Preparation method and application of astragalus degradation polysaccharide

Abstract

The application relates to the technical field of traditional Chinese medicine processing, and discloses a preparation method and application of degraded astragalus polysaccharide, which adopts ultrasonic-assisted H2O2-Vc system to degrade astragalus polysaccharide (APS) to obtain degraded astragalus polysaccharide (DAPS). H2O2 can oxidize Vc to quickly generate hydroxyl radicals, and the degradation rate of the polysaccharide is obviously improved. Meanwhile, the ultrasonic-assisted oxidative degradation can combine the advantages of the two methods and play a synergistic role. The DAPS can delay the life of CL4176 nematodes, improve the paralysis of the CL4176 nematodes, improve the movement ability, heat stress ability and oxidative stress ability of the nematodes, and can be applied to the preparation of drugs for treating Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine processing, and more particularly to a preparation method and application of degradation Astragalus polysaccharides. BACKGROUND

[0002] Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. is a common medicinal plant, mainly produced in Shanxi, Gansu, Heilongjiang and other places. As a traditional Chinese medicine, Astragalus membranaceus (Fisch.) Bge. has the effects of tonifying qi and consolidating the exterior, enhancing immunity, and resisting bacteria and inflammation. Astragalus membranaceus (Fisch.) Bge. mainly contains polysaccharides, saponins, flavonoids and other chemical components, among which the application of Astragalus polysaccharides (APS) is more common. APS has various physiological activities such as antioxidant, anti-inflammatory, antibacterial and immunomodulatory activities. However, natural APS is difficult to penetrate multiple cell membrane barriers due to its large molecular weight, high viscosity and poor water solubility, and cannot enter the body to exert functional activity. Therefore, degrading APS to prepare low molecular weight Degradation Astragalus polysaccharides (DAPS) is of great significance to improve the biological activity of APS.

[0003] Degradation polysaccharides mainly include chemical degradation, physical degradation and enzymatic degradation, and chemical degradation includes oxidation, acid and base degradation. In recent years, H2O2 oxidation method is a commonly used method for polysaccharide degradation. Hydroxyl radicals generated by the decomposition of H2O2 are used to oxidize and break the glycosidic bond between polysaccharides. This method is green and controllable. However, the process of polysaccharide degradation by spontaneous generation of hydroxyl radicals from H2O2 is slow.

[0004] Alzheimer disease (AD) is a severe neurodegenerative disease accompanied by loss of memory and cognitive ability, eventually leading to disability and dependency. The main features of AD are extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFT), and the plaques are composed of aggregates of beta amyloid (Aβ) and deposits of protofibrils, while NFT is produced by hyperphosphorylated microtubule-associated protein Tau. The pathogenesis of AD is complex and still unclear. There are several competing hypotheses, including genetics, amyloid hypothesis, cholinergic hypothesis, free radical damage hypothesis, calcium homeostasis imbalance hypothesis and apoptosis hypothesis. Caenorhabditis elegans (C.elegans), simply called nematode, belongs to the phylum Nematoda and the class Nematoda, and is one of the important model organisms for studying material anti-aging. Because it is very suitable for elucidating the pharmacological effects and various molecular mechanisms of AD treatment drugs, it has been widely used in the field of AD research. CL4176 uses a muscle cell-specific promoter myo-3 to control the expression of Aβ, which causes the appearance of convulsive symptoms in the phenotype of the nematode, replicates the symptoms and pathological changes of AD, and thus can clearly distinguish the transgenic nematode.

[0005] Therefore, how to provide a preparation method of Astragalus polysaccharide with high extraction rate and purity and also having resistance to Aβ protein toxicity in CL4176 C.elegans is a difficult problem to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the present application provides a preparation method and application of Astragalus polysaccharide, which degrades Astragalus polysaccharide (APS) to obtain Astragalus polysaccharide (DAPS) by using an ultrasonic-assisted H2O2-Vc system. H2O2 can oxidize Vc to rapidly generate hydroxyl radicals, and the degradation rate of polysaccharide is obviously improved. At the same time, ultrasonic-assisted oxidative degradation can combine the advantages of the two methods and play a synergistic role. The DAPS of the present application can delay the lifespan of CL4176 nematodes, improve the paralysis of CL4176 nematodes, and improve the movement ability, heat stress ability and oxidative stress ability of the nematodes.

[0007] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0008] A preparation method of Astragalus polysaccharide, comprising the following steps:

[0009] (1) adding an ethanol solution to Astragalus membranaceus for reflux degreasing, filtering and drying in the shade to obtain degreased Astragalus membranaceus, then soaking in deionized water, and performing microwave extraction and filtering to obtain a filtrate;

[0010] (2) adding anhydrous ethanol to the filtrate to perform alcohol precipitation, centrifuging, redissolving the precipitate, reducing pressure concentration, freeze-drying to obtain crude astragalus polysaccharide;

[0011] (3) configuring the crude astragalus polysaccharide into an aqueous solution, adding papain to perform enzymolysis, then centrifuging and suction-filtering to obtain supernatant, adding anhydrous ethanol to perform alcohol precipitation, centrifuging, redissolving the precipitate, reducing pressure concentration, freeze-drying to obtain astragalus polysaccharide;

[0012] (4) configuring the astragalus polysaccharide into an aqueous solution, adjusting the pH of the solution, then adding H2O2 and Vc, degrading the astragalus polysaccharide through ultrasonic-assisted H2O2 combined with Vc to obtain astragalus degraded polysaccharide.

[0013] Preferably, in step (1), the ratio of defatted astragalus to deionized water is 1:10-14 (g·mL -1 ); the volume concentration of the ethanol solution is 75%.

[0014] Preferably, in step (1), the power of microwave extraction is 500-600 W, the temperature is 70-90℃, the time is 2-3 h, and the number of times is 3.

[0015] Preferably, in steps (2) and (3), anhydrous ethanol is added to a volume concentration of 85%; the alcohol precipitation temperature is 4℃, and the time is 48 h; the centrifugation condition is 3500-4000 r·min -1 for 15-20 min.

[0016] The above technical solution has the beneficial effect that the largest molecular weight astragalus polysaccharide can be obtained under the alcohol precipitation condition, and the alcohol precipitation concentration reaches 85%, and no astragalus polysaccharide is precipitated.

[0017] Preferably, in step (3), the concentration of the aqueous solution is 10-15 mg·mL -1 , and the mass ratio of astragalus crude polysaccharide to papain is 4-6:1.

[0018] The above technical solution has the beneficial effect that the optimal removal of protein in astragalus crude polysaccharide does not damage the structure of astragalus crude polysaccharide, and the extraction rate of polysaccharide is maximized.

[0019] Preferably, in step (3), the enzymolysis condition is first enzymolysis at 55℃ for 3 h, and then heating to 95℃ for 10 min to inactivate the enzyme.

[0020] Preferably, in step (4), the concentration of the aqueous solution is 10-15 mg·mL -1 , the pH of the solution is adjusted to 3-4, the concentration of H2O2 is 7.5-10 mmol·L -1 , and the mass ratio of H2O2 to VC is 1:0.8-1.

[0021] The beneficial effects of the above technical solution are that H2O2 can oxidize Vc to quickly generate hydroxyl radicals, and the degradation rate of polysaccharide is obviously improved, and the ultrasonic-assisted oxidative degradation can combine the advantages of the two methods and play a synergistic role.

[0022] Preferably, the temperature of the ultrasonic in step (4) is 60-70 DEG C, the power is 800-1000 W, and the degradation time is 3-4 h.

[0023] A degraded polysaccharide of Astragalus membranaceus prepared by the above method.

[0024] The application of the degraded polysaccharide of Astragalus membranaceus in preparing a drug for treating Alzheimer's disease.

[0025] The application adopts the Alzheimer's disease nematode model of the Caenorhabditis elegans CL4176 model, and the DAPS prepared by the application is used for drug administration, and the results show that the DAPS can delay the lifespan of the CL4176 nematode, improve the paralysis of the CL4176 nematode, and improve the movement ability, heat stress ability and oxidative stress ability of the nematode.

[0026] According to the above technical solution, compared with the prior art, the application provides a preparation method and application of a degraded polysaccharide of Astragalus membranaceus, and has the following excellent effects:

[0027] (1) The polysaccharide degradation method of the application adopts the free radical combined ultrasonic degradation method, which can not only solve the problems of poor solubility and low bioavailability of macromolecular polysaccharide of Astragalus membranaceus, but also improve the purity and bioactivity of the polysaccharide of Astragalus membranaceus.

[0028] (2) The preparation method disclosed by the application has the advantages of convenient operation, time saving, high efficiency and no pollution.

[0029] (3) The degraded polysaccharide of Astragalus membranaceus prepared by the application has the effect of reducing the toxicity of Aβ in the CL4176 nematode. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description, and obviously, the drawings in the following description are only embodiments of the application, and those skilled in the art can obtain other drawings according to the provided drawings without creating any creative labor.

[0031] Figure 1 HPGPC spectrum of the DAPS prepared for Example 1.

[0032] Figure 2GC-MS chromatograms of mixed monosaccharide standard derivatives (mix) and monosaccharide component fractions of DAPS prepared in Example 1, wherein: 1. Rha; 2. Ara; 3. Fuc; 4. Xyl; 5. Man; 6. Glc; 7. Gal.

[0033] Figure 3 Effects of APS and DAPS prepared in Example 1 on the lifespan of CL4176 nematodes.

[0034] Figure 4 Effects of APS and DAPS prepared in Example 1 on the paralysis rate of CL4176 nematodes.

[0035] Figure 5 Effects of APS and DAPS prepared in Example 1 on the oxidative stress and heat stress capacity of CL4176 nematodes.

[0036] Figure 6 Effects of APS and DAPS prepared in Example 1 on the movement capacity of CL4176 nematodes. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0038] Example 1

[0039] A preparation method of Astragalus polysaccharides, comprising the following steps:

[0040] Astragalus membranaceus 2.5 kg is weighed into a round-bottom flask, 25 L of 75% ethanol is added for degreasing by reflux for 2 hours, which is repeated three times, the filter residue is dried in the shade after filtration, deionized water is added at a solid-liquid ratio of 1:12 (g·mL -1 ), and microwave extraction is performed at 80°C and a microwave power of 600 W for 2 hours. The filtrate is obtained after filtration, the extraction is performed three times, the filtrate is combined, the filtrate is concentrated to 3 L, anhydrous ethanol is added to reach an ethanol volume concentration of 85%, stirring is performed, alcohol precipitation is performed at 4°C for 48 hours, centrifugation is performed at 4000 r·min -1 for 10 minutes, the precipitate is redissolved, vacuum concentration is performed, and freeze-drying is performed to obtain Astragalus membranaceus crude polysaccharides. The Astragalus membranaceus crude polysaccharide powder is configured into a 15 mg·mL -1 aqueous solution, papain is added to the aqueous solution to make the concentration of papain 3 mg·mL -1 , uniform stirring is performed, enzymolysis is performed at 55°C for 1 hour, and then heating is performed to 95°C for 10 minutes to inactivate the enzyme, and centrifugation is performed at 4000 r·min-1 The supernatant was taken, anhydrous ethanol was added to reach an ethanol volume concentration of 85%, stirred, and then placed at 4 DEG C for 48 h for alcohol precipitation, and centrifuged at 4000 r·min -1 for 10 min, and the precipitate was redissolved, concentrated under reduced pressure, and freeze-dried to obtain the astragalus polysaccharide (APS); the APS was configured into a 10 mg·mL -1 aqueous solution, 1 mol·L -1 of HCL solution was added to adjust the pH to 4, H2O2 was added to make the concentration of H2O2 9 mmol·L -1 , Vc was added to make the mass ratio of H2O2 to Vc 1:1, ultrasonic-assisted H2O2 combined with Vc degradation was carried out at 70 DEG C for 3 h, the ultrasonic power was 900 W, then anhydrous ethanol was added to reach an ethanol volume concentration of 85%, stirred, and then placed at 4 DEG C for 48 h for alcohol precipitation, and centrifuged at 4000 r·min -1 for 10 min, and the precipitate was redissolved, concentrated under reduced pressure, and freeze-dried to obtain the astragalus polysaccharide (APS); the APS was configured into a 10 mg·mL

[0041] The polysaccharide extraction rate formula is:

[0042] The content of the polysaccharide was determined by the phenol-sulfuric acid method, 1 mg·mL -1 of glucose solution was prepared, 0.2, 0.4, 0.6, 0.8, and 1.0 mL was accurately transferred into 10 mL stoppered test tubes, 1 mL of distilled water was added, 1 mL of 6% phenol solution was added, stirred, 5 mL of concentrated sulfuric acid was slowly added dropwise, stirred, heated at 75 DEG C for 10 min, cooled to room temperature, and the absorbance (A) was determined at 490 nm, a standard curve was drawn with the glucose concentration (mg·mL -1 ) as the x-axis and the A value as the y-axis, and a glucose standard curve regression equation y = 2.6773x + 0.0896 was obtained, R 2 = 0.9993, 1 mg·mL -1 of polysaccharide sample solution was taken, three parallel experiments were carried out according to the above method, the average value was taken, the absorbance A was determined, and the sugar content was calculated by substituting the standard curve.

[0043] The extraction rate of the DAPS obtained in Example 1 was 10.74%, the sugar content was 82.17%, and the protein content was 2.19%.

[0044] In addition, in order to further illustrate the non-obviousness of the process parameter conditions in the technical solutions disclosed in the present application, the inventors optimized the feed liquid ratio of defatted astragalus and deionized water, the mass ratio of astragalus crude polysaccharide to papain, and the mass ratio of H2O2 to Vc, and the specific contents are as follows.

[0045] Examples 2-4

[0046] A preparation method of degraded astragalus polysaccharide, comprising the following steps:

[0047] Respectively, take 3 parts of defatted astragalus 100g, soak in 1L, 1.2L, 1.4L deionized water, under the microwave power of 600W, 80℃, microwave extraction 2h, extraction 3 times, combine the filtrate, add anhydrous ethanol to the ethanol volume concentration of 85%, stir evenly, 4℃ under static 48h alcohol precipitation, under 4000r·min -1 Centrifugal 10min, take the precipitate, redissolve, reduce pressure concentration, freeze-drying, get astragalus polysaccharide. Astragalus polysaccharide powder is configured into 15mg·mL -1 Water solution, add papain to the water solution, so that the concentration of papain is 3mg·mL -1 , enzymolysis 1h at 55℃, then heated to 95℃ to inactivate the enzyme for 10min, centrifugal 10min under 4000r·min -1 , take the supernatant, add anhydrous ethanol to the ethanol volume concentration of 85%, 4℃ under static 48h alcohol precipitation, centrifugal 10min under 4000r·min -1 , take the precipitate, redissolve, reduce pressure concentration, freeze-drying, get APS, APS is configured into 10mg·mL -1 Water solution, add 1mol·L -1 HCL solution to adjust pH to 4, add H2O2, so that the concentration of H2O2 is 9mmol·L -1 , add Vc to make the mass ratio of H2O2 and Vc 1:1, 70℃ ultrasonic assisted H2O2 combined with Vc degradation 3h, ultrasonic power is 900W, add anhydrous ethanol to the ethanol volume concentration of 85%, 4℃ under static 48h, centrifugal 10min under 4000r·min -1 , take the precipitate, redissolve, reduce pressure concentration, freeze-drying, get DAPS1, DAPS2, DAPS3.

[0048] According to the different ratio of defatted astragalus and deionized water, the extraction rate of DAPS1 is 8.64%, the sugar content is 75.63%, and the protein content is 2.58%; the extraction rate of DAPS2 is 10.74%, the sugar content is 82.17%, and the protein content is 2.19%; the extraction rate of DAPS3 is 9.46%, the sugar content is 77.92%, and the protein content is 2.31%. Therefore, the volume ratio of astragalus and deionized water is 1:12 for preparation.

[0049] Examples 5-7

[0050] A preparation method of degraded astragalus polysaccharide, comprising the following steps:

[0051] Three portions of defatted Astragalus membranaceus (100g each) were weighed and soaked in 1.2L of deionized water. Microwave extraction was performed at 80℃ and 60W for 2 hours, repeated three times. The filtrates were combined, and anhydrous ethanol was added until the ethanol concentration reached 85%. The mixture was stirred and allowed to stand at 4℃ for 48 hours for alcohol precipitation. The precipitate was then determined at 4000 rpm. -1 Centrifuge for 10 min, reconstitute the precipitate, concentrate under reduced pressure, and freeze-dry to obtain crude Astragalus polysaccharide. Prepare a 15 mg / mL solution of crude Astragalus polysaccharide powder. -1 Aqueous solution: Papain was added to the aqueous solution to make the papain concentration 2 mg / mL. -1 3 mg·mL -1 4 mg·mL -1 Enzymatic hydrolysis was performed at 55℃ for 1 hour, followed by enzyme inactivation at 95℃ for 10 minutes, and then at 4000 rpm. -1 Centrifuge for 10 min, collect the supernatant, add anhydrous ethanol until the ethanol concentration reaches 85%, let stand at 4°C for 48 h for alcohol precipitation, and then precipitate at 4000 r·min. -1 Centrifuge for 10 min, reconstitute the precipitate, concentrate under reduced pressure, freeze dry to obtain APS, and prepare a 10 mg / mL solution. -1 Add 1 mol·L to the solution. -1 The pH of the HCl solution was adjusted to 4, and H2O2 was added to make the H2O2 concentration 9 mmol·L⁻¹. -1 Add vitamin C to make the mass ratio of H2O2 to Vc 1:1. Perform ultrasonic-assisted degradation of H2O2 and Vc at 70℃ for 3 hours with an ultrasonic power of 900W. Add anhydrous ethanol until the ethanol volume concentration reaches 85%. Let stand at 4℃ for 48 hours, and then degrade the H2O2 and Vc at 4000 r·min. -1 Centrifuge for 10 min, reconstitute the precipitate, concentrate under reduced pressure, and freeze dry to obtain DAPS4, DAPS5, and DAPS6.

[0052] The extraction ratio of Astragalus membranaceus crude polysaccharide to papain was optimized based on different mass ratios: DAPS4 had an extraction rate of 11.43%, a sugar content of 78.43%, and a protein content of 4.11%; DAPS5 had an extraction rate of 10.74%, a sugar content of 82.17%, and a protein content of 2.19%; and DAPS6 had an extraction rate of 10.38%, a sugar content of 80.12%, and a protein content of 2.27%. Therefore, the optimal mass ratio of Astragalus membranaceus crude polysaccharide to papain is 5:1.

[0053] Examples 8-10

[0054] A method for preparing degradation of Astragalus membranaceus includes the following steps:

[0055] Take 3 parts of defatted Astragalus membranaceus 100g respectively, soak in 1.2L deionized water, microwave extraction at 600W, 80℃ for 2h, 3 times, combine the filtrate, add anhydrous ethanol to the ethanol volume concentration of 85%, stir evenly, stand at 4℃ for 48h, centrifuge at 4000r·min -1 10min, take the precipitate, redissolve, reduce pressure concentration, freeze-drying, get Astragalus membranaceus crude polysaccharide. Astragalus membranaceus crude polysaccharide powder is configured into 15mg·mL -1 Water solution, add papain to the water solution, so that the concentration of papain is 3mg·mL -1 , enzymolysis at 55℃ for 1h, then heat to 95℃ to inactivate the enzyme for 10min, centrifuge at 4000r·min -1 10min, take the supernatant, add anhydrous ethanol to the ethanol volume concentration of 85%, stand at 4℃ for 48h, centrifuge at 4000r·min -1 10min, take the precipitate, redissolve, reduce pressure concentration, freeze-drying, get APS, configure APS into 10mg·mL -1 Water solution, adjust pH to 4 by adding 1mol·L -1 HCL solution, add H2O2 to make the concentration of H2O2 9mmol·L -1 , add Vc respectively to make the mass ratio of H2O2 to Vc 1:0.8, 1:0.9, 1:1, then ultrasonic-assisted H2O2 combined with Vc degradation at 70℃ for 3h, ultrasonic power is 900W, add anhydrous ethanol to the ethanol volume concentration of 85%, stand at 4℃ for 48h, centrifuge at 4000r·min -1 10min, take the precipitate, redissolve, reduce pressure concentration, freeze-drying, get DAPS7, DAPS8, DAPS9.

[0056] According to the mass ratio of H2O2 to Vc, the extraction rate of DAPS7 is 9.02%, the sugar content is 80.11%, and the protein content is 2.87%; the extraction rate of DAPS8 is 10.37%, the sugar content is 80.92%, and the protein content is 2.31%; the extraction rate of DAPS9 is 10.74%, the sugar content is 82.17%, and the protein content is 2.19%. Therefore, the mass ratio of H2O2 to Vc is preferably 1:1 for preparation.

[0057] And, in order to highlight the excellent effect of the technical scheme disclosed in the present application compared with the prior art, the following comparative experiments are carried out:

[0058] Comparative example 1

[0059] The method of preparing Astragalus polysaccharide degradation polysaccharide according to the method of Example 1, the difference is that Astragalus polysaccharide is not subjected to microwave-assisted extraction, and other steps are the same as Example 1, to obtain Astragalus polysaccharide degradation polysaccharide DAPS-A.

[0060] Using the method of Comparative Example 1, DAPS-A is prepared with an extraction rate of 8.02% and a sugar content of 75.63%. It can be seen that the extraction rate and sugar content of Astragalus polysaccharide degradation polysaccharide extracted by microwave-assisted extraction are significantly higher than those of Astragalus polysaccharide degradation polysaccharide not subjected to microwave-assisted extraction.

[0061] Comparative Example 2

[0062] The method of preparing Astragalus polysaccharide degradation polysaccharide according to the method of Example 1, the difference is that Astragalus polysaccharide is not subjected to microwave-assisted extraction, and other steps are the same as Example 1, to obtain Astragalus polysaccharide degradation polysaccharide DAPS-A.

[0063] Table 1 DAPS and DAPS-B~D extraction rate and sugar content

[0064]

[0065] Enzymatic method, trifluorotrichloroethane method, Sevage method, TCA method can remove protein in polysaccharide to some extent. Protein removal is mainly for the separation and purification of polysaccharide, and then the physicochemical properties of polysaccharide are studied, which provides basis for the pharmacological activity research and deep development of Astragalus polysaccharide degradation polysaccharide. Therefore, while removing protein from polysaccharide, the loss of polysaccharide is minimized. Papain is used to hydrolyze protein, making it into polypeptide or more complete amino acid with relatively small molecular weight. Then, most of the macromolecular polysaccharide is precipitated, and most of the polypeptide and amino acid with relatively small molecular weight is retained in the solution.

[0066] Comparative Example 3

[0067] The method of preparing Astragalus polysaccharide degradation polysaccharide according to the method of Example 1, the difference is that Astragalus polysaccharide is not subjected to microwave-assisted extraction, and other steps are the same as Example 1, to obtain Astragalus polysaccharide degradation polysaccharide DAPS-A.

[0068] Table 2 DAPS and DAPS-E~G extraction rate, sugar content and molecular weight

[0069]

[0070] Therefore, it can be concluded that the extraction rate and sugar content of Astragalus polysaccharides degraded by ultrasound-assisted H2O2 combined with Vc degradation are significantly higher than those degraded by ultrasound, H2O2, and H2O2 combined with Vc. This proves that ultrasound-assisted H2O2 combined with Vc degradation can combine the advantages of both methods, utilizing the hydroxyl radicals generated by H2O2 decomposition to oxidize and break the interglycosidic bonds of polysaccharides. This degradation condition is mild and the process is controllable. The degree of degradation can be controlled by controlling the quality of H2O2 and Vc added during the reaction, and no harmful chemical byproducts are generated during the degradation process. However, the polysaccharide degradation process caused by the spontaneous generation of hydroxyl radicals by H2O2 is slow. When Vc is added to the H2O2 system, H2O2 can oxidize Vc and rapidly generate hydroxyl radicals, but this also results in low degradation efficiency. Using two or more degradation methods for composite degradation of polysaccharides can effectively degrade polysaccharides while improving degradation efficiency. Therefore, this invention simultaneously uses ultrasound-assisted H2O2 combined with Vc oxidation to degrade polysaccharides, which has the advantages of simple operation, high efficiency and environmental protection, and significantly improves the degradation rate of polysaccharides.

[0071] Experiment 1

[0072] The DAPS obtained in Example 1 were analyzed: the molecular weight (Mw) of the DAPS was determined by high-performance gel permeation chromatography (HPGPC); the DAPS was prepared into a solution of a certain concentration, centrifuged, and the supernatant was filtered through a 0.45 μm filter membrane; the instrument used was an Agilent 1260 high-performance liquid chromatograph; the detector was a UM4800 evaporative light detector; the column was an Ultrahydrogel™ linear (7.8 × 300 mm); the mobile phase was ultrapure water; and the flow rate was 0.8 mL / min. -1 Temperature: 80℃; Carrier gas: N2 2 Gas flow rate: 2 L / min -1 Injection volume: 10 μL. High-performance gel size exclusion chromatography (HPLC) results are as follows: Figure 1 As shown, the average molecular weight is 41.23 kDa. The monosaccharide composition was determined using an Agilent 8890A-5977B chromatograph. Rhamnose, arabinose, fucose, xylose, mannose, glucose, and galactose were used as standards. 10 mg of sample was accurately weighed and placed in an ampoule, 10 mg of hydroxylamine hydrochloride and 1 mL of pyridine were added, mixed, and reacted at 90 °C for 30 min. After cooling to room temperature, 1 mL of acetic anhydride was added, mixed, and reacted at 90 °C for 30 min. After cooling to room temperature, the mixture was filtered through a 0.22 μm microporous membrane and analyzed by GC-MS. Column: Agilent HP-5MS (30 m × 250 μm × 0.25 μm); Instrument conditions: column gas flow rate: 1 mL / min. -1; carrier gas: He; injection port temperature: 250℃; injection volume: 1 μL; temperature program: 100℃ for 2 min, increased to 220℃ at 10℃·min -1 held for 5 min, solvent delay 3 min. The ion source was electron impact, the collision energy was 70 eV, and the mass scan range was m / s 40-600. The results of the GC-MS detection of the monosaccharide composition are shown in Table 1. Figure 2 As shown in Table 1, the APS prepared in Example 1 is composed of five monosaccharides, i.e., rhamnose, xylose, mannose, glucose and galactose, and the molar ratio is 0.08:0.19:0.09:0.45:0.19.

[0073] To further illustrate the application of the Astragalus polysaccharide prepared by the present application in reducing the deposition of Aβ in C. elegans, the inventors also conducted the following experiments, and the specific content is as follows:

[0074] Experiment 2

[0075] The CL4176 strain of C. elegans was incubated at 16℃, and E. coli OP50 was used as food. An appropriate amount of APS and DAPS prepared in Example 1 was dissolved in ultrapure water to obtain a solution with a mass concentration of 2 mg·mL -1 . The mother liquor was filtered through a sterile filter head, and then E. coli OP50 was added to prepare solutions with mass concentrations of 0.50 mg·mL -1 , 1.00 mg·mL -1 and 2.00 mg·mL -1 , respectively, each of which was recorded as a low, medium or high dose group. 100 μL of each solution was placed on an NGM plate, and three plates were prepared for each mass concentration.

[0076] The lifespan experiment included a blank group (Control) and low, medium and high dose groups of APS and DAPS prepared in Example 1. The CL4176 eggs after synchronization were placed in the blank group and each concentration group, 30 worms per plate. The survival of the worms was recorded every 3 days until all the worms died. The head and tail of the worms were gently touched with a pick, and if there was no response within 30 seconds, the worm was determined to be dead. The results showed that, compared with the blank group, the Astragalus polysaccharides prepared in Example 1 with different concentrations had a certain effect on prolonging the lifespan of the worms (P<0.05), and the effect was more obvious than that of the undegraded APS in each dose group. Among them, the APS and DAPS (1 mg·mL -1 ) groups had the best effect, and the subsequent experiments used a drug concentration of 1 mg·mL -1 . The results are shown in Figure 3 .

[0077] Experiment 3

[0078] Paralysis experiment: a blank group (Control) and Astragalus polysaccharides and Astragalus polysaccharides prepared in Example 1 (1 mg·mL -1The synchronized CL4176 nematode eggs were placed in the control group and the treatment group, 30 eggs per plate, and cultured at 16℃ for 24 hours. They were then transferred to another plate and cultured at 16℃ for another 12 hours, followed by incubation at 25℃ for 36 hours. Afterwards, the eggs were observed under a microscope every 2 hours, and the number of paralyzed nematodes was recorded (paralysis was defined as the nematode's head and body being gently touched several times with a needle, with no movement in any part of the body except the head). The results showed that, compared to the control group, both the Astragalus polysaccharide prepared in Example 1 and the Astragalus degraded polysaccharide had certain anti-nematode paralysis effects. Furthermore, the Astragalus degraded polysaccharide exhibited a better inhibitory effect on CL4176 nematode paralysis than the Astragalus polysaccharide not degraded using the method in Example 1. (See attached figures). Figure 4 .

[0079] Experiment 4

[0080] The heat stress experiment included a control group and a group containing Astragalus polysaccharide and Astragalus degraded polysaccharide prepared in Example 1 (1 mg / mL). -1 The synchronized CL4176 nematode eggs were placed in the control group and the treatment group, 30 eggs per plate, and cultured in an incubator at 16℃ for 3 days. The temperature was then increased to 35℃, and the survival of the nematodes was observed every hour until all nematodes died. The results showed that, compared with the control group, both the Astragalus polysaccharide prepared in Example 1 and the degraded Astragalus polysaccharide could reduce the heat stress damage to the nematodes. Furthermore, the degraded Astragalus polysaccharide showed a significantly higher anti-heat stress effect than the undegraded Astragalus polysaccharide. (See attached figures). Figure 5 .

[0081] The oxidative stress experiment included a control group and a group of Astragalus polysaccharides and Astragalus degraded polysaccharides prepared in Example 1 (1 mg / mL). -1 CL4176 nematodes synchronized to stage L4 were placed in the control group and the treatment group, 30 nematodes per plate, and treated at 16°C for two days. The experimental nematodes were then transferred to NGM plates containing 240 μM juglone. Nematode survival was observed every 1 hour until all nematodes died. Results showed that, compared to the control group, both the Astragalus polysaccharide prepared in Example 1 and the Astragalus-degraded polysaccharide could reduce the oxidative stress damage experienced by the nematodes. The Astragalus polysaccharide, after degradation using the method in Example 1, significantly improved the antioxidant capacity of CL4176 nematodes. (See attached figures). Figure 5 .

[0082] Experiment 5

[0083] Exercise capacity testing experiment: A blank group (Control) and a group consisting of Astragalus polysaccharide and Astragalus degraded polysaccharide prepared in Example 1 (1 mg / mL) were set up. -1). The synchronized CL4176 eggs were placed in the blank group and each concentration of the drug group, 10 per plate, and cultured in a 16℃ incubator, and the culture dish was replaced every 2d. When the worms grew to 10d, an appropriate amount of M9 buffer solution was added, and the number of sinusoidal movements of the worms within 30s was detected under a microscope to determine the change in the movement ability of CL4176 nematodes. The results show that the Astragalus polysaccharide degradation can significantly improve the movement ability of CL4176 nematodes at the 4th day (P<0.01), and the Astragalus polysaccharide can improve the movement ability of CL4176 nematodes to a certain extent, but there is no significant difference. The results are shown in Figure 6 .

[0084] The above experimental results show that the Astragalus polysaccharide prepared by the present application has high extraction rate and sugar content, low protein content, and can prolong the lifespan of nematodes to a certain extent, resist nematode paralysis, reduce heat stress and oxidative stress damage, improve the movement ability, reduce the deposition of Aβ in CL4176 Caenorhabditis elegans, and reduce the toxicity of Aβ in nematodes. The comprehensive data show that the inhibition of Aβ toxicity in CL4176 nematodes by the Astragalus polysaccharide prepared by the present application is higher than that of the polysaccharide not degraded by the present method.

[0085] The embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple, and the related parts can be referred to the method part.

[0086] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing Astragalus membranaceus degraded polysaccharide, characterized in that, Includes the following steps: (1) Add ethanol solution to Astragalus membranaceus for reflux defatting, filter and air dry to obtain defatted Astragalus membranaceus, then add deionized water for soaking, microwave extraction and filtration to obtain filtrate; (2) Add anhydrous ethanol to the filtrate for alcohol precipitation, centrifuge, redissolve the precipitate, concentrate under reduced pressure, freeze dry, and obtain crude Astragalus polysaccharide; (3) Prepare an aqueous solution of crude Astragalus polysaccharide, add papain for enzymatic hydrolysis, then centrifuge and filter to obtain the supernatant, add anhydrous ethanol for alcohol precipitation, centrifuge, redissolve the precipitate, concentrate under reduced pressure, freeze dry to obtain Astragalus polysaccharide; (4) Prepare an aqueous solution of Astragalus polysaccharide, adjust the pH of the solution, and then add H2O2 and Vc. Degrade Astragalus polysaccharide by ultrasound-assisted H2O2 combined with Vc to obtain Astragalus degraded polysaccharide. The ratio of defatted Astragalus membranaceus to deionized water in step (1) is 1:10~14 g∙mL. -1 The volume concentration of the ethanol solution is 75%. The microwave extraction in step (1) uses a power of 500-600 W, a temperature of 70-90℃, a time of 2-3 h, and is performed 3 times. The concentration of the aqueous solution in step (3) is 10~15 mg∙mL. -1 The mass ratio of crude polysaccharide of Astragalus membranaceus to papain is 4~6:1; The concentration of the aqueous solution in step (4) is 10~15 mg∙mL. -1 Adjust the solution pH to 3-4 and the H2O2 concentration to 7.5-10 mmol·L. -1 The mass ratio of H2O2 to VC is 1:0.8 ~1; The temperature of the ultrasound in step (4) is 60~70℃, the power is 800~1000W, and the degradation time is 3~4 h; The average molecular weight of the Astragalus degraded polysaccharide is 41.23 kDa; The monosaccharide component of the Astragalus degraded polysaccharide consists of rhamnose, xylose, mannose, glucose, and galactose in a molar ratio of 0.08:0.19:0.09:0.45:0.

19.

2. The method for preparing Astragalus degraded polysaccharide according to claim 1, characterized in that, In steps (2) and (3): anhydrous ethanol is added until the ethanol volume concentration is 85%; the alcohol precipitation temperature is 4℃ and the time is 48 h; the centrifugation conditions are 3500~4000 r∙min -1 Centrifuge for 15-20 minutes.

3. The method for preparing Astragalus degraded polysaccharide according to claim 1, characterized in that, The enzymatic hydrolysis conditions described in step (3) are as follows: first, enzymatic hydrolysis is carried out at 55°C for 3 h, and then the enzyme is inactivated by heating to 95°C for 10 min.

4. A degraded polysaccharide of Astragalus membranaceus prepared by the method according to any one of claims 1-3.

5. The application of the Astragalus degraded polysaccharide according to claim 4 in the preparation of a drug for treating Alzheimer's disease.

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