A method for preparing and using a low molecular weight polysaccharide

Abstract

This invention belongs to the field of traditional Chinese medicine processing technology, specifically relating to a method for preparing and applying low molecular weight polysaccharides. Taking Astragalus membranaceus as an example, this invention employs ultrasound-assisted H2O2-VC... 2+ -Fe 2+ The system degrades Astragalus polysaccharide (APS) to obtain low molecular weight Astragalus polysaccharide (DAPS). Compared with existing low molecular weight polysaccharide preparation processes, the low molecular weight polysaccharide preparation process of this invention is simple, has a wide range of applications, measurable molecular weight, high yield, and strong reproducibility. The results of the embodiments of this invention show that the low molecular weight Astragalus polysaccharide prepared by this method can prolong the lifespan of CL4176 nematodes, improve the paralysis of CL4176 nematodes, and enhance the nematode's motility and stress resistance. Therefore, the low molecular weight Astragalus polysaccharide prepared by this invention can be applied to the preparation of novel drugs for prolonging lifespan, improving paralysis, and enhancing motility and stress resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a preparation method and application of low-molecular-weight polysaccharides. BACKGROUND

[0002] With the trend of accelerated pace of life and population aging, chronic diseases such as decreased exercise and stress capacity have become a focus of health problems. Under great life pressure, adults are prone to symptoms such as mental tension, slow thinking and depression, resulting in decreased exercise capacity, stress capacity and memory, accompanied by complications such as falls and infections, and severe cases can affect life span. The characteristics of these chronic diseases are abnormal accumulation of intracellular and extracellular neurofibrils, leading to synaptic degeneration, neuronal cell death and gliosis, and exacerbating the neurodegenerative process. Traditional Chinese medicine polysaccharide components have the advantages of multiple pathways and multiple targets, and can inhibit oxidative stress, neuroinflammation, cell apoptosis, regulate autophagy and improve energy metabolism.

[0003] Caenorhabditis elegans (C.elegans) is a nematode belonging to the phylum Nematoda and the class Nematoda, and is one of the important model organisms for studying material anti-aging and oxidative stress. It has a short lifespan, simple cell model, rapid reproduction, stable behavioral response and sensitive and reliable results, and can exhibit a series of characteristics similar to mammals. Protein skinhead-1 (skn-1) has consistency in function with nuclear factor E2 related factor 2 (Nrf2) protein in mammals, and can exert similar antioxidant effects in vivo.

[0004] Radix Astragali is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.), a traditional Chinese medicine plant for tonifying the spleen, invigorating the middle, tonifying qi and ascending yang, and tonifying kidney qi. Radix Astragali contains polysaccharides, saponins, flavonoids and other chemical components, and has anti-inflammatory, anti-fibrosis and anti-aging activities. Among them, Astragalus polysaccharide (APS) is one of the main chemical components extracted from the dried root of Radix Astragali, and is widely used. It has good immune-enhancing, antioxidant and anti-inflammatory effects. Natural polysaccharides have the characteristics of large molecular weight, high viscosity and poor water solubility. A large molecular weight can cause obstacles in crossing the cell membrane, making it difficult to enter the cells and exert biological activity. Compared with polysaccharides, oligosaccharides have the characteristics of small molecular weight, simple structure, and being beneficial to human absorption and utilization.

[0005] At present, low molecular weight polysaccharides can be obtained by degradation, and the polysaccharide degradation methods mainly include physical degradation method, chemical degradation method and biological degradation method. In recent years, H2O2 oxidation method is a polysaccharide degradation method which is applied more frequently, and the glycosidic bond of polysaccharide is oxidized and broken by using the hydroxyl radicals generated by the decomposition of H2O2, and the method is green and controllable. However, the polysaccharide degradation process by the spontaneous generation of hydroxyl radicals from H2O2 is slow.

[0006] Therefore, it is urgent to develop a method for rapidly and efficiently degrading polysaccharides. SUMMARY

[0007] The purpose of the present application is to provide a preparation method and application of low molecular weight polysaccharides, and the method provided by the present application has simple preparation process, wide application range, high yield and strong reproducibility.

[0008] In order to achieve the above purpose, the present application provides the following technical scheme:

[0009] The present application provides a preparation method of low molecular weight polysaccharides, which specifically comprises the following steps:

[0010] S1, the raw material is refluxed with ethanol to remove grease, deionized water is added to the residue, ultrasonic extraction is carried out, and crude polysaccharide is obtained by alcohol precipitation;

[0011] S2, after the crude polysaccharide aqueous solution is enzymatically hydrolyzed, dialysis and alcohol precipitation are carried out, and polysaccharide is obtained;

[0012] S3, the polysaccharide is degraded by ultrasonic-assisted H2O2-VC 2+ -Fe 2+ system, and degraded polysaccharide is obtained;

[0013] S4, the degraded polysaccharide is eluted by DEAE-650M cellulose chromatographic column with distilled water to obtain low molecular weight polysaccharide;

[0014] The H2O2-VC 2+ -Fe 2+ system includes H2O2, VC and ferric sulfate.

[0015] Preferably, the raw material is Astragalus membranaceus, Acanthopanax or Semecarpus.

[0016] Preferably, the ratio of the residue to deionized water in step S1 is 1:(14-18)g / mL.

[0017] Preferably, the enzyme used in step S2 is subtilisin, and the concentration of subtilisin in the enzyme solution is 2-4mg / mL.

[0018] Preferably, the mass ratio of H2O2, VC and ferric sulfate added in step S3 is 1:1:(0.8-1.2).

[0019] The present application also provides a low molecular weight astragalus polysaccharide prepared by the above preparation method.

[0020] Preferably, the monosaccharide components of the low molecular weight astragalus polysaccharide include Glc and Gal, with a molar ratio of 1:0.054.

[0021] The present application also provides the use of the low molecular weight astragalus polysaccharide prepared by the above preparation method or the above low molecular weight astragalus polysaccharide, characterized in that the low molecular weight astragalus polysaccharide has the ability to prolong the lifespan of CL4176 nematodes, improve the anti-paralysis effect, movement ability and / or oxidative stress of the CL4176 nematodes.

[0022] The present application also provides the use of the low molecular weight astragalus polysaccharide prepared by the above preparation method or the above low molecular weight astragalus polysaccharide in the preparation of a product and / or a medicine for prolonging lifespan.

[0023] The present application also provides the use of the low molecular weight astragalus polysaccharide prepared by the above preparation method or the above low molecular weight astragalus polysaccharide in the preparation of a product and / or a medicine for improving paralysis.

[0024] The present application has the following advantages:

[0025] (1) The present application uses a composite degradation method to degrade polysaccharides, which not only solves the problems of poor solubility and low bioavailability of high molecular weight polysaccharides, but also improves the purity of polysaccharides and the biological activity of polysaccharides.

[0026] (2) The preparation method disclosed by the present application has the advantages of simple preparation process, wide application range, low molecular weight, measurable molecular weight, high yield and strong reproducibility.

[0027] (3) The low molecular weight astragalus polysaccharide DAPS prepared by the preparation method of the present application can prolong the lifespan of CL4176 nematodes, improve the paralysis of CL4176 nematodes, and enhance the movement ability and stress resistance of the nematodes. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0029] Figure 1 HPGPC spectrum of DAPS prepared by the present application;

[0030] Figure 2The GC-MS chromatograms of the mixed monosaccharide standard derivatives (mixed standard) and the monosaccharide composition parts of the DAPS prepared in Example 1 are as follows: 1. Man; 2. Rha; 3. GlcA; 4. GalA; 5. Glc; 6. Gal; 7. Xyl; 8. Ara; 9. Fuc;

[0031] Figure 3 Effects of APS and DAPS prepared in Example 1 on the lifespan of CL4176 nematodes;

[0032] Figure 4 Effects of APS and DAPS prepared in Example 1 on the paralysis rate of CL4176 nematodes;

[0033] Figure 5 Effects of APS and DAPS prepared in Example 1 on the oxidative stress and heat stress capacity of CL4176 nematodes;

[0034] Figure 6 Effects of APS and DAPS prepared in Example 1 on the movement ability of CL4176 nematodes. DETAILED DESCRIPTION

[0035] The present application provides a preparation process of low molecular weight polysaccharide, which specifically comprises: adding an ethanol solution to the raw material for reflux degreasing, filtering and drying in the shade to obtain degreased material, then adding deionized water at a solid-liquid ratio of 1:14-18 g / mL, preferably 1:16 g / mL, ultrasonic extraction, and filtering to obtain a filtrate; adding anhydrous ethanol to the filtrate for alcohol precipitation, reducing pressure concentration, and freeze-drying to obtain crude polysaccharide.

[0036] The crude polysaccharide is configured into an aqueous solution, the pH of the solution is adjusted, and subtilisin is added for enzymatic hydrolysis to make the concentration of subtilisin in the solution between 2-4 mg / mL, preferably 3 mg / mL. Centrifugation and suction filtration are performed to obtain supernatant, dialysis is performed, anhydrous ethanol is added for alcohol precipitation, reducing pressure concentration is performed, and freeze-drying is performed to obtain polysaccharide.

[0037] The polysaccharide is configured into an aqueous solution, the pH of the solution is adjusted, and then H2O2, VC and ferric sulfate are added. The mass ratio of H2O2, VC and ferric sulfate is 1:1:(0.8-1.2), preferably 1:1:1.2. The polysaccharide is degraded by ultrasonic-assisted H2O2-VC 2+ -Fe 2+ degradation. The degraded polysaccharide is resuspended with distilled water, the supernatant is taken and loaded, and the DEAE-650M cellulose chromatographic column is used to separate and purify the degraded polysaccharide to obtain low molecular weight polysaccharide.

[0038] The raw material in the above preparation method includes but is not limited to Astragalus membranaceus, Acanthopanax or Semecarpus.

[0039] The embodiment of the present application provides a preparation process of low molecular weight astragalus polysaccharide by using the preparation method. 2+ -Fe 2+ The system degrades astragalus polysaccharide (APS) to obtain low molecular weight astragalus polysaccharide (DAPS), and VC reduces Fe 3+ to Fe 2+ H2O2 can oxidize VC to generate hydroxyl radicals rapidly, and H2O2 and Fe 2+ perform Fenton reaction to generate hydroxyl radicals, hydrogen abstraction reaction occurs, the glycosidic bond is broken, and the degradation rate of polysaccharide is obviously improved. Meanwhile, the ultrasonic-assisted oxidative degradation can combine the advantages of the two methods and play a synergistic role.

[0040] The preparation process of the low molecular weight astragalus polysaccharide specifically comprises the following steps: adding an ethanol solution to astragalus to perform reflux degreasing, filtering and drying in the dark to obtain degreased astragalus, then adding deionized water to soak according to a material-liquid ratio of 1:14-18 g / mL, preferably 1:16 g / mL, ultrasonic extraction and filtering to obtain a filtrate; adding anhydrous ethanol to the filtrate to perform alcohol precipitation, reducing pressure concentration, and freeze-drying to obtain astragalus crude polysaccharide.

[0041] The astragalus crude polysaccharide is configured into an aqueous solution, the pH of the solution is adjusted, and the bacillus subtilis protease is added to perform enzymolysis, so that the concentration of the bacillus subtilis protease in the solution is between 2-4 mg / mL, and preferably 3 mg / mL. Centrifugation, suction filtration, dialysis, alcohol precipitation with anhydrous ethanol, reducing pressure concentration and freeze-drying are performed to obtain astragalus polysaccharide (APS).

[0042] The APS is configured into an aqueous solution, the pH of the solution is adjusted, and then H2O2, VC and ferric sulfate are added, and the mass ratio of H2O2, VC and ferric sulfate is 1:1:(0.8-1.2), preferably 1:1:1.2. Ultrasonic-assisted H2O2-VC 2+ -Fe 2+ degrades astragalus polysaccharide to obtain astragalus degraded polysaccharide. The astragalus degraded polysaccharide is redissolved in distilled water, the supernatant is taken and loaded, and the supernatant is separated and purified by passing through a DEAE-650M cellulose chromatographic column with distilled water to obtain low molecular weight astragalus polysaccharide (DAPS).

[0043] The low molecular weight astragalus polysaccharide prepared by the present application has an average molecular weight of 42.34 kDa, and the monosaccharide structure mainly comprises Glc and Gal, and the molar ratio is 1:0.054. The growth state of Caenorhabditis elegans is studied by using the low molecular weight astragalus polysaccharide prepared by the present application, and the research results show that the DAPS can prolong the life of the nematode to a certain extent, improve the antioxidant stress resistance and movement ability of CL4176 nematodes, and has a significant anti-nematode paralysis effect.

[0044] Further, the low molecular weight astragalus polysaccharide prepared by the method provides a new direction for researching medicines and / or products for inhibiting neuroinflammation and improving exercise capacity.

[0045] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the present application.

[0046] The production process, experimental method or detection method involved in the embodiments of the present application are all conventional methods in the prior art without special description, and the name and / or abbreviation thereof all belong to the conventional name in the field, which is very clear and definite in the related application field, and the person skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the conventional conditions or the conditions suggested by the manufacturer.

[0047] The various instruments, equipment, raw materials or reagents used in the embodiments of the present application do not have special restrictions on the source, and are conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional method well known to the person skilled in the art.

[0048] Example 1

[0049] A preparation method of low molecular weight astragalus polysaccharide, comprising the following steps:

[0050] 2kg of astragalus is weighed into a round-bottom flask, 25L of 75% ethanol is added for degreasing reflux for 2h, which is repeated for 3 times, the filter residue is dried in the dark after filtration, deionized water is added at a solid-liquid ratio of 1:16g / mL, extraction is carried out at 85℃ under ultrasonic power of 700W for 3h, the filtrate is obtained after filtration, the extraction is carried out for 3 times, the filtrate is combined, the filtrate is concentrated to 3L, anhydrous ethanol is added to reach an ethanol volume concentration of 85%, stirring is uniform, alcohol precipitation is carried out at 4℃ for 48h, centrifugation is carried out at 4200r / min for 20min, the precipitate is redissolved, vacuum concentration is carried out, and freeze-drying is carried out to obtain astragalus crude polysaccharide.

[0051] The astragalus crude polysaccharide powder is configured into a 15mg / mL aqueous solution, the pH of the solution is adjusted to 9-10, the aqueous solution is added with bacillus subtilis protease to make the concentration of the bacillus subtilis protease 3mg / mL, uniform stirring is carried out, enzyme hydrolysis is carried out at 50℃ for 1h, then heating is carried out to 95℃ for 10min to inactivate the enzyme, centrifugation is carried out at 4200r / min for 20-30min, the supernatant is reserved, dialysis is carried out for 48h, then anhydrous ethanol is added to reach an ethanol volume concentration of 85%, stirring is uniform, alcohol precipitation is carried out at 4℃ for 48h, centrifugation is carried out at 4200r / min for 20min, the precipitate is redissolved, vacuum concentration is carried out, and freeze-drying is carried out to obtain astragalus polysaccharide (APS).

[0052] The APS is configured into a 15 mg / mL aqueous solution, 1 mol / L HCL solution is added to adjust the pH to 4, H2O2 is added to make the concentration of H2O2 9 mmol / L, Vc and ferric sulfate are added to make the mass ratio of H2O2, VC and ferric sulfate 1:1:1.2, and the ultrasonic-assisted H2O2-VC is carried out at 70 DEG C. 2+ -Fe 2+ The degradation is carried out for 3 hours, the ultrasonic power is 900 W, then anhydrous ethanol is added to make the volume concentration of ethanol 85%, stirring is uniform, standing is carried out at 4 DEG C for 48 hours, centrifugation is carried out at 4200 r / min for 20 min, the precipitate is redissolved, vacuum concentration is carried out, and freeze-drying is carried out to obtain the degraded polysaccharide of Astragalus membranaceus, which is called degraded polysaccharide of Astragalus membranaceus 3 g.

[0053] The polysaccharide extraction rate formula is as follows:

[0054] The content of the polysaccharide is determined by the phenol-sulfuric acid method, 1 mg / mL glucose solution is prepared, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the solution are accurately transferred into 10 mL test tubes with stoppers, 1 mL of distilled water is added, 1 mL of 6% phenol solution is added, stirring is uniform, 5 mL of concentrated sulfuric acid is slowly added dropwise, stirring is uniform, heating is carried out at 75 DEG C for 10 min, cooling is carried out to room temperature, the absorbance (A) is determined at 490 nm, a standard curve is drawn with the glucose concentration as the x-axis and the A value as the y-axis, and the glucose standard curve regression equation is y = 2.6685x + 0.0787, R = 0.9994, 1 mL of the polysaccharide sample solution with a concentration of 1 mg / mL is prepared, three parallel experiments are carried out according to the above method, the average value is taken, the absorbance A is determined, and the sugar content is calculated by substituting the standard curve. 2

[0055] The extraction rate of the DAPS obtained in Example 1 is 11.24%, the sugar content is 82.51%, and the protein content is 2.12%.

[0056] 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 optimize the feed liquid ratio of defatted Astragalus membranaceus and deionized water, the mass ratio of Astragalus membranaceus crude polysaccharide and subtilisin, and the mass ratio of H2O2, VC and ferric sulfate, and the specific contents are as follows.

[0057] Example 2

[0058] A preparation method of low molecular weight Astragalus polysaccharide, comprising the following steps:

[0059] ​Take 3 parts of defatted Astragalus membranaceus 100g, respectively, soak in 1.4L, 1.6L, 1.8L deionized water, under the ultrasonic power of 700W, 85℃, extract 3h, extract 3 times, combine the filtrate, add anhydrous ethanol to the ethanol volume concentration of 85%, stir evenly, 4℃ under static 48h alcohol precipitation, centrifuged at 4200r / min for 20min, take the precipitate redissolution, reduced pressure concentration, freeze-drying, Astragalus membranaceus polysaccharide.

[0060] Astragalus membranaceus polysaccharide powder is configured into a 15mg / mL aqueous solution, the pH of the solution is adjusted to 9-10, Bacillus subtilis protease is added to the aqueous solution to make the concentration of Bacillus subtilis protease 3mg / mL, stirred evenly, enzymolysis at 50℃ for 1h, then heated to 95℃ to inactivate the enzyme for 10min, centrifuged at 4200r / min for 20min, the supernatant is reserved, dialyzed for 48h, then anhydrous ethanol is added to the ethanol volume concentration of 85%, stirred evenly, 4℃ under static 48h alcohol precipitation, centrifuged at 4200r / min for 20min, take the precipitate redissolution, reduced pressure concentration, freeze-drying, Astragalus polysaccharide (APS) is obtained.

[0061] APS is configured into a 15mg / mL aqueous solution, 1mol / L HCL solution is added to adjust the pH to 4, H2O2 is added to make the concentration of H2O2 9mmol / L, Vc and ferric sulfate are added to make the mass ratio of H2O2, VC and ferric sulfate 1:1:1.2, ultrasonic assisted H2O2-VC 2+ -Fe 2+ degradation at 70℃ for 3h, the ultrasonic power is 900W, then anhydrous ethanol is added to the ethanol volume concentration of 85%, stirred evenly, 4℃ under static 48h, centrifuged at 4200r / min for 20min, take the precipitate redissolution, reduced pressure concentration, freeze-drying, Astragalus degradation polysaccharide is obtained, 3g of Astragalus degradation polysaccharide is added to distilled water to redissolve, centrifuged, the supernatant is taken and loaded, adsorbed for 2h, eluted by DEAE-650M cellulose chromatography column with distilled water to obtain DAPS1, DAPS2 and DAPS3.

[0062] According to the different ratios of defatted Astragalus membranaceus and deionized water, the extraction rate of DAPS1 is 9.44%, the sugar content is 78.23%, and the protein content is 2.55%; the extraction rate of DAPS2 is 11.24%, the sugar content is 82.51%, and the protein content is 2.12%; the extraction rate of DAPS3 is 10.41%, the sugar content is 78.93%, and the protein content is 2.37%. Therefore, the volume ratio of Astragalus membranaceus to deionized water is preferably 1:16 for preparation.

[0063] Example 3

[0064] A method for preparing a low molecular weight Astragalus polysaccharide, comprising the following steps:

[0065] Take 3 parts of defatted Astragalus membranaceus 100 g, respectively, and soak in 1.6 L of deionized water. Extract at 85°C under ultrasonic power of 700 W for 3 h, 3 times. Combine the filtrates, add anhydrous ethanol to a volume concentration of 85%, stir well, and stand at 4°C for 48 h. Centrifuge at 4200 r / min for 20 min, redissolve the precipitate, reduce pressure, and freeze-dry to obtain Astragalus membranaceus crude polysaccharide.

[0066] Prepare an aqueous solution of Astragalus membranaceus crude polysaccharide powder at 15 mg / mL, adjust the pH of the solution to 9-10, add Bacillus subtilis protease to the aqueous solution to make the concentration of Bacillus subtilis protease 2 mg / mL, 3 mg / mL, and 4 mg / mL, stir well, and enzymatically hydrolyze at 50°C for 1 h. Then heat to 95°C to inactivate the enzyme for 10 min, centrifuge at 4200 r / min for 20 min, retain the supernatant, dialyze for 48 h, then add anhydrous ethanol to a volume concentration of 85%, stir well, and stand at 4°C for 48 h. Centrifuge at 4200 r / min for 20 min, redissolve the precipitate, reduce pressure, and freeze-dry to obtain Astragalus polysaccharide (APS).

[0067] Prepare an aqueous solution of APS at 15 mg / mL, adjust the pH to 4 by adding 1 mol / L HCL solution, add H2O2 to make the concentration of H2O2 9 mmol / L, and add Vc and ferric sulfate to make the mass ratio of H2O2, Vc, and ferric sulfate 1:1:1.2. Ultrasonically assist H2O2-VC 2+ -Fe 2+ degradation at 70°C for 3 h, with ultrasonic power of 900 W. Then add anhydrous ethanol to a volume concentration of 85%, stir well, stand at 4°C for 48 h, centrifuge at 4200 r / min for 20 min, redissolve the precipitate, reduce pressure, and freeze-dry to obtain Astragalus degradation polysaccharide. Take 3.00 g of Astragalus degradation polysaccharide, redissolve with distilled water, centrifuge, take the supernatant, and adsorb for 2 h. Elute the DEAE-650M cellulose chromatography column with distilled water to obtain DAPS4, DAPS5, and DAPS6.

[0068] Optimize according to the mass ratio of Astragalus membranaceus crude polysaccharide to Bacillus subtilis protease: the extraction rate of DAPS4 is 10.53%, the sugar content is 77.43%, and the protein content is 4.23%; the extraction rate of DAPS5 is 11.24%, the sugar content is 82.51%, and the protein content is 2.12%; and the extraction rate of DAPS6 is 9.38%, the sugar content is 80.21%, and the protein content is 2.31%. Therefore, the mass ratio of Astragalus membranaceus crude polysaccharide to Bacillus subtilis protease is preferably 3:1 for preparation.

[0069] Example 4

[0070] A preparation method of low molecular weight astragalus polysaccharide, comprising the following steps:

[0071] Respectively, take 3 parts of defatted astragalus 100g, soak in 1.6L deionized water, under the ultrasonic power of 700W, 85℃, extract for 3h, extract for 3 times, combine the filtrate, add anhydrous ethanol to the ethanol volume concentration of 85%, stir evenly, 4℃ under static 48h alcohol precipitation, centrifuge at 4200r / min for 20min, take the precipitate redissolution, reduce pressure concentration, freeze drying, get astragalus polysaccharide.

[0072] The astragalus polysaccharide powder is configured into 15mg / mL aqueous solution, the solution pH is adjusted to 9-10, the aqueous solution is added with bacillus subtilis protease, so that the concentration of bacillus subtilis protease is 3mg / mL, and then stirred uniformly, and then enzymolysis is carried out at 50℃ for 1h, and then heated to 95℃ for 10min to inactivate the enzyme, and then centrifuged at 4200r / min for 20min, and then the supernatant is reserved, and then dialyzed for 48h, and then added with anhydrous ethanol to the ethanol volume concentration of 85%, and then stirred evenly, and then placed at 4℃ for 48h alcohol precipitation, and then centrifuged at 4200r / min for 20min, and then the precipitate is redissolved, and then reduced pressure concentration, and then freeze dried to obtain astragalus polysaccharide (APS).

[0073] The APS is configured into 15mg / mL aqueous solution, 1mol / L HCL solution is added to adjust the pH to 4, H2O2 is added to make the concentration of H2O2 9mmol / L, Vc and ferric sulfate are added to make the mass ratio of H2O2, VC and ferric sulfate 1:1:0.8, 1:1:1 and 1:1:1.2, and then ultrasonic assisted H2O2-VC 2+ -Fe 2+ Degradation is carried out at 70℃ for 3h, the ultrasonic power is 900W, and then anhydrous ethanol is added to the ethanol volume concentration of 85%, and then stirred evenly, and then placed at 4℃ for 48h, and then centrifuged at 4200r / min for 20min, and then the precipitate is redissolved, and then reduced pressure concentration, and then freeze dried to obtain DAPS7, DAPS8 and DAPS9.

[0074] According to the mass ratio of H2O2, VC and Fe 2+ , the DAPS7 has an extraction rate of 10.32%, a sugar content of 79.11% and a protein content of 2.77%; the DAPS8 has an extraction rate of 11.24%, a sugar content of 82.51% and a protein content of 2.12%; and the DAPS9 has an extraction rate of 10.24%, a sugar content of 81.12% and a protein content of 2.23%. Therefore, the mass ratio of H2O2, VC and ferric sulfate is preferably 1:1:1.2 for preparation.

[0075] In addition, in order to highlight the excellent effect of the disclosed technical scheme of the present application compared with the prior art, the following comparative experiments are carried out:

[0076] Comparative Example 1

[0077] A preparation method of low molecular weight astragalus polysaccharide, comprising the following steps:

[0078] Take 2 kg of astragalus in a round-bottom flask, add 25 L of 75% ethanol to reflux for 2 h, repeat 3 times, dry the filter residue, add deionized water at a solid-liquid ratio of 1:16 g / mL, extract at 85°C under ultrasonic power of 700 W for 3 h, filter to obtain the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 3 L, add anhydrous ethanol to a volume concentration of 85%, stir evenly, stand at 4°C for 48 h, centrifuge at 4200 r / min for 20 min, redissolve the precipitate, reduce pressure and concentrate, freeze-dry to obtain crude astragalus polysaccharide.

[0079] Prepare a 15 mg / mL aqueous solution of crude astragalus polysaccharide powder, adjust the pH of the solution to 9-10, add bacillus subtilis protease to the aqueous solution to make the concentration of bacillus subtilis protease 3 mg / mL, stir evenly, and enzymatically hydrolyze at 50°C for 1 h, then heat to 95°C to inactivate the enzyme for 10 min, centrifuge at 4200 r / min for 20-30 min, retain the supernatant, dialyze for 48 h, then add anhydrous ethanol to a volume concentration of 85%, stir evenly, stand at 4°C for 48 h, centrifuge at 4200 r / min for 20 min, redissolve the precipitate, reduce pressure and concentrate, freeze-dry to obtain astragalus polysaccharide (APS).

[0080] Prepare a 15 mg / mL aqueous solution of APS, adjust the pH to 4 by adding 1 mol / L HCL solution, add H2O2 to make the concentration of H2O2 9 mmol / L, add Vc and ferric sulfate to make the mass ratio of H2O2, VC, and ferric sulfate 1:1:1.2, and then hydrolyze at 70°C for 3 h, then add anhydrous ethanol to a volume concentration of 85%, stir evenly, stand at 4°C for 48 h, centrifuge at 4200 r / min for 20 min, redissolve the precipitate, reduce pressure and concentrate, freeze-dry to obtain astragalus polysaccharide degradation product, take 3 g of astragalus polysaccharide degradation product, dissolve with distilled water, centrifuge, take the supernatant, and adsorb for 2 h, then elute with a DEAE-650M cellulose chromatography column to obtain DAPS-A. 2+ -Fe 2+

[0081] The polysaccharide extraction rate formula is:

[0082] ​The content of polysaccharide is determined by phenol-sulfuric acid method. 1 mg / mL glucose solution is prepared, 0.2, 0.4, 0.6, 0.8, 1.0 mL is precisely transferred into 10 mL test tube with stopper, 1 mL of distilled water is added, 1 mL of 6% phenol solution is added, shaken, 5 mL of concentrated sulfuric acid is slowly added dropwise, shaken, heated at 75 ℃ for 10 min, cooled to room temperature, and its absorbance (A) is determined at 490 nm. The standard curve is drawn with glucose concentration as x-axis and A value as y-axis, and the glucose standard curve regression equation is y = 2.6685x + 0.0787, R = 0.9994. 1 mg / mL polysaccharide sample solution is prepared, 1 mL is taken, and the above method is used for three parallel experiments, and the average value is taken to determine the absorbance A, and the sugar content is calculated by substituting the standard curve. 2 =0.9994, 1 mg / mL polysaccharide sample solution is prepared, 1 mL is taken, and the above method is used for three parallel experiments, and the average value is taken to determine the absorbance A, and the sugar content is calculated by substituting the standard curve.

[0083] The DAPS-A extraction rate is 8.11%, and the sugar content is 74.61%. It can be seen that the extraction rate and sugar content of the DAPS subjected to ultrasonic assisted degradation are obviously higher than those of the DAPS not subjected to ultrasonic assisted degradation.

[0084] Comparative Example 2

[0085] A preparation method of low molecular weight astragalus polysaccharide, comprising the following steps:

[0086] 2 kg of astragalus is weighed into a round-bottom flask, 25 L of 75% ethanol is added, and defatting is carried out under reflux for 2 h, which is repeated for 3 times. After filtration, the filter residue is dried in the dark. Deionized water is added at a solid-liquid ratio of 1:16 g / mL. At 85 ℃, the ultrasonic power is 700 W, and the extraction is carried out for 3 h. The filtrate is filtered, and the extraction is carried out for 3 times. The filtrates are combined, and the filtrate is concentrated to 3 L. Anhydrous ethanol is added to reach an ethanol volume concentration of 85%. After stirring, it is placed at 4 ℃ for 48 h for alcohol precipitation. Centrifugation is carried out at 4200 r / min for 20 min. The precipitate is redissolved, concentrated under reduced pressure, and freeze-dried to obtain astragalus crude polysaccharide.

[0087] The astragalus crude polysaccharide powder is configured into a 15 mg / mL aqueous solution. The Sevage method is used to remove protein, and centrifugation is carried out at 4200 r / min for 20-30 min. The supernatant is reserved, dialysis is carried out for 48 h, and then anhydrous ethanol is added to reach an ethanol volume concentration of 85%. After stirring, it is placed at 4 ℃ for 48 h for alcohol precipitation. Centrifugation is carried out at 4200 r / min for 20 min. The precipitate is redissolved, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0088] The APS is configured into a 15 mg / mL aqueous solution. 1 mol / L HCL solution is added to adjust the pH to 4. H2O2 is added to make the H2O2 concentration 9 mmol / L. Vc and ferric sulfate are added to make the mass ratio of H2O2, VC, and ferric sulfate 1:1:1.2. Ultrasonic assisted H2O2-VC-Fe is carried out at 70 ℃. 2+ -Fe2+ Degradation 3h, the ultrasonic power is 900W, then add anhydrous ethanol to the ethanol volume concentration of 85%, stirring, 4℃ for 48h, centrifugation at 4200r / min for 20min, take the precipitate redissolved, reduced pressure concentration, freeze-drying, get Astragalus polysaccharide degradation, called Astragalus polysaccharide degradation 3g, dissolved with distilled water, centrifugation, take the supernatant sample, adsorbed 2h, eluted with distilled water through DEAE-650M cellulose chromatography column to get DAPS-B.

[0089] The polysaccharide extraction rate formula is:

[0090] The content of polysaccharide is determined by phenol-sulfuric acid method, prepare 1mg / mL glucose solution, accurately transfer 0.2, 0.4, 0.6, 0.8, 1.0mL to 10mL stoppered test tube, add distilled water to 1mL, add 6% phenol solution 1mL, shake well, slowly drop 5mL of concentrated sulfuric acid, shake well, heat at 75℃ for 10min, cool to room temperature, measure its absorbance (A) at 490nm, draw the standard curve with glucose concentration as x axis and A value as y axis, get the glucose standard curve regression equation: y=2.6685x+0.0787, R 2 =0.9994, prepare 1mg / mL polysaccharide sample solution, take 1mL, parallel three times according to the above method, take the average value, measure its absorbance A, and calculate the sugar content by substituting the standard curve.

[0091] The preparation of DAPS-B extraction rate is 12.56%, the sugar content is 76.32%, and the protein content is 5.34%. It can be seen that the protein removal rate of Sevage method is lower than that of enzyme method, and the removal process is very complex, and the polysaccharide loss rate is also high.

[0092] Comparative example 3

[0093] A preparation method of a low molecular weight Astragalus polysaccharide, comprising the following steps:

[0094] Take 2kg of Astragalus to a round bottom flask, add 25L of 75% ethanol to defat for 2h, repeat 3 times, filter the residue and dry in the shade, add deionized water at a solid-liquid ratio of 1:16g / mL, extract at 85℃ under ultrasonic power of 700W for 3h, filter to get filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 3L, add anhydrous ethanol to the ethanol volume concentration of 85%, stir, stand at 4℃ for 48h, alcohol precipitation, centrifugation at 4200r / min for 20min, take the precipitate redissolved, reduced pressure concentration, freeze-drying, get Astragalus crude polysaccharide.

[0095] The crude astragalus polysaccharide powder is configured into a 15 mg / mL aqueous solution, the pH of the solution is adjusted to 9-10, the aqueous solution is added with bacillus subtilis protease to make the concentration of the bacillus subtilis protease 3 mg / mL, and then stirred uniformly, and subjected to enzymolysis at 50°C for 1 h, and then heated to 95°C for 10 min to inactivate the enzyme, and then centrifuged at 4200 r / min for 20-30 min, and then the supernatant is reserved, and subjected to dialysis for 48 h, and then added with anhydrous ethanol to make the volume concentration of the ethanol 85%, and then stirred uniformly, and then placed at 4°C for 48 h for alcohol precipitation, and then centrifuged at 4200 r / min for 20 min, and then the precipitate is redissolved, and then subjected to vacuum concentration, and then subjected to freeze-drying to obtain astragalus polysaccharide (APS).

[0096] The APS is configured into a 15 mg / mL aqueous solution, and then added with 1 mol / L HCL solution to adjust the pH to 4, and then added with H2O2 to make the concentration of the H2O2 9 mmol / L, and then added with Vc and iron sulfate to make the mass ratio of the H2O2, Vc and iron sulfate 1:1:1.2, and then subjected to ultrasonic-assisted H2O2-Vc-Fe3+ degradation at 70°C. 2+ -Fe 2+ The degradation is performed for 3 h with an ultrasonic power of 900 W, and then added with anhydrous ethanol to make the volume concentration of the ethanol 85%, and then stirred uniformly, and then placed at 4°C for 48 h, and then centrifuged at 4200 r / min for 20 min, and then the precipitate is redissolved, and then subjected to vacuum concentration, and then subjected to freeze-drying to obtain astragalus degraded polysaccharide, and then 3 g of the astragalus degraded polysaccharide is dissolved with distilled water, and then centrifuged, and then the supernatant is taken and subjected to adsorption for 2 h, and then subjected to DEAE-650M cellulose chromatography column elution with 0.1 mol / L NaCl and 0.2 mol / L NaCl in sequence to obtain DAPS-C and DAPS-D.

[0097] The polysaccharide extraction rate formula is:

[0098] The content of the polysaccharide is determined by the phenol-sulfuric acid method, 1 mg / mL glucose solution is prepared, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the solution is accurately transferred into 10 mL stoppered test tubes, 1 mL of distilled water is added, 1 mL of 6% phenol solution is added, stirred uniformly, 5 mL of concentrated sulfuric acid is slowly added dropwise, stirred uniformly, heated at 75°C for 10 min, cooled to room temperature, and then the absorbance (A) is determined at 490 nm, and then a standard curve is drawn with the glucose concentration as the x-axis and the A value as the y-axis, and then a glucose standard curve regression equation y=2.6685x+0.0787, R=0.9994 is obtained, 1 mg / mL polysaccharide sample solution is prepared, 1 mL of the solution is taken, and then the above method is used for three parallel experiments, and then the average value is taken, and then the absorbance A is determined, and then the sugar content is calculated by substituting the standard curve. 2

[0099] ​The DAPS-C extraction rate was 9.75%, the sugar content was 80.51%, and the molecular weight was 6853.263 Da; the DAPS-D extraction rate was 10.12%, the sugar content was 81.34%, and the molecular weight was 39934.83 Da. Thus, it can be seen that the molecular weight of the DAPS eluted by distilled water is significantly lower than that of the DAPS eluted by NaCl.

[0100] Example 5: Molecular weight determination and monosaccharide composition analysis

[0101] The DAPS obtained in Example 1 was detected: the molecular weight Mw of the DAPS was determined by high-performance gel permeation chromatography (HPGPC), and 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 for sample injection was an Agilent 1260 high-performance liquid chromatograph; the detector was a UM4800 evaporative light detector; the chromatographic column was an UltrahydrogelTMlinear (7.8 x 300 mm); the mobile phase was ultrapure water; the flow rate was 0.8 mL / min; the temperature was 80°C; the carrier gas was N2; the gas flow rate was 2 L / min; and the sample injection volume was 10 μL.

[0102] The high-performance gel exclusion chromatography detection results are shown in Figure 1 , and the average molecular weight is 42.34 kDa.

[0103] Agilent 8890A-5977B was used to determine the monosaccharide composition. Rhamnose, arabinose, fucose, xylose, mannose, glucose, and galactose were used as standard samples. 10 mg of the sample was accurately weighed into an ampoule, 10 mg of hydroxylamine hydrochloride and 1 mL of pyridine were added, mixed, reacted at 90°C for 30 min, cooled to room temperature, 1 mL of acetic anhydride was added, mixed, reacted at 90°C for 30 min, cooled to room temperature, filtered through a 0.22 μm microporous filter, and analyzed by GC-MS. The chromatographic column was an Agilent HP-5MS (30 m x 250 μm x 0.25 μm); the instrument conditions were as follows: the chromatographic column gas flow rate was 1 mL / min; the carrier gas was He; the injection port temperature was 250°C; the injection volume was 1 μL; the temperature program was 100°C for 2 min, increased to 220°C at a rate of 10°C / min, and maintained for 5 min, with a solvent delay of 3 min. The ion source was an electron impact ion source, the collision energy was 70 eV, and the mass scan range was m / s 40-600.

[0104] The GC-MS detection results of the monosaccharide composition are shown in Figure 2 , which is mainly composed of Glc and Gal, with a molar ratio of 1:0.054.

[0105] In order to further illustrate the application of the low molecular weight astragalus polysaccharide prepared by the present application in prolonging life and improving exercise capacity, the inventors also carried out the following experiments, and the specific content is as follows.

[0106] Example 6 Life experiment of Caenorhabditis elegans CL4176

[0107] The CL4176 strain of nematodes was cultured at a constant temperature of 16°C, and Escherichia coli OP50 was used as food. An appropriate amount of APS and DAPS prepared in Example 1 was dissolved in ultrapure water to form a 2 mg / mL mother liquor, and after filtration with a sterile filter head, the Escherichia coli OP50 was added to form a solution with a mass concentration of 0.50 mg / mL, 1.00 mg / mL and 2.00 mg / mL, respectively, which were recorded as low, medium and high dose groups, respectively. 100 μL of each was placed on an NGM plate, and 3 plates were prepared for each mass concentration.

[0108] A blank group (Control) and low, medium and high dose groups of APS and DAPS prepared in Example 1 were set. The CL4176 eggs after synchronization were placed in the blank group and each concentration group, 30 per plate, and the survival of the nematodes was recorded every 3 days until all died. The head and tail of the nematodes were gently touched with a pick, and if there was no reaction within 30 seconds, it was determined to be dead.

[0109] The results are shown in Table 1. Figure 3 As shown in Table 1, compared with the blank group, different concentrations of DAPS prepared in Example 1 had a certain effect on prolonging the life of nematodes (P<0.05), and the effect was more obvious than that of the non-degraded APS groups. Among them, the APS and DAPS (1 mg / mL) groups had the best effect, and 1 mg / mL was selected as the administration concentration for subsequent experiments.

[0110] Example 7 Paralysis experiment of Caenorhabditis elegans CL4176

[0111] A blank group (Control) and APS and DAPS (1 mg / mL) prepared in Example 1 were set. The CL4176 eggs after synchronization were placed in the blank group and the drug administration group, 30 per plate, and cultured at 16°C for 24 h, then transferred to another plate and cultured at 16°C for 12 h, and then transferred to a 25°C incubator for temperature culture for 36 h. Then, observation was carried out every 2 h under a microscope, and the number of paralyzed nematodes was recorded (the head and body of the nematodes were gently touched with a pick several times, and the body parts other than the head were considered paralyzed if they did not move).

[0112] The results are shown in Table 2. Figure 4 As shown in Table 2, compared with the blank group, APS and DAPS prepared in Example 1 had a certain effect on inhibiting the paralysis of nematodes, and the effect of DAPS on inhibiting the paralysis of CL4176 nematodes was better than that of APS which was not degraded by the method of Example 1.

[0113] Example 8 Stress experiment of Caenorhabditis elegans CL4176

[0114] The heat stress experiment was set up with a blank group (Control) and APS and DAPS (1 mg / mL) prepared according to Example 1. The synchronized CL4176 eggs were placed in the blank group and the drug groups, 30 per plate, and cultured in a 16°C incubator for 3 days, and then the temperature was raised to 35°C. The survival of the nematodes was observed every 1 h until all the nematodes died.

[0115] The results, as shown in Table 1, showed that, compared with the blank group, APS and DAPS prepared according to Example 1 could weaken the heat stress damage to the nematodes, and the anti-heat stress effect of DAPS was significantly higher than that of APS. Figure 5 The oxidative stress experiment was set up with a blank group (Control) and APS and DAPS (1 mg / mL) prepared according to Example 1. The CL4176 nematodes synchronized to the L4 stage were placed in the blank group and the drug groups, 30 per plate, and treated at 16°C for two days. Then the experimental nematodes were transferred to NGM plates with 240 μM juglone, and the survival of the nematodes was observed every 1 h until all the nematodes died.

[0116] The results, as shown in Table 2, showed that, compared with the blank group, APS and DAPS prepared according to Example 1 could weaken the oxidative stress damage to the nematodes, and the anti-oxidative stress ability of the CL4176 nematodes was significantly improved after the degradation of astragalus polysaccharides according to the method of Example 1.

[0117] Figure 5 Example 9 Movement ability experiment of Caenorhabditis elegans CL4176

[0118] The blank group (Control) and APS and DAPS (1 mg / mL) prepared according to Example 1 were set up. The synchronized CL4176 eggs were placed in the blank group and the drug groups, 10 per plate, and cultured in a 16°C incubator, and the culture dishes were replaced every 2 days. When the worms grew to 10 days, an appropriate amount of M9 buffer solution was added, and the number of sine movements of the worms in 30 s was detected under a microscope to determine the change in the movement ability of the CL4176 nematodes.

[0119] The results, as shown in Table 3, showed that DAPS could significantly improve the movement ability of the CL4176 nematodes on the 4th day (P<0.01), and APS could improve the movement ability of the CL4176 nematodes to a certain extent, but there was no significant difference.

[0120] Figure 6

[0121] ​​​The experimental results show that the DAPS prepared by the application has low molecular weight, high sugar content and low protein content, and can prolong the life of CL4176 nematodes, reduce paralysis, reduce stress, and improve the movement ability to a certain extent.

[0122] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A low molecular weight astragalus polysaccharide, characterized in that, The monosaccharide components of the low molecular weight astragalus polysaccharide include Glc and Gal in a molar ratio of 1:0.054; the average molecular weight of the low molecular weight astragalus polysaccharide is 42.34 kDa. The preparation method of the low molecular weight astragalus polysaccharide specifically includes the following steps: S1. Astragalus was defatted by reflux with ethanol, deionized water was added to the filter residue, ultrasonic extraction was performed, and crude polysaccharide of Astragalus was obtained by alcohol precipitation. S2. Astragalus polysaccharide was obtained by enzymatic hydrolysis, dialysis, and alcohol precipitation. S3, Astragalus polysaccharide, processed with ultrasound-assisted H2O2-VC 2+ -Fe 2+ The system degrades to obtain degraded Astragalus polysaccharides. S4. The degraded Astragalus polysaccharide was eluted with distilled water through a DEAE-650M cellulose column to obtain low molecular weight Astragalus polysaccharide. The H2O2-VC 2+ -Fe 2+ The system includes H2O2, VC and ferric sulfate; The enzyme used in step S2 is subtilisin, and the concentration of subtilisin in the enzymatic hydrolysis solution is 2-4 mg / mL. The mass ratio of H2O2, VC and ferric sulfate is 1:1:(0.8-1.2).

2. The low molecular weight astragalus polysaccharide according to claim 1, characterized in that, In step S1, the ratio of filter residue to deionized water is 1:(14-18) g / mL.

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