A polysaccharide of acenthophanaculata and a preparation method and application thereof

Abstract

The application discloses a 100,000-level acanthopanax senticosus polysaccharide and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The 100,000-level acanthopanax senticosus polysaccharide is prepared from acanthopanax senticosus by adopting an ultrasonic-enzyme-assisted aqueous two-phase method. The polysaccharide is obtained by separation and purification. The polysaccharide is selectively separated and enriched in situ by using the difference between the distribution coefficients of two phases in the aqueous two-phase system. Meanwhile, the ultrasonic mechanical crushing and cavitation effect effectively destroy the cell wall structure, accelerate the dissolution and mass transfer process of the polysaccharide. The enzyme-assisted extraction has the advantages of high efficiency, environmental friendliness, easy operation and the like. The preparation process of the 100,000-level acanthopanax senticosus polysaccharide realizes efficient extraction and synchronous purification of the polysaccharide, has the characteristics of mild operation condition, high bioactivity retention rate, green environmental protection and easy industrialization amplification, and can enhance the anti-stress ability and anti-paralysis ability of CL4176 nematodes.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and more specifically to a 100,000-grade Acanthopanax senticosus polysaccharide that can inhibit abnormal Aβ aggregation and is rich in mannose, its preparation method, and its application in enhancing anti-stress and anti-paralysis capabilities. Background Technology

[0002] Acanthopanax senticosus (Rupr. et Maaxim.) Harms, a plant in the Araliaceae family, is a dried rhizome. It is warm in nature, pungent and slightly bitter in taste, and enters the spleen, kidney, and heart meridians. It has the effects of invigorating qi and strengthening the spleen, tonifying the kidneys and calming the mind. Therefore, it is commonly used in folk medicine for symptoms such as qi deficiency and fatigue, insomnia, and lower back and knee pain. Chemical composition studies show that Acanthopanax senticosus contains polysaccharides, glycosides, flavonoids, lignans, and organic acids, among which lignans or phenylpropanoids are the main chemical components. Modern pharmacological studies have shown that Acanthopanax senticosus has anti-inflammatory, anti-cancer, neuroprotective, antioxidant, immunomodulatory, and cardiovascular protective effects. Acanthopanax senticosus polysaccharides (ASAP) are one of the main components of Acanthopanax senticosus, possessing significant biological activity. ASAP plays an important role in physiological processes such as immune response, oxidative stress defense, apoptosis regulation, and tissue and organ protection, and is widely used in pharmaceuticals, functional foods, and dietary supplements. Further rational development and utilization of Acanthopanax senticosus resources are needed, but problems such as low extraction rate and severe polysaccharide loss during separation and purification have not yet been solved.

[0003] Caenorhabditis elegans (C. elegans) is a tiny, transparent model organism with a filamentous body that is thick at the head and thin at the tail. Under laboratory conditions, it feeds on Escherichia coli OP50 and possesses advantages such as rapid reproduction, short lifespan, high genetic homology with humans, and ease of culture. The paralytic behavior and stress response of the CL4176 strain of nematode can be directly observed under a microscope, and its paralysis and stress phenotypes can mimic relevant physiological processes in higher animals. Therefore, it is widely used in drug screening and activity evaluation, demonstrating significant scientific value and application prospects in promoting medical research and new drug discovery.

[0004] In recent years, with the continuous changes in the living environment and the accelerating pace of life, social groups generally experience high levels of psychological stress, leading to an increase in negative emotions such as anxiety and depression. Prolonged stress can easily trigger abnormal psychological functions such as low mood, slowed thinking, and impaired learning and memory. In severe cases, it can manifest as a decline in the body's stress response capacity, and even an increased risk of paralysis, significantly impacting daily life and work efficiency. Studies have shown a close correlation between decreased stress response capacity and paralysis tendency, specifically manifested as sluggish responses to external stimuli, limited mobility, and increased susceptibility to paralysis. Traditional Chinese medicine (TCM) demonstrates unique advantages in improving these pathological states, providing an important research direction for developing novel drugs targeting stress regulation and anti-paralysis. Therefore, establishing an efficient preparation method for Acanthopanax senticosus polysaccharide and clarifying its anti-stress and anti-paralysis capabilities in CL4176 nematodes has become an urgent research need.

[0005] Therefore, providing a 100,000-level Acanthopanax senticosus polysaccharide, its preparation method, and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a 100,000-grade Acanthopanax senticosus polysaccharide, its preparation method, and its application.

[0007] This invention uses Acanthopanax senticosus as raw material and employs an ultrasonic-enzyme-assisted aqueous two-phase extraction (UEA-ATPE) method to extract crude Acanthopanax senticosus polysaccharide (ASAP). After separation and purification, a 100,000-level Acanthopanax senticosus polysaccharide is obtained. The mechanical and cavitation effects of ultrasound are utilized to instantly break down cells and accelerate molecular motion. Enzymes break down the physical barriers to the dissolution of active ingredients. Furthermore, the difference in partition between the two phases allows for the timely transfer and enrichment of the dissolved active ingredients, disrupting the dissolution equilibrium. The combination of aqueous two-phase extraction, ultrasonic-assisted extraction, and enzyme-assisted extraction methods works synergistically. The 100,000-level Acanthopanax senticosus polysaccharide prepared by this invention can enhance the stress resistance and anti-paralysis ability of nematodes and can be applied to the preparation of novel drugs that enhance stress resistance and anti-paralysis capabilities.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing 100,000-grade Acanthopanax senticosus polysaccharide includes the following steps: (1) Add petroleum ether to Acanthopanax senticosus, and use Soxhlet extraction to degrease the Acanthopanax senticosus raw material by reflux. After filtration, air dry naturally to obtain degreased Acanthopanax senticosus.

[0010] (2) Add ammonium sulfate solid to purified water in portions, and stir continuously until the solid is completely dissolved to prepare an ammonium sulfate aqueous solution with a mass volume concentration of 20%-60% (w / v); add polyethylene glycol to the above ammonium sulfate aqueous solution, and control the amount of polyethylene glycol added to be 5%-25% (w / v). After thorough mixing, seal and let stand to complete the pre-separation of the system and obtain a stable polyethylene glycol-ammonium sulfate aqueous two-phase extraction system.

[0011] (3) Add the defatted Acanthopanax senticosus obtained in step (1) to the polyethylene glycol-ammonium sulfate aqueous two-phase extraction system prepared in step (2), and control the material-liquid ratio of defatted Acanthopanax senticosus to polyethylene glycol-ammonium sulfate aqueous two-phase extraction system to be 1:15-35 g / mL; use ultrasonic-assisted extraction treatment, filter after extraction to remove plant solid residue, and collect the aqueous two-phase mixed extract containing the target polysaccharide.

[0012] (4) Add the compound enzyme preparation to the aqueous two-phase mixed extract obtained in step (3) for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme activity is inactivated by high temperature, and insoluble impurities are removed by centrifugation to obtain a clear aqueous two-phase mixed extract after enzymatic hydrolysis. The amount of the compound enzyme preparation added is 4% to 6% of the aqueous two-phase mixed extract. The compound enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:2, wherein the cellulase activity is 20,000 U / g and the pectinase activity is 30,000 U / g.

[0013] (5) Transfer the clear enzymatic hydrolysis solution obtained in step (4) into a separatory container and let it stand to separate into layers. The system spontaneously forms two phases: the upper layer is a polyethylene glycol-rich phase and the lower layer is an ammonium sulfate-rich phase. The target polysaccharide is enriched in the ammonium sulfate-rich phase. Discard the upper polyethylene glycol-rich phase and collect the lower ammonium sulfate-rich phase. Filter with filter paper to remove trace suspended impurities and obtain a clear polysaccharide-rich salt phase solution.

[0014] (6) Place the polysaccharide-enriched salt phase solution obtained in step (5) into a vacuum concentration device for low-temperature vacuum concentration. Temperature: 45-55℃, vacuum degree: -0.085 to -0.095 MPa, low-temperature vacuum concentration for 90-150 min. During the concentration process, ammonium sulfate in the system reaches a supersaturated state due to the reduction of solvent and crystallizes out. Maintain the system at 50-60℃ for 10-20 min, filter while hot, and completely remove the precipitated ammonium sulfate crystals and trace amounts of insoluble impurities. Collect the clear filtrate to obtain a completely desalted polysaccharide concentrate, avoiding interference from the high-salt system on subsequent alcohol precipitation and purification processes.

[0015] (7) Slowly add anhydrous ethanol to the polysaccharide concentrate obtained in step (6), with the ratio of anhydrous ethanol to polysaccharide concentrate being 4:1. After stirring and mixing thoroughly, let it stand for alcohol precipitation to allow the water-soluble Acanthopanax senticosus polysaccharide to fully precipitate. After the alcohol precipitation is completed, centrifuge and collect the polysaccharide precipitate at the bottom. Redissolve the obtained polysaccharide precipitate thoroughly with distilled water, and then concentrate it again under reduced pressure to remove the residual ethanol solvent. Finally, freeze dry it under vacuum to remove the water in the system and obtain Acanthopanax senticosus crude polysaccharide solid powder.

[0016] (8) Take the solid powder of Acanthopanax senticosus crude polysaccharide prepared in step (7), add distilled water, stir thoroughly to reconstitute, and add papain to remove protein; the ratio of solid powder of Acanthopanax senticosus crude polysaccharide to distilled water is 1:20~30 g / mL; the amount of papain added is 0.8%~1.5% (w / v) of the solid powder of Acanthopanax senticosus crude polysaccharide in distilled water; the enzyme activity of papain is 800000 U / g; use papain to deproteinize the supernatant to degrade free protein impurities in crude polysaccharide. After the deproteinization treatment is completed, inactivate enzyme, centrifuge, centrifuge and filter to remove denatured protein residue to obtain deproteinized polysaccharide solution.

[0017] (9) The deproteinized polysaccharide solution obtained in step (8) is passed into a membrane separation device for molecular weight sieving and classification. The polysaccharide components with a molecular weight range of 50-150 KDa are accurately retained by the retention sieving method, and small molecule polysaccharides, inorganic salt residues and macromolecular impurities are removed to obtain a 100,000 Dalton grade Acanthopanax senticosus polysaccharide (ASAP) component solution.

[0018] (10) Take the polysaccharide supernatant after membrane fractionation and use a DEAE-650M cellulose ion exchange column for adsorption elution separation and purification; further purify the target polysaccharide component by gradient elution and collect the main peak eluent.

[0019] (11) The target eluent collected by column chromatography was concentrated under reduced pressure and freeze-dried to finally obtain 100,000 grade Acanthopanax senticosus polysaccharide.

[0020] Furthermore, the Soxhlet extraction method described in step (1) involves reflux defatting with a Soxhlet extractor 2-3 times, with each defatting session lasting 2-3 hours. The standing time mentioned in step (2) is 30 min at a constant temperature of 25 ℃; The ultrasonic-assisted extraction parameters in step (3) are: ultrasonic power 150~250 W, extraction temperature 50~60 ℃, and ultrasonic extraction time 40~60 min; The enzymatic hydrolysis described in step (4) involves controlling the temperature of the system to 65℃ and hydrolyzing at a constant temperature for 40 min; after the enzymatic hydrolysis is completed, the temperature is raised to 100℃ to inactivate the enzyme for 10 min, and after inactivation of the enzyme, the system is centrifuged at 5000 r / min for 10 min, and the supernatant is collected. In step (7), the alcohol precipitation environment temperature is 4℃, the alcohol precipitation standing time is 48 h, the centrifugation speed is 3500 r / min, and the centrifugation time is 10 min. In step (8), the deproteinization temperature is 50 °C and the enzymatic hydrolysis time is 50 min; the enzyme inactivation is performed at 100 °C for 10 min; and the centrifugation is performed at 5000 r / min for 5 min.

[0021] Furthermore, the membrane separation elements of the membrane separation equipment described in step (9) are 150 kDa and 50 kDa, respectively.

[0022] Further, the adsorption time of the chromatographic column in step (10) is 2 h; gradient elution is performed using an anhydrous ethanol-distilled water system, with gradient concentrations set sequentially as 5%, 10%, 15%, 20%, and 25% (v / v) anhydrous ethanol; the elution flow rate is 2.0 mL / min.

[0023] Furthermore, the method prepares 100,000-grade Acanthopanax senticosus polysaccharide (high-purity Acanthopanax senticosus polysaccharide with a molecular weight of 50~150 KDa).

[0024] Furthermore, the application of the 100,000-grade Acanthopanax senticosus polysaccharide in the preparation of drugs that inhibit abnormal Aβ aggregation, resist oxidative stress, resist heat stress, and resist paralysis.

[0025] This invention uses CL4176 Caenorhabditis elegans as a model and applies a 100,000-grade Acanthopanax senticosus polysaccharide to the nematode. The results show that the 100,000-grade Acanthopanax senticosus polysaccharide prepared in this invention can enhance the stress resistance and anti-paralysis ability of the nematode.

[0026] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a 100,000-grade Acanthopanax senticosus polysaccharide, its preparation method and application, which has the following beneficial effects: (1) The present invention uses an ultrasonic-enzyme-assisted aqueous two-phase method to extract 100,000-grade Acanthopanax senticosus polysaccharide, which solves the bottleneck of low extraction rate and poor purity of the existing process; the reaction conditions of this method are mild, the extraction is fast and efficient.

[0027] (2) The preparation method of the present invention has the advantages of simple operation, high extraction rate and purity, time-saving and high efficiency, measurable molecular weight and mild system.

[0028] (3) The 100,000-grade quinacrine polysaccharide prepared in this invention can enhance the stress resistance and paralysis resistance of CL4176 Caenorhabditis elegans. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 The morphology of the ASAP 2 prepared for this experiment is shown in the scanning electron microscope image.

[0031] Figure 2 The IR spectrum of ASAP 2 prepared according to the present invention.

[0032] Figure 3 The ASAP 2 Congo Red spectrum prepared for this invention.

[0033] Figure 4 HPGPC spectra of ASAP 1-5 prepared for this invention.

[0034] Figure 5 The image shows the HPLC chromatogram of a mixed monosaccharide standard, where 1: ManA; 2: Man; 3: Rha; 4: GlcA; 5: GalA; 6: Glc; 7: Gal; 8: Ara; 9: Fuc.

[0035] Figure 6 The HPLC chromatogram is shown for the ASAP 2 monosaccharide standard derivative prepared in this invention; where A, G, and G are respectively mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), glucose (Glc), galactose (Gal), arabinose (Ara), and fucose (Fuc).

[0036] Figure 7 The effects of ASAP and ASAP 1-5 prepared for this invention on ROS in CL4176 nematodes.

[0037] Figure 8 The effects of ASAP and ASAP 1-5 prepared for this invention on Aβ deposition in CL4176 nematodes.

[0038] Figure 9 The effects of ASAP and ASAP 1-5 prepared for this invention on oxidative stress in CL4176 nematodes.

[0039] Figure 10 The effects of ASAP and ASAP 1-5 prepared for this invention on heat stress in CL4176 nematodes.

[0040] Figure 11The effects of ASAP and ASAP 1-5 prepared for this invention on the anti-paralysis ability of CL4176 nematode. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1 A method for preparing Acanthopanax senticosus polysaccharide includes the following steps: 3 kg of Acanthopanax senticosus raw material was weighed and placed in a round-bottom flask. 50 L of petroleum ether was added, and the material was degreased by reflux using Soxhlet extraction. Each degreasing process lasted 2 hours, and the process was repeated 3 times. After degreasing, solid and liquid were separated, and the solid filter residue was collected and air-dried to completely remove residual petroleum ether solvent, yielding degreased Acanthopanax senticosus raw material. 1000 mL of purified water was added to a 5000 mL beaker, and 200 g of ammonium sulfate solid was accurately weighed. The mixture was continuously shaken and stirred until completely dissolved to prepare an ammonium sulfate aqueous solution with a mass-volume concentration of 20% (w / v). Polyethylene glycol was then slowly added to the above ammonium sulfate aqueous solution, and the amount of polyethylene glycol added was adjusted to achieve a system mass-volume concentration of 16% (w / v). After thorough mixing, the system was sealed and allowed to stand at 25 ℃ for 30 min to reach a stable phase equilibrium state, thus preparing a stable polyethylene glycol-ammonium sulfate aqueous two-phase extraction system. All defatted Acanthopanax senticosus raw materials were added to the prepared aqueous two-phase extraction system, and the ratio of defatted Acanthopanax senticosus to the aqueous two-phase extraction system was controlled at 1:25 g / mL. Ultrasonic-assisted extraction was used, with an ultrasonic power of 250 W, an extraction temperature of 55 ℃, and an ultrasonic extraction time of 50 min. After extraction, the plant solid residue was removed by filtration, and the aqueous two-phase mixed extract containing the target polysaccharide was collected. A compound enzyme preparation was added to the aqueous two-phase mixed extract. The compound enzyme preparation consisted of 20000 U / g cellulase and 30000 U / g pectinase in a mass ratio of 1:2, and the amount of compound enzyme preparation added was 5% of the material system. The system was kept at a constant temperature of 65 ℃ for 40 min for enzymatic hydrolysis. After enzymatic hydrolysis, the temperature was raised to 100 ℃ for 10 min to inactivate the enzyme, followed by centrifugation at 5000 r / min for 10 min to remove insoluble impurities. The clear aqueous two-phase mixed extract after enzymatic hydrolysis was collected. After centrifugation and enzyme inactivation, the aqueous two-phase mixture was transferred to a separatory container and allowed to stand for separation. The system spontaneously formed two stable phases: an upper layer rich in polyethylene glycol and a lower layer rich in ammonium sulfate. The target polysaccharide of Acanthopanax senticosus was enriched in the ammonium sulfate-rich phase. The upper polyethylene glycol phase containing impurities was discarded, and the lower ammonium sulfate-rich phase was collected. The phase was filtered to remove trace suspended impurities, resulting in a clear polysaccharide-enriched salt phase solution. The obtained polysaccharide-enriched salt phase solution was placed in a vacuum concentration apparatus for low-temperature vacuum concentration at 50℃ and a vacuum degree of -0.090 MPa for 120 min. During the concentration process, the ammonium sulfate in the system reached a supersaturated state due to the reduction of solvent, and crystals precipitated. The system was kept warm (55℃ for 12 min) and filtered while hot to remove the precipitated ammonium sulfate crystals and trace insoluble impurities. The clear filtrate was collected and further concentrated to a system volume of 100 mL to obtain a completely desalted polysaccharide concentrate.Anhydrous ethanol was slowly added to the fully desalted polysaccharide concentrate, controlling the volume ratio of anhydrous ethanol to polysaccharide concentrate to be 4:1. After thorough mixing, the mixture was placed in a 4℃ environment for alcohol precipitation for 48 h. After the alcohol precipitation was completed, the mixture was centrifuged at 3500 r / min for 10 min, and the bottom polysaccharide precipitate was collected. The obtained polysaccharide precipitate was fully reconstituted with an appropriate amount of distilled water, and then concentrated again under reduced pressure to remove residual ethanol solvent. Finally, the mixture was freeze-dried under vacuum to remove water from the system, and the crude polysaccharide solid powder of Acanthopanax senticosus was obtained. Take the prepared Acanthopanax senticosus crude polysaccharide solid powder, add distilled water at a material-to-liquid ratio of 1:25 g / mL (crude polysaccharide powder: distilled water), stir thoroughly to reconstitute, centrifuge to remove insoluble impurities, and collect the supernatant; use papain with an enzyme activity of 800,000 U / g to deproteinize the supernatant, controlling the deproteinization temperature at 50 ℃ and the enzymatic hydrolysis time at 50 min; the amount of papain added is 1.0% (w / v) of the crude polysaccharide distilled water reconstituted solution; after deproteinization, inactivate the enzyme at 100 ℃ for 10 min, centrifuge at 5000 r / min for 5 min to remove protein and obtain a clear deproteinized polysaccharide solution. The deproteinized polysaccharide solution was passed into a membrane separation device, and molecular weight sieving and fractionation were performed sequentially using membrane separation elements with specifications of 150 kDa, 50 kDa, 10 kDa, and 3 kDa, resulting in five polysaccharide solutions with different molecular weights. The polysaccharide fraction with a molecular weight cutoff of 50–150 kDa was selected as the 100,000 Dalton grade Acanthopanax senticosus polysaccharide (ASAP) fraction solution. The supernatant of the polysaccharide after membrane fractionation was collected and purified by adsorption and elution using a DEAE-650M cellulose ion exchange column. After sample loading, static adsorption was performed for 2 h, followed by gradient elution using an anhydrous ethanol-distilled water system at an elution flow rate of 2.0 mL / min. The gradient concentrations were successively set to 5%, 10%, 15%, 20%, and 25% (v / v) anhydrous ethanol, with each ethanol concentration having an elution volume of 2–3 column volumes. The gradient elution was performed at a constant rate throughout, and the eluent with a single symmetrical main peak was collected based on the real-time monitoring results of the chromatographic elution peaks. The eluent of the target main peak collected by column chromatography was concentrated under reduced pressure and freeze-dried to finally obtain five molecular weight homogeneous Asarum polysaccharides (ASAP 1-5); where ASAP 1: >150 KDa; ASAP 2: 50-150 KDa; ASAP 3: 10-50 KDa; ASAP 4: 3-10 KDa; ASAP 5: <3KD.

[0043] The purified ASAP 2 was subjected to scanning electron microscopy (SEM) morphology characterization. After gold sputtering pretreatment, the dried polysaccharide samples were placed under a scanning electron microscope at 2000x magnification to observe the microscopic surface morphology, particle morphology, and structural characteristics of different polysaccharide components. The differences in microstructure of the samples were clearly recorded. The results are shown in [Figure number missing]. Figure 1Fourier transform infrared (FTIR) spectroscopy was used to detect the infrared spectrum of ASAP 2. The dried and purified polysaccharide powder was mixed with dried KBr powder at a ratio of 1:200, ground, and pressed into tablets. The spectra were scanned in the wavenumber range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹. The characteristic infrared absorption peaks of the polysaccharide were measured and recorded, and the functional group structure of the polysaccharide was analyzed. The infrared spectral results are shown below. Figure 2 The helical conformation characteristics of ASAP 2 were investigated using the Congo red staining method. A 2 mg / mL polysaccharide solution was prepared and mixed with Congo red reagent. After standing for reaction, the UV absorption spectra of the mixture at different wavelengths were scanned and measured. The shift in the maximum absorption wavelength was analyzed to determine whether the polysaccharide exhibits a triple-helix spatial conformation. The Congo red spectral results are shown below. Figure 3 .

[0044] The formula for polysaccharide extraction rate is: Polysaccharide extraction rate (%) = Polysaccharide sample mass / Acanthopanax senticosus raw material mass × 100%.

[0045] Sugar content was determined using the phenol-sulfuric acid method, expressed as glucose concentration (mg·mL). -1 Plot a standard curve with A as the x-axis and A as the y-axis. The regression equation for the glucose standard curve is: y = 0.7224x + 0.1011, R0. 2 =0.9929, glucose concentration in the range of 0.2~1.0 mg·mL -1 The linear relationship was good within the range. Prepare 1 mg / mL solution. -1 The Acanthopanax senticosus polysaccharide solution was tested in three parallel trials according to the above method. The average value was taken, and its absorbance A was measured. The absorbance A was then substituted into the standard curve to calculate the sugar content.

[0046] The extraction rate of ASAP obtained in Example 1 was 77.45%, and the sugar content was 78.26%.

[0047] The extraction rate of ASAP 1 was 78.55%, and the sugar content was 80.43%.

[0048] The extraction rate of ASAP 2 was 77.55%, and the sugar content was 78.12%.

[0049] The extraction rate of ASAP 3 was 77.23%, and the sugar content was 77.85%.

[0050] The extraction rate of ASAP 4 was 76.81%, and the sugar content was 77.43%.

[0051] The extraction rate of ASAP 5 was 76.55%, and the sugar content was 76.92%.

[0052] In addition, to further illustrate the non-obviousness of the process parameters in the technical solution of the present invention, the inventors have optimized the mass fraction of the defatted Acanthopanax senticosus and ammonium sulfate system, the amount of compound enzyme preparation added, and the proportion of compound enzyme preparation, as detailed in Examples 2-4.

[0053] Example 2 A method for preparing Acanthopanax senticosus polysaccharide includes the following steps: Three equal-weight portions of defatted Acanthopanax senticosus raw material, 100 g each, were weighed. All other process parameters were kept the same as in Example 1, except for the ammonium sulfate concentration in the aqueous two-phase system. Three gradients of 20%, 40%, and 50% (w / v) were set to prepare three corresponding polyethylene glycol-ammonium sulfate aqueous two-phase extraction systems. The polyethylene glycol concentration was fixed at 16% (w / v), and the extraction-to-liquid ratio was 1:25 g / mL. The three portions of defatted Acanthopanax senticosus raw material were added to the three aqueous two-phase systems with different salt concentrations, respectively. Ultrasonic-assisted extraction was performed using an ultrasonic power of 250 W, an extraction temperature of 55 ℃, and an ultrasonic time of 50 min. After extraction, the residue was removed by filtration. The same ratio of compound enzyme preparation (cellulase to pectinase mass ratio 1:2, total addition 5%) was added to each filtrate. Enzymatic hydrolysis was performed at 65 ℃ for 40 min, followed by enzyme inactivation at 100 ℃ for 10 min, and centrifugation at 5000 r / min for 10 min. The clarified hydrolysate was collected. The enzymatic hydrolysates were subjected to a series of steps, including static separation, collection of the salt-rich phase, concentration under reduced pressure to remove salt, precipitation with 4 volumes of anhydrous ethanol at 4°C for 48 h, centrifugation at 3500 r / min for 10 min, reconstitution, and lyophilization, to obtain three groups of crude polysaccharides from Acanthopanax senticosus. These crude polysaccharides were then purified using papain for deproteinization, membrane fractionation (50 and 150 kDa), and a DEAE-650M cellulose column to obtain three groups of refined Acanthopanax senticosus polysaccharides, labeled ASAP-1, ASAP-2, and ASAP-3, respectively.

[0054] Based on the optimization results of different ammonium sulfate concentrations: ASAP-1 (20% ammonium sulfate) had an extraction rate of 77.32% and a polysaccharide purity of 78.15%; ASAP-2 (40% ammonium sulfate) had an extraction rate of 75.86% and a polysaccharide purity of 76.82%; and ASAP-3 (50% ammonium sulfate) had an extraction rate of 74.29% and a polysaccharide purity of 75.67%. Therefore, an ammonium sulfate concentration of 20% (w / v) in the aqueous two-phase system was preferred for preparation.

[0055] Example 3 A method for preparing Acanthopanax senticosus polysaccharide includes the following steps: Three equal-weight portions of defatted Acanthopanax senticosus raw material, 100 g each, were weighed. The ammonium sulfate concentration in the aqueous two-phase system was fixed at 20% (w / v), the polyethylene glycol concentration at 16% (w / v), the extraction material-to-liquid ratio at 1:25 g / mL, and all pretreatment process parameters were consistent with Example 1, except for the total amount of the compound enzyme preparation added, setting three gradients of 4%, 5%, and 6%. The three portions of defatted Acanthopanax senticosus raw material were added to the same aqueous two-phase system and extracted using ultrasonic power of 250 W, extraction temperature of 55 ℃, and ultrasonic time of 50 min. The corresponding amounts of the compound enzyme preparation (cellulase:pectinase = 1:2) were added to the three filtrates, with the enzyme activity parameters unchanged. The mixture was hydrolyzed at 65 ℃ for 40 min, inactivated at 100 ℃ for 10 min, and centrifuged at 5000 r / min for 10 min. The clarified hydrolysates were collected from each group. The enzyme hydrolysates were sequentially subjected to phase separation, desalting, alcohol precipitation, centrifugation, and freeze-drying to obtain three groups of crude polysaccharides. Then, through a unified deproteinization, membrane fractionation (50, 150 KDa), and column chromatography purification process, refined Acanthopanax senticosus polysaccharides ASAP-4, ASAP-5, and ASAP-6 were obtained.

[0056] Based on the optimization results of different compound enzyme addition amounts: ASAP-4 (addition amount 4%) had an extraction rate of 75.18% and a polysaccharide purity of 76.43%; ASAP-5 (addition amount 5%) had an extraction rate of 77.36% and a polysaccharide purity of 78.19%; and ASAP-6 (addition amount 6%) had an extraction rate of 76.25% and a polysaccharide purity of 77.26%. Therefore, a compound enzyme preparation with an addition amount of 5% is preferred.

[0057] Example 4 A method for preparing Acanthopanax senticosus polysaccharide includes the following steps: Three equal portions of defatted Acanthopanax senticosus raw material, 100 g each, were weighed. The ammonium sulfate concentration in the aqueous two-phase system was fixed at 20% (w / v), the polyethylene glycol concentration at 16% (w / v), the extraction material-to-liquid ratio at 1:25 g / mL, and all pretreatment process parameters were consistent with Example 1. Only the ratio of the compound enzyme preparation was changed, with three gradients of cellulase to pectinase mass ratios of 1:1, 1:2, and 2:1, respectively. The three portions of defatted Acanthopanax senticosus raw material were added to the same aqueous two-phase system and extracted using ultrasonic power of 250 W, extraction temperature of 55 ℃, and ultrasonic time of 50 min. The corresponding ratio of compound enzyme preparation was added to the three filtrates, with the amount of compound enzyme preparation fixed at 5%, and the enzyme activity parameters unchanged. Enzymatic hydrolysis was performed at 65 ℃ for 40 min, followed by enzyme inactivation at 100 ℃ for 10 min, and centrifugation at 5000 r / min for 10 min. The clarified enzymatic hydrolysates were collected from each group. Each group of enzymatic hydrolysates was sequentially subjected to phase separation, desalting, alcohol precipitation, centrifugation, and lyophilization to obtain three groups of crude polysaccharides. Then, through a unified deproteinization, membrane fractionation (50, 150 KDa), and column chromatography purification process, three groups of refined Acanthopanax senticosus polysaccharides were obtained, which were labeled ASAP-7, ASAP-8, and ASAP-9, respectively.

[0058] Based on the optimized ratios of different compound enzyme preparations: ASAP-7 (1:1 ratio) achieved an extraction rate of 76.13% and a polysaccharide purity of 77.08%; ASAP-8 (1:2 ratio) achieved an extraction rate of 77.41% and a polysaccharide purity of 78.22%; and ASAP-9 (2:1 ratio) achieved an extraction rate of 75.47% and a polysaccharide purity of 76.54%. Therefore, the preferred compound enzyme preparation is prepared by mixing cellulase and pectinase in a mass ratio of 1:2.

[0059] Furthermore, to highlight the superior effects of the technical solutions disclosed in this invention compared with the prior art, comparative experiments were conducted, see Comparative Examples 1-3.

[0060] Comparative Example 1 The method of Example 1 was used to prepare 100,000 grade Acanthopanax senticosus polysaccharide, except that Acanthopanax senticosus was not defatted with petroleum ether before extraction. Other steps were the same as in Example 1, and ASPA-A was prepared.

[0061] Using the method of Comparative Example 1, ASAP-A was prepared with an extraction rate of 64.25% and a sugar content of 68.71%. This shows that the extraction rate and sugar content of ASAP defatted with petroleum ether are significantly higher than those of ASAP not defatted with petroleum ether.

[0062] Comparative Example 2 The method of Example 1 was used to prepare 100,000 grade Acanthopanax senticosus polysaccharide, except that Acanthopanax senticosus was not extracted through a polyethylene glycol-ammonium sulfate system. Other steps were the same as in Example 1, and ASAP-B was prepared.

[0063] Using the method of Comparative Example 2, ASAP-B was prepared with an extraction rate of 65.18% and a sugar content of 70.24%. This shows that the extraction rate and sugar content of ASAP extracted by the polyethylene glycol-ammonium sulfate system are significantly higher than those of ASAP that has not been extracted by polyethylene glycol-ammonium sulfate system.

[0064] Comparative Example 3 The method of Example 1 was used to prepare 100,000 grade Acanthopanax senticosus polysaccharides, with the difference that cellulase and pectinase were used for enzymatic hydrolysis, respectively, while other steps were the same as in Example 1, and Acanthopanax senticosus polysaccharides APAP-C and ASAP-D were prepared.

[0065] Using the method described in Comparative Example 3, the extraction rate of ASAP-C was 62.37% and the sugar content was 67.58%; the extraction rate of ASAP-D was 63.12% and the sugar content was 68.15%. This shows that the extraction rate and sugar content of ASAP obtained by the compound enzyme hydrolysis method were significantly higher than those obtained by the single enzyme hydrolysis method.

[0066] To verify the bioactivity of the 100,000-grade Acanthopanax senticosus polysaccharide prepared in this invention, the following experiment was conducted using CL4176 nematode as a model to evaluate its effectiveness.

[0067] Experimental Example 1 The molecular weight of ASAP 1-5 obtained in Example 1 was determined by high-performance gel permeation chromatography (HPGPC). ASAP 1-5 was prepared into 2 mg / mL solutions. -1 The solution was filtered through a 0.22 µm filter membrane before injection. The detection results are as follows: Figure 4 As shown, the HPGPC chromatograms of ASAP 1-5 exhibit a single symmetrical chromatographic peak.

[0068] Experiment Example 2 Accurately weigh 2 mg each of mannuronic acid (ManA), mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), arabinose (Ara), and fucose (Fuc) into a hydrolysis tube and mix. Add 400 μL of 0.5 mol·L⁻¹ -1 PMP (1-phenyl-3-methyl-5-pyrazolone) solution and 0.3 mol·L -1 NaOH solution was shaken well and reacted in a 70℃ water bath for 0.5 h. After the reaction was completed, 400 μL of 0.3 mol·L⁻¹ NaOH solution was added. - 1 Neutralize with HCl solution, then add 2 mL of chloroform for extraction, centrifuge, repeat three times, and filter the aqueous layer through a 0.22 μm filter membrane before injection for analysis. Results are shown below. Figure 5 .

[0069] Another 5 mg of ASAP2 was placed in a hydrolysis tube, and 2 mL of trifluoroacetic acid (TFA) solution was added. The mixture was reacted at 120 °C for 5 h for complete hydrolysis. The TFA was removed by rotary evaporation under reduced pressure at 60 °C, and the solution was reconstituted with deionized water. 100 μL of the sample solution was then processed using the same derivatization method as the monosaccharide standard described above (addition of PMP solution, NaOH derivatization reaction, hydrochloric acid neutralization, chloroform extraction, centrifugation, and filtration through a 0.22 μm filter membrane). After extraction, centrifugation, and membrane filtration, the sample was injected for analysis. The composition of ASAP2 was determined by comparing the peak times with those of the mixed monosaccharide standard.

[0070] Figure 5 The ingredients 1-9 are mannuronic acid (ManA), mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), arabinose (Ara), and fucose (Fuc).

[0071] Figure 6 This is an HPLC chromatogram of the ASAP2 monosaccharide standard derivative. The ASAP2 monosaccharide composition is mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), glucose (Glc), galactose (Gal), arabinose (Ara), and fucose (Fuc).

[0072] To further illustrate the application of the Acanthopanax senticosus polysaccharide prepared by this invention in terms of ROS scavenging ability, Aβ deposition delay, oxidative stress resistance, and anti-paralysis ability, the inventors also conducted a mechanism study, the details of which are as follows: Experimental Example 3 CL4176 nematode is a temperature-sensitive AD model nematode. 16 ℃ is its permissible culture temperature, under which the nematode can maintain normal growth without significant abnormal Aβ expression or oxidative damage, while completing polysaccharide pretreatment. Raising the temperature to 25 ℃ can specifically induce a large amount of Aβ1-42 protein expression in nematode muscle tissue, inducing oxidative stress and abnormal ROS accumulation in the body, successfully constructing an Aβ-mediated oxidative damage model. The blank treatment without polysaccharide intervention serves as the model control group.

[0073] Synchronized L4-stage nematodes were exposed to different NGM media containing E. coli OP50 bacterial suspension (1:1 ratio of OP50 bacterial suspension + corresponding polysaccharide solution), ASAP at equal concentrations (2 mg / mL), and ASAP 1–5 single-component polysaccharide solutions, and cultured at 16°C for 5 days. After culture, the nematodes were washed three times with sterile M9 buffer to remove residual culture medium. Thirty nematodes from each group were randomly transferred to media containing E. coli OP50 and cultured at 25°C for 48 h. ROS levels in CL4176 nematodes were measured. An agar solution containing 2% agar was prepared using ultrapure water and dissolved at high temperature until the agar was completely dissolved. The agar solution was poured into blank plates, ensuring a thickness of no more than 2 mm. After complete cooling and solidification, the thinnest part of the solidified plate was cut off and placed on a clean, transparent glass slide for later use. Reactive oxygen species (ROS) specific labeling of nematodes was performed using a 2,7'-dichlorodihydrofluorescein diethyl ester (HDCF-DA, 0.1 mM) fluorescent probe: after washing with M9 buffer, the nematodes were co-incubated with HDCF-DA solution at 37°C for 3 h in the dark, followed by incubation at 1000 r·min⁻¹. -1 After centrifuging at low speed for 2 minutes and washing three times to remove uninternalized probes, the worms were placed on a 2% agar plate. Intestinal autofluorescence was captured by fluorescence microscopy, and the average fluorescence intensity was quantified by ImageJ. The level of ROS was determined by the fluorescence intensity.

[0074] The results showed that, compared with the model group, the ROS levels in nematodes were reduced after administration of the Acanthopanax senticosus polysaccharide prepared in Example 1, indicating that the Acanthopanax senticosus polysaccharide prepared in Example 1 can scavenge reactive oxygen species in nematodes and reduce the toxicity of Aβ to nematodes. Among them, the ASAP 2 group had a stronger ability to scavenge ROS, as shown in the results below. Figure 7 .

[0075] Experiment Example 4 At a permissible temperature of 16 ℃, CL4176 nematodes showed no obvious Aβ pathological damage, could grow normally, and completed polysaccharide drug pretreatment. After being transferred to a temperature of 25 ℃ for culture, the temperature-sensitive transgenic pathway of nematodes was activated, inducing the continuous and massive deposition of Aβ1-42 protein in muscle tissue, causing protein aggregation toxicity damage, and constructing a stable Aβ deposition pathological model.

[0076] Synchronized L4-stage nematodes were exposed to different NGM media containing E. coli OP50 bacterial suspension (1:1 ratio of OP50 bacterial suspension + corresponding polysaccharide solution), ASAP at equal concentrations (2 mg / mL), and single-component polysaccharide solutions of ASAP 1–5, and cultured at 16°C for 5 days. After culture, the nematodes were washed three times with sterile M9 buffer to remove residual culture medium. Thirty nematodes from each group were randomly transferred to a medium containing E. coli OP50 and cultured at 25°C for 48 h. Nematodes from different experimental groups were collected using M9 buffer, and residual OP50 on the nematode surface was washed off at 1000 r·min. -1 Centrifuge for 3 min, repeat three times, and discard the supernatant. Add 200 μL of 4% paraformaldehyde fixative to each centrifuge tube and fix at 4℃ for 24 h. After fixation, wash with PBS buffer, add 200 μL of 5% β-mercaptoethanol, 1% Triton X100, and 125 mM TrisHCl solution to each tube, and incubate at 37℃ for 24 h. Wash with PBS buffer containing 1% Tween 20, centrifuge and discard the supernatant, repeat the washing three times, add 200 μL of 0.125% ThT staining for 5 min, shaking the incubator to ensure full contact between the insects and the staining solution. After staining, wash three times with 50% ethanol for destaining, and finally rinse with PBS buffer until the solution is clear. Prepare slides and photograph under a fluorescence microscope.

[0077] The results showed that after temperature-induced Aβ expression in CL4176 nematodes, compared with the model group, the Aβ deposition in the nematode tissue of the treatment group was significantly reduced, and the survival status was better than that of the blank group. When Aβ deposition in CL4176 nematodes increased, it easily caused muscle paralysis and death. This result indicates that the Acanthopanax senticosus polysaccharide prepared by Example 1 reduced Aβ deposition in nematodes, delayed the paralysis process, and reduced the toxicity caused by Aβ deposition. Among them, the ASAP 2 group had the strongest effect in delaying Aβ deposition in nematodes. The results are shown in […]. Figure 8 .

[0078] Experimental Example 5 This experiment employed a temperature-independent chemical oxidative stress model. Polysaccharide pretreatment at a constant temperature of 16 °C avoided temperature stress interference and ensured the full action of the drug. Subsequently, exogenous H2O2 strong oxidant stimulation was used to disrupt the redox balance in nematodes, induce acute oxidative stress injury, and construct an oxidative damage model.

[0079] L4 stage nematodes, after synchronization treatment, were exposed to different NGM media containing equal proportions of E. coli OP50 bacterial suspension (1:1 ratio of OP50 bacterial suspension + corresponding polysaccharide solution), equal concentrations (2 mg / mL) of ASAP, and single-component polysaccharide solutions of ASAP 1–5, and cultured at 16°C for 24 h. After culture, the nematodes were washed three times with sterile M9 buffer to remove residual culture medium. Thirty nematodes from each group were randomly transferred to an oxidation induction system containing 5% H2O2 solution and placed in a 16°C incubator for oxidative stress testing. Nematode mortality was observed and recorded every 2 hours. No response to external mechanical stimulation was used as the mortality criterion. Individuals with abnormalities such as loss, adhesion, vaginal rupture, or difficulty in oviposition were excluded until all nematodes died. To ensure the reliability of the experimental results, this experiment was conducted in triplicate. The results showed that, compared with the model group, the survival curves of the treatment groups shifted to the right after administration of the Acanthopanax senticosus polysaccharide prepared in Example 1. This indicates that the survival ability of CL4176 nematodes under strong oxidative conditions was significantly improved after pretreatment with the Acanthopanax senticosus polysaccharide prepared in Example 1, thus improving the survival status of the nematodes. Among them, the ASAP 2 group showed a significant increase in antioxidant stress resistance. The results are shown in […]. Figure 9 .

[0080] Experimental Example 6 First, nematode culture and polysaccharide pretreatment were completed at a suitable temperature of 16 ℃ to eliminate basal temperature damage. Then, an acute heat stress injury model was constructed by raising the temperature.

[0081] L4 stage nematodes, after synchronization treatment, were exposed to different NGM media containing E. coli OP50 bacterial suspension (1:1 ratio of OP50 bacterial suspension + corresponding polysaccharide solution), ASAP at equal concentrations (2 mg / mL), and ASAP 1–5 single-component polysaccharide solutions, and cultured at 16°C for 24 h. After culture, the nematodes were washed three times with sterile M9 buffer to remove residual culture medium. Thirty nematodes from each group were randomly transferred to media containing E. coli OP50 and placed in a 37°C incubator for acute heat stress treatment. Nematode mortality was observed and recorded every 2 hours. Unresponsiveness to external mechanical stimuli was used as the mortality criterion. Individuals with abnormalities such as loss, adhesion, vaginal rupture, or difficulty in oviposition were excluded until all nematodes died. To ensure the reliability of the experimental results, this experiment was conducted in triplicate. The results showed that, compared with the model group, the survival curves of the treatment groups shifted to the right after administration of the Acanthopanax senticosus polysaccharide prepared in Example 1. This indicates that the acute heat stress defense ability of CL4176 nematodes pretreated with the Acanthopanax senticosus polysaccharide prepared in Example 1 was significantly enhanced, and the average survival time was prolonged. Among them, the heat stress resistance of ASAP2 was significantly improved. The results are shown in […]. Figure 10 .

[0082] Experimental Example 7 Pre-culture at 16 ℃ can achieve effective intervention with polysaccharides while maintaining the normal physiological state of nematodes and no obvious pathological phenotype. Subsequent temperature increase can stably induce CL4176 nematodes to specifically express Aβ protein, causing nerve and muscle damage and producing a paralysis phenotype, thereby constructing an Aβ paralysis pathological model.

[0083] L4 stage nematodes, after synchronization treatment, were exposed to different NGM media containing equal proportions of E. coli OP50 bacterial suspension and corresponding polysaccharide solution (1:1 ratio), equal concentrations (2 mg / mL) of ASAP, and single-component polysaccharide solutions of ASAP 1–5, and cultured at 16°C for 24 h. After culture, the nematodes were washed three times with sterile M9 buffer to remove residual culture medium. Thirty nematodes from each group were randomly transferred to media containing E. coli OP50 and incubated at 25°C to induce a paralysis phenotype in CL4176 nematodes. The paralysis status of the nematodes was observed and recorded every 2 hours. The criteria for paralysis were either no longer moving or only slight head shaking after stimulation with a nematode pick. Abnormal individuals such as those that were lost, adhered to the wall, had vaginal rupture, or had difficulty laying eggs were excluded until all nematodes were paralyzed. To ensure the reliability of the experimental results, this experiment was conducted in triplicate. The results showed that, compared with the model group, administration of the Acanthopanax senticosus polysaccharide prepared in Example 1 could delay the onset of paralysis and exert a preventive and delaying effect on Aβ toxicity. Among them, ASAP 2 showed the best anti-paralysis effect. The results are shown in […]. Figure 11 .

[0084] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing 100,000-grade Acanthopanax senticosus polysaccharide, characterized in that, Includes the following steps: (1) Add petroleum ether to Acanthopanax senticosus, and use Soxhlet extraction to degrease the Acanthopanax senticosus raw material by reflux. After filtration, air dry to obtain defatted Acanthopanax senticosus. (2) Add ammonium sulfate solid to purified water in portions, and stir and shake continuously until the solid is completely dissolved to prepare an ammonium sulfate aqueous solution with a mass volume concentration of 20%-60% (w / v); add polyethylene glycol to the above ammonium sulfate aqueous solution, controlling the amount of polyethylene glycol added to be 5%-25% (w / v), mix thoroughly, seal, and let stand to obtain a polyethylene glycol-ammonium sulfate aqueous two-phase extraction system; (3) Add the defatted Acanthopanax senticosus obtained in step (1) to the polyethylene glycol-ammonium sulfate aqueous two-phase extraction system prepared in step (2), and control the material-liquid ratio of defatted Acanthopanax senticosus to polyethylene glycol-ammonium sulfate aqueous two-phase extraction system to be 1:15-35 g / mL; use ultrasonic-assisted extraction treatment, filter after extraction to remove plant solid residue, and collect the aqueous two-phase mixed extract containing the target polysaccharide; (4) Add the compound enzyme preparation to the aqueous two-phase mixed extract obtained in step (3) for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme activity is inactivated by high temperature and centrifugation is performed to obtain a clear aqueous two-phase mixed extract after enzymatic hydrolysis. The amount of the compound enzyme preparation added is 4% to 6% of the aqueous two-phase mixed extract. The compound enzyme preparation is composed of cellulase and pectinase in a mass ratio of 1:2, wherein the cellulase activity is 20,000 U / g and the pectinase activity is 30,000 U / g. (5) Transfer the clear enzymatic hydrolysis solution obtained in step (4) into a separatory container and let it stand to separate into layers. The system spontaneously forms two phases: the upper layer is a polyethylene glycol-rich phase and the lower layer is an ammonium sulfate-rich phase. Collect the lower ammonium sulfate-rich phase, filter it, and obtain a clear polysaccharide-rich salt phase solution. (6) Place the polysaccharide-enriched salt phase solution obtained in step (5) into a vacuum concentration device for low-temperature vacuum concentration. Temperature: 45-55℃, vacuum degree: -0.085 to -0.095 MPa, low-temperature vacuum concentration for 90-150 min. During the concentration process, ammonium sulfate in the system reaches a supersaturated state due to the reduction of solvent and crystallizes out. Maintain the system at 50-60℃ for 10-20 min, filter while hot, collect the clear filtrate, and obtain a completely desalted polysaccharide concentrate. (7) Slowly add anhydrous ethanol to the polysaccharide concentrate obtained in step (6), the ratio of anhydrous ethanol to polysaccharide concentrate is 4:1, stir and mix thoroughly, and let stand for alcohol precipitation; after alcohol precipitation, centrifuge and collect the bottom polysaccharide precipitate; fully reconstitute the obtained polysaccharide precipitate with distilled water, concentrate under reduced pressure again to remove residual ethanol solvent, and finally freeze dry under vacuum to obtain Acanthopanax senticosus crude polysaccharide solid powder; (8) Take the Acanthopanax senticosus crude polysaccharide solid powder prepared in step (7), add distilled water, stir thoroughly to reconstitute, and add papain to remove protein; the material-to-liquid ratio of Acanthopanax senticosus crude polysaccharide solid powder to distilled water is 1:20~30 g / mL; the amount of papain added is 0.8%~1.5% (w / v) of the Acanthopanax senticosus crude polysaccharide solid powder reconstituted in distilled water; the papain enzyme activity is 800000 U / g; after the deproteinization treatment is completed, inactivate the enzyme, centrifuge, and obtain the deproteinized polysaccharide solution; (9) The deproteinized polysaccharide solution obtained in step (8) is passed into a membrane separation device for molecular weight sieving and classification. The polysaccharide components with a molecular weight range of 50-150 KDa are accurately retained by the retention sieving method to obtain a 100,000 Dalton grade Acanthopanax senticosus polysaccharide component solution. (10) Take the polysaccharide supernatant after membrane fractionation and purify it by adsorption elution using a DEAE-650M cellulose ion exchange column; The target polysaccharide component was further purified by gradient elution, and the main peak eluent was collected. (11) The target eluent collected by column chromatography was concentrated under reduced pressure and freeze-dried to finally obtain 100,000 grade Acanthopanax senticosus polysaccharide.

2. The method for preparing 100,000-grade Acanthopanax senticosus polysaccharide according to claim 1, characterized in that, The Soxhlet extraction method described in step (1) involves reflux degreasing with a Soxhlet extractor 2-3 times, with each degreasing session lasting 2-3 hours. The standing time mentioned in step (2) is a constant temperature standing time of 25 ℃ for 30 min; The ultrasonic-assisted extraction parameters in step (3) are: ultrasonic power 150~250 W, extraction temperature 50~60 ℃, and ultrasonic extraction time 40~60 min; The enzymatic hydrolysis described in step (4) involves controlling the temperature of the system to 65℃ and hydrolyzing at a constant temperature for 40 min; after the enzymatic hydrolysis is completed, the temperature is raised to 100℃ to inactivate the enzyme for 10 min, and after inactivation of the enzyme, the system is centrifuged at 5000 r / min for 10 min, and the supernatant is collected. In step (7), the alcohol precipitation environment temperature is 4℃, the alcohol precipitation standing time is 48 h, the centrifugation speed is 3500 r / min, and the centrifugation time is 10 min. In step (8), the deproteinization temperature is 50 °C and the enzymatic hydrolysis time is 50 min; the enzyme inactivation is performed at 100 °C for 10 min; and the centrifugation is performed at 5000 r / min for 5 min.

3. The method for preparing 100,000-grade Acanthopanax senticosus polysaccharide according to claim 1, characterized in that, The membrane separation elements of the membrane separation equipment mentioned in step (9) are 150 kDa and 50 kDa, respectively.

4. The method for preparing 100,000-grade Acanthopanax senticosus polysaccharide according to claim 1, characterized in that, The adsorption time of the chromatographic column in step (10) is 2 h; gradient elution is performed using an anhydrous ethanol-distilled water system, with gradient concentrations set to 5%, 10%, 15%, 20%, and 25% (v / v) anhydrous ethanol; the elution flow rate is 2.0 mL / min.

5. The 100,000-grade Acanthopanax senticosus polysaccharide prepared by the method according to any one of claims 1 to 4.

6. The use of the 100,000-grade Acanthopanax senticosus polysaccharide according to claim 5 in the preparation of drugs that inhibit abnormal Aβ aggregation, resist oxidative stress, resist heat stress, and resist paralysis.

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