / min, and kept for 3 min. The mass spectrometry system uses a quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) of American Aiglent Company, equipped with an electron impact ion source (EI) and a MassHunter workstation. The analyte is detected in full scan (SCAN) mode using an electron impact ion source (EI), and the mass scan range (m / z) is 50-350. The total ion chromatogram of the sample is shown in Figure 1. Figure 1 .

[0051] (2) Analysis of the monosaccharide composition of aspartate homogeneous polysaccharide ACP by ion chromatography:

[0052] A, fucrose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid and mannuronic acid mixed monosaccharide standard as control.

[0053] B. Precisely weigh 5 mg of the sample into an ampoule, add 3M trifluoroacetic acid reagent TFA 2 mL, hydrolyze at 120°C for 3 h; accurately pipette the acid hydrolysis solution into a tube and dry it by nitrogen blowing, add 5 mL of water and mix well by vortex, pipette 200 μL into 800 μL of deionized water, centrifuge at 12000 rpm for 5 min; take the supernatant into an ion chromatograph (ICS5000) for detection and analysis. The chromatographic conditions are: Dionex Carbopac T PA20 chromatographic column (3x150 mm), column temperature 30°C, sample size 25 μL, flow rate: 0.3 mL / min, mobile phase: A: H2O, B: 15 mM NaOH, C: 15 mM NaOH & 100 mM NaOAC, detector: electrochemical detector.

[0054] The results show that the asparagus polysaccharide ACP is mainly composed of fucose, rhamnose, arabinose, galactose, glucose, xylose and fructose in a molar ratio of 2.854:0.022:0.048:12.185:81.837:0.574:2.481.

[0055] (3) According to the chemical shifts of 13C and 1H of each sugar residue in the sample, combined with the analysis of the structure and connection mode of the polysaccharide by HMBC and NOESY spectrum, since the cross peak signal of the HMBC spectrum is relatively weak, the connection order of each residue in the polysaccharide is mainly judged and speculated by the NOESY spectrum: sugar residue A-H1 exists cross peak δ4.61 / 4.06ppm with residue A-H4, exists cross peak δ4.61 / 4.05ppm with residue C-H4, and exists cross peak δ4.61 / 3.48ppm with residue D-H4. Sugar residue B-H1 exists cross peak δ5.19 / 4.06ppm with residue A-H4, and exists cross peak δ5.19 / 3.74ppm with residue C-H6. Sugar residue C-H1 exists cross peak δ5.13 / 4.06ppm with residue A-H4, and sugar residue D-H1 exists cross peak δ4.86 / 4.06ppm with residue A-H4. Therefore, according to the analysis of one-dimensional nuclear magnetic and two-dimensional nuclear magnetic information and methylation results, it is inferred that the polysaccharide is mainly composed of →4)-β-D-Glcp-(1→, a small amount of →4,6)-α-D-Galp-(1→ and →3,4)-α-D-Glcp-(1→, etc. connected to form a main chain, and the branch chain is mainly composed of α-D-Glcp-(1→ connected to the O-6 position of sugar residue →4,6)-α-D-Galp-(1→ and the O-3 position of sugar residue →3,4)-α-D-Galp-(1→. The analysis result is shown in Table 1:

[0056] Table 1 Each sugar residue in the asparagus polysaccharide of the application 1 H and 13 C chemical shifts

[0057]

[0058] The aspartic uniform polysaccharide prepared by the application is also subjected to a series of structural characterization, including: methylation test is used to analyze sugar residues, and nuclear magnetic spectrum is used to analyze and determine the connection mode of glycosidic bond. Thus, the structure of the aspartic uniform polysaccharide ACP prepared by the application is obtained as follows:

[0059]

[0060] The application experiment of the aspartic uniform polysaccharide prepared by the application in Alzheimer's disease is as follows:

[0061] 1. Materials and methods

[0062] 1.1 Experimental materials: experimental animals include 18 male SAMP8 mice and 16 anti-aging control mice SAMR1, with a body weight of about 25.0 g, purchased from the First Affiliated Hospital of Tianjin University of Chinese Medicine. License No.: SCXK(Jin)2015-0003. The mice are fed at a density of 6 per cage, and they can freely obtain food and water. Donepezil hydrochloride, specification: 5 mg per tablet, approval number: GMP H20010723, batch number: 19090005, produced by Weisheng Pharmaceutical Co., Ltd. ACP is prepared by the application. The antibodies used in the experiment are purchased from Cell Signaling Technology (CST) company.

[0063] 1.2 Experimental animal grouping and administration: the mice are randomly divided into a wild type control group (SAMR1 mice), a control group (SAMP8 mice), an ACP group (SAMP8 mice treated with 6.75 mg / kg / d ACP) and a positive control group (SAMP8 mice treated with 1.667 mg / kg / d donepezil hydrochloride). After two months of gavage, the mice are anesthetized with 10% chloral hydrate, and the mouse heart is flushed with 0.9% normal saline. The rat brain is removed and split along the midline. One half brain is fixed in 4% paraformaldehyde (pH 7.4) phosphate buffer at 4℃ for 24 hours, and vibration sectioning is performed. The hippocampus and cortex are separated from the other hemisphere, quickly frozen in liquid nitrogen, and stored in a refrigerator at -80℃ for Western blot analysis. All animal studies are performed according to the approved protocols of the Animal Protection and Use Committee of Henan University of Chinese Medicine.

[0064] 1.3 Immunohistochemistry: Phosphate-buffered saline (PBS) solution was placed in a 24-well plate. Next, the olfactory bulb and cerebellum were cut off. These tissues were then implanted into an implant machine. After sectioning, the brain tissue was fixed on a tissue tray with the brain tissue facing upwards and the back facing downwards. The section thickness was set to 4 pm. About 10 brain sections were placed in each well after sectioning and stored in a refrigerator at 4 °C. The desired brain sections were selected and placed in a 24-well plate at a density of 3-5 sections per well. The embedding agent and preservative were removed by rinsing with PBS for 5 minutes. Subsequently, PBS was aspirated and 0.5% Triton (diluted with PBS) was added. Incubation was performed at 37 °C for 1 h, and PBS was shaken and washed 3 times (5 min / wash). After washing, the samples were blocked with 5% goat serum (diluted with PBS) and shaken for 1.5 hours at room temperature. The serum was then discarded, and about 100-150 pL of primary antibody (diluted with 5% goat serum according to the antibody instructions) was directly added to each well. The samples were incubated at 37 °C for 1 h and then placed in a 4 °C refrigerator overnight. After that, the primary antibody was removed, and the samples were washed with PBS three times (5 minutes each time). About 100-150 pL of fluorescent secondary antibody (diluted with PBS according to the manufacturer's instructions) was added to each well. The plate was wrapped with tin foil to prevent light exposure and shaken for 2 hours at room temperature. Subsequently, the samples were washed with PBS 3 times (5 minutes each time). Finally, about 50 pL of ready-made 4', 6-diamidino-2-phenylindole (DAPI) solution was added to each well. After shaking for 5 minutes at room temperature, the samples were washed with PBS three times (5 minutes each time). After completing these steps, the samples were observed and photographed for analysis using a fluorescence microscope.

[0065] 1.4 Western blotting experiments: Western blotting experiments were used to detect the expression levels of amyloid precursor protein (APP), VCAM-1, macrophage inflammatory protein (MIP)-1β, CCR5, ERK1 / 2, and phosphorylated ERK (p-ERK1 / 2) in the brain. The brain tissues of mice in each group were homogenized. After centrifugation, the proteins were extracted from the tissues and quantified using a BCA protein quantification kit. The target proteins were separated using 12% polyacrylamide gels and transferred to PVDF membranes for immunoblotting analysis. β-Actin was used as an internal reference to detect the expression of inflammatory mediators and signal transduction components in the brain homogenate of mice in each group: VCAM-1 (1:2000), CCR5 (1:1000), and anti-phosphorylated and anti-total ERK1 / 2 mitogen-activated protein kinase (MAPK) kinase (1:1000) and c-Jun n-terminal kinase (JNK) MAPK kinase IgG (1:1000). The secondary antibody was goat anti-rabbit or goat anti-mouse peroxidase-conjugated IgG antibody (both 1:5000). Chemiluminescence enhancer (catalog number NEL103E001EA; PerkinElmer, Inc, Waltham, Massachusetts, USA) was used for signal detection. Quantity One software version 4.62 (Bio-Rad Laboratories, Inc, Hercules, California, USA) was used for densitometric analysis. At least 6 animals were used for each Western blotting study, and representative data are presented.

[0066] 2. Experimental results:

[0067] 1) The prepared aspartic uniform polysaccharide ACP can reduce the deposition of Aβ in the brain of SAMP8 mice (for details, see Figure 8 ).

[0068] 2) The prepared aspartic uniform polysaccharide ACP can reduce the expression of VCAM-1, MIP-1β, and CCR5 in the brain of SAMP8 mice (for details, see Figure 9 ).

[0069] Through experiments, the present application explores the effect of ACP on the content of Aβ in the brain of SAMP8 mice. Immunohistochemical experiments were used to detect the expression of Aβ in the brain of mice in each group, and Western blotting experiments were used to detect the expression of APP in the brain of mice in each group. Compared with the control group, the total Aβ deposition and APP expression in the hippocampus of model mice were significantly increased. Compared with SAMP8 control mice, the content of Aβ in the brain of the ACP treatment group was significantly reduced (for details, see Figure 8 A), and the expression of APP was also reduced (for details, see Figure 8B). These results indicated that ACP treatment significantly affected the deposition of Aβ in the brain of SAMP8 mice. In addition, the deposition of Aβ in AD promotes the release of chemotactic factors and adhesion molecules by endothelial cells. These factors disrupt the blood-brain barrier, allowing fibrinogen, immunoglobulin and leukocytes to enter the brain parenchyma, causing neuronal apoptosis, while activating microglia and astrocytes, thus triggering a sustained neuroinflammatory response. Western blotting and immunofluorescence analysis showed that the expression of VCAM-1 was significantly increased in the model group; while the expression of VCAM-1 was significantly reduced in the ACP treatment group (see details in Figure 9 A and 9B). In addition, the expression levels of MIP-1β and CCR5 in the model mice were significantly increased compared with the control group. However, compared with the SAMP8 model group, the expression of MIP-1β and CCR5 in the ACP treatment group was significantly reduced (see details in Figure 9 C and 9D). These findings suggest that VCAM-1, MIP-1β and CCR5 are involved in the pathogenesis of AD, and the effect of ACP on cognitive impairment may be related to the reduction of related neuroinflammatory mediators.

[0070] In summary, the aspartic uniform polysaccharide ACP prepared in the present application has great potential in the prevention and treatment of AD, and is a potential new drug for the treatment of AD. Therefore, the present application provides a basis for the development of a potential new drug for the prevention and treatment of AD, and promotes the industrialization process of asparagus.

Claims

1. A method for preparing asparagine uniform polysaccharide, characterized by, The preparation method comprises the following steps: a. crushing raw asparagus, adding 15-20 times of hydrochloric acid solution to decoct, cooling and filtering the obtained decocting liquid, and obtaining asparagus polysaccharide extract; b. adjusting the pH of the asparagus polysaccharide extract to 6-8 by using NaOH solution, then evaporating and concentrating to obtain a flow extract, and then alcohol precipitating the flow extract to obtain alcohol-precipitated crude polysaccharide; c. removing protein from the alcohol-precipitated crude polysaccharide by using the Sevag method to obtain total asparagus polysaccharide; d. sequentially separating and purifying the total asparagus polysaccharide by using an ion exchange column and a gel column, then concentrating, freezing and drying to obtain uniform asparagus polysaccharide; The filler used in the ion exchange column is DEAE-agarose gel FF; the filler used in the gel column is Superdex-200; the elution gradient of the ion exchange column chromatography is 0 mol / L, 0.2 mol / L, 0.4 mol / L and 2.0 mol / L NaCl eluent, 10 mL test tube is used to collect the eluent label, the absorbance value is measured at 490 nm by using the phenol-sulfuric acid method, a scatter plot is drawn, and the eluent of 0-20 Tube is collected; the concentration of the NaCl eluent is 0-2 mol / L, and the solution corresponding to the peak of 155-170 min is collected after purification by the Superdex-200 gel column.

2. The method for preparing homogeneous asparagus polysaccharide according to claim 1, characterized in that: The concentration of the hydrochloric acid solution in step a is 0.05-0.2 mol / L; the temperature during decoction is controlled at 80-90°C, and the decoction times are 2-3 times.

3. The method for preparing homogeneous asparagus polysaccharide according to claim 1, characterized in that: The concentration of the NaOH solution in step b is 0.5-2 mol / L; The specific process of alcohol precipitation is as follows: dispersing and dissolving the flow extract by using distilled water, adding 95% industrial ethanol to a concentration of 80-95% after dissolving, standing for 20-30 h, centrifuging to obtain a precipitate, and evaporating the ethanol to obtain alcohol-precipitated crude polysaccharide.

4. The method for preparing homogeneous asparagus polysaccharide according to claim 1, characterized in that, The specific process of the Sevag method for removing protein in step c is as follows: adding 1-3 times the volume of distilled water to the alcohol-precipitated crude polysaccharide, adding a chloroform-n-butanol mixed solution in an amount of 1 / 5 of the solution, extracting, placing on a shaking bed for 15-20 min, then transferring to a separatory funnel and standing, taking the upper liquid after standing, centrifuging for 1-2 min to remove residual protein precipitate; repeating the operation for 3-5 times to obtain a crude asparagus polysaccharide solution after removing protein, and sequentially concentrating under reduced pressure, drying to obtain total asparagus polysaccharide.

5. The method for preparing homogeneous asparagus polysaccharide according to claim 4, characterized in that: The mixing volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixed solution is 3-5:

1.

6. The use of the uniform asparagus polysaccharide in claim 1 in the preparation of a drug for treating Alzheimer's disease.

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