Preparation method and application of low-molecular-weight polysaccharide

Abstract

The invention belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a preparation method and application of low-molecular-weight polysaccharide. Astragalus membranaceus is taken as an example, and an ultrasonic-assisted H2O2-V2 +-Fe < 2 + > system is adopted to degrade astragalus polysaccharide (APS) to obtain the low-molecular-weight astragalus polysaccharide (DAPS). Compared with an existing low-molecular-weight polysaccharide preparation technology, the low-molecular-weight polysaccharide preparation technology has the advantages of being simple, wide in application range, measurable in molecular weight, high in yield and high in reproducibility. Results of the embodiment of the invention show that the low-molecular-weight astragalus polysaccharide prepared by the preparation method can prolong the life of CL4176 nematodes, improve the paralysis condition of the CL4176 nematodes and enhance the exercise ability and stress ability of the nematodes; furthermore, the prepared low-molecular-weight astragalus polysaccharide can be applied to preparation of novel medicines for prolonging the service life, improving paralysis, enhancing the athletic ability and stress ability and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a preparation method and application of low molecular weight polysaccharide. Background Art

[0002] With the trend of accelerated pace of life and aging population, chronic diseases such as decreased motor and stress capacity have become health issues of key concern. Under the tremendous pressure of life, adults are prone to mental tension, slow thinking and depression, resulting in decreased motor ability, stress capacity and memory, accompanied by complications such as falls and infections, which can seriously affect life expectancy. These chronic diseases are characterized by abnormal accumulation of intracellular and extracellular neurofibrils, leading to synaptic degeneration, neuronal cell death and gliosis, exacerbating neurodegenerative processes. Traditional Chinese medicine polysaccharide components have the advantages of multiple pathways and multiple targets, which can inhibit oxidative stress, neuroinflammation, cell apoptosis, regulate autophagy and improve energy metabolism.

[0003] Caenorhabditis elegans (C.elegans), also known as nematodes, belongs to the class Nematoda of the phylum Nematode and is one of the important model organisms for studying the anti-aging and oxidative stress effects of substances. It has the characteristics of short lifespan, simple cell pattern, rapid reproduction, stable behavioral response, and sensitive and reliable results. It can exhibit a series of characteristics similar to those of mammals. Among them, the BZIP domain protein 1 (protein skinhead-1, skn-1) and the mammalian nuclear factor E2 related transcription factor 2 (nuclear factor E2 related factor 2, Nrf2) protein are functionally consistent and can play a similar anti-oxidative stress role in vivo.

[0004] Astragalus is the dried root of Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.), a plant of the Leguminosae family. It is a traditional Chinese medicinal plant that can strengthen the spleen and replenish the middle, replenish qi and raise yang, and replenish kidney qi. Astragalus contains chemical components such as polysaccharides, saponins, and flavonoids, and has multiple pharmacological activities such as anti-inflammatory, anti-fibrotic, and anti-aging. Among them, Astragalus polysacharin (APS) is one of the main chemical components extracted from the dried root of Astragalus, a traditional Chinese medicine. It is widely used and has good immune enhancement, antioxidant, and anti-inflammatory effects. Natural polysaccharides have the characteristics of large molecular weight, high viscosity, and poor water solubility. Too large molecular weight will lead to obstacles in the process of crossing the cell membrane, making it difficult to enter the cell to exert biological activity. Compared with polysaccharides, oligosaccharides have the characteristics of small molecular weight, simple structure, and are beneficial to human absorption and utilization.

[0005] At present, low molecular weight polysaccharides can be obtained through degradation. The main methods of polysaccharide degradation are physical degradation, chemical degradation, and biodegradation. In recent years, H2O2 oxidation is a widely used polysaccharide degradation method. The hydroxyl radicals generated by H2O2 decomposition are used to oxidatively break the glycosidic bonds between polysaccharides. This method is green, environmentally friendly, and has strong controllability. However, the polysaccharide degradation process of spontaneously generated hydroxyl radicals by H2O2 is slow.

[0006] Therefore, it is urgent to develop a method for rapidly and efficiently degrading polysaccharides. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation method and application of low molecular weight polysaccharides. The method provided by the present invention has not only a simple preparation process and a wide range of applications, but also a high yield and strong reproducibility.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing a low molecular weight polysaccharide, which specifically comprises the following steps:

[0010] S1, adding ethanol to the raw material for reflux degreasing, adding deionized water to the filter residue, ultrasonic extraction, and alcohol precipitation to obtain crude polysaccharide;

[0011] S2, the crude polysaccharide aqueous solution is enzymatically hydrolyzed, dialyzed, and precipitated with alcohol to obtain polysaccharides;

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

[0013] S4, eluting the degraded polysaccharide through a DEAE-650M cellulose chromatography column with distilled water to obtain a low molecular weight polysaccharide;

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

[0015] Preferably, the raw material is astragalus, Acanthopanax senticosus or Caulis Piperis d'Azurariae.

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

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

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

[0019] The present invention also provides a low molecular weight astragalus polysaccharide, which is obtained by the above preparation method.

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

[0021] The present invention also provides a low molecular weight astragalus polysaccharide prepared by the above preparation method or the use of 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 CL4176 nematodes.

[0022] The present invention also provides the low molecular weight astragalus polysaccharide prepared by the above preparation method or the use of the low molecular weight astragalus polysaccharide in preparing products and / or medicines for prolonging life.

[0023] The present invention also provides the low molecular weight astragalus polysaccharide prepared by the above preparation method or the use of the low molecular weight astragalus polysaccharide in preparing products and / or medicines for improving paralysis.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention adopts a composite degradation method to degrade polysaccharides, which can not only solve the shortcomings of poor solubility and low bioavailability of large molecular weight polysaccharides, but also improve the purity of polysaccharides and enhance the biological activity of polysaccharides.

[0026] (2) The preparation method disclosed in the present invention 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 invention 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 THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

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

[0030] Figure 2GC-MS spectra of the monosaccharide components of the mixed monosaccharide standard derivative (mixed standard) and DAPS prepared in Example 1, wherein: 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 life span of CL4176 nematodes;

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

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

[0034] Figure 6 The effects of APS and DAPS prepared in Example 1 on the motility of CL4176 nematodes. DETAILED DESCRIPTION

[0035] The invention provides a preparation process of a low molecular weight polysaccharide, which specifically comprises: adding an ethanol solution to a raw material for reflux degreasing, filtering, and shade drying to obtain a defatted product, then adding deionized water for immersion at a material-liquid ratio of 1:14-18 g / mL, preferably 1:16 g / mL, ultrasonically extracting, filtering, and obtaining a filtrate; adding anhydrous ethanol to the filtrate for alcohol precipitation, concentrating under reduced pressure, and freeze-drying to obtain a crude polysaccharide.

[0036] The crude polysaccharide is prepared into an aqueous solution, the pH of the solution is adjusted, and subtilisin is added for enzymolysis, so that the concentration of subtilisin in the solution is between 2-4 mg / mL, preferably 3 mg / mL. The supernatant is obtained by centrifugation and suction filtration, dialyzed, anhydrous ethanol is added for alcohol precipitation, concentrated under reduced pressure, and freeze-dried to obtain the polysaccharide.

[0037] The polysaccharide is prepared into an aqueous solution, the pH of the solution is adjusted, and then H2O2, VC and ferrous sulfate are added, and the mass ratio of H2O2, VC and ferrous sulfate is 1:1:(0.8-1.2), preferably 1:1:1.2. 2+ -Fe 2+ Degrade the polysaccharide to obtain the degraded polysaccharide. Add distilled water to re-dissolve the degraded polysaccharide, take the supernatant for sample loading, and separate and purify it by passing it through a DEAE-650M cellulose chromatography column with distilled water to obtain a low molecular weight polysaccharide.

[0038] The raw materials in the above preparation method include but are not limited to astragalus, Acanthopanax senticosus or Piper mesenteroides.

[0039] The present invention provides a preparation process of low molecular weight astragalus polysaccharide by using the above preparation method. The low molecular weight astragalus polysaccharide is prepared by using astragalus as raw material and using ultrasound-assisted H2O2-VC 2+ -Fe 2+ The system degraded astragalus polysaccharide (APS) to obtain low molecular weight astragalus polysaccharide (DAPS), and VC converted Fe 3+ Reduction to Fe 2+ , H2O2 can oxidize VC and quickly generate hydroxyl free radicals. 2+ The Fenton reaction produces hydroxyl radicals, which cause hydrogen abstraction reactions, leading to faster glycosidic bond breakage and significantly increased polysaccharide degradation rate. At the same time, ultrasound-assisted oxidative degradation can combine the advantages of the two methods and work synergistically.

[0040] The preparation process of the low molecular weight astragalus polysaccharide of the present invention specifically comprises: adding ethanol solution to astragalus for reflux defatting, filtering, and drying in the shade to obtain defatted astragalus, then adding deionized water for immersion at a solid-liquid ratio of 1:14-18 g / mL, preferably 1:16 g / mL, ultrasonic extraction, filtering, and obtaining a filtrate; adding anhydrous ethanol to the filtrate for alcohol precipitation, concentrating under reduced pressure, and freeze-drying to obtain crude astragalus polysaccharide.

[0041] The crude astragalus polysaccharide is prepared into an aqueous solution, the pH value of the solution is adjusted, and subtilisin is added for enzymolysis, so that the concentration of subtilisin in the solution is between 2-4 mg / mL, preferably 3 mg / mL. The supernatant is obtained by centrifugation and suction filtration, dialyzed, anhydrous ethanol is added for alcohol precipitation, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0042] APS is prepared into an aqueous solution, the pH of the solution is adjusted, and then H2O2, VC and ferrous sulfate are added. The mass ratio of H2O2, VC and ferrous sulfate is 1:1:(0.8-1.2), preferably 1:1:1.2. 2+ -Fe 2+ Degrade Astragalus polysaccharide to obtain degraded Astragalus polysaccharide. Add distilled water to re-dissolve the degraded Astragalus polysaccharide, take the supernatant and apply it to a DEAE-650M cellulose column for separation and purification, and obtain low molecular weight Astragalus polysaccharide (DAPS).

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

[0044] Furthermore, the low molecular weight astragalus polysaccharide prepared by the present invention provides a new direction for studying drugs and / or products that can inhibit neuroinflammation, improve motor ability, etc.

[0045] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0047] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0048] Example 1

[0049] A method for preparing low molecular weight astragalus polysaccharide comprises the following steps:

[0050] Weigh 2 kg of Astragalus into a round-bottom flask, add 25 L of 75% ethanol and reflux for degreasing for 2 hours, repeat 3 times, filter and dry the residue in the shade, add deionized water at a solid-liquid ratio of 1:16 g / mL, extract for 3 hours at 85 ° C and an ultrasonic power of 700 W, filter to obtain the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 3 L, add anhydrous ethanol to the ethanol volume concentration of 85%, stir well, stand at 4 ° C for 48 hours for alcohol precipitation, centrifuge at 4200 r / min for 20 minutes, take the precipitate and redissolve it, concentrate under reduced pressure, and freeze-dry to obtain crude Astragalus polysaccharide.

[0051] The crude astragalus polysaccharide powder is prepared into a 15 mg / mL aqueous solution, and the pH of the solution is adjusted to 9-10. Subtilisin is added to the aqueous solution to make the concentration of subtilisin 3 mg / mL, and stirred evenly. It is enzymatically hydrolyzed at 50°C for 1 hour, and then heated to 95°C for 10 minutes to inactivate the enzyme, centrifuged at 4200 r / min for 20-30 minutes, and the supernatant is retained. It is dialyzed for 48 hours, and then anhydrous ethanol is added to the ethanol volume concentration to reach 85%, stirred evenly, and allowed to stand at 4°C for 48 hours for alcohol precipitation. It is centrifuged at 4200 r / min for 20 minutes, and the precipitate is taken for redissolution, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0052] APS was prepared into a 15 mg / mL aqueous solution, and a 1 mol / L HCL solution was added to adjust the pH to 4. H2O2 was added to make the H2O2 concentration 9 mmol / L. Vc and ferrous sulfate were added to make the mass ratio of H2O2, VC, and ferrous sulfate 1:1:1.2. Ultrasonic-assisted H2O2-VC was heated at 70 °C. 2+ -Fe 2+ Degrade for 3 hours with an ultrasonic power of 900 W, then add anhydrous ethanol to a volume concentration of 85%, stir well, let stand at 4°C for 48 hours, centrifuge at 4200 r / min for 20 minutes, take the precipitate for redissolution, concentrate under reduced pressure, and freeze-dry to obtain degraded astragalus polysaccharide, weighing 3 g of degraded astragalus polysaccharide, dissolve it with distilled water, centrifuge it, take the supernatant for sample, adsorb for 2 hours, and elute it with distilled water through a DEAE-650M cellulose column to obtain DAPS.

[0053] The polysaccharide extraction rate formula is:

[0054] The content of polysaccharide was determined by phenol-sulfuric acid method. A 1 mg / mL glucose solution was prepared. 0.2, 0.4, 0.6, 0.8, and 1.0 mL were accurately pipetted into a 10 mL stoppered test tube. Distilled water was added to 1 mL. 1 mL of 6% phenol solution was added and shaken. 5 mL of concentrated sulfuric acid was slowly added and shaken. The solution was heated at 75°C for 10 min and cooled to room temperature. The absorbance (A) was measured at 490 nm. A standard curve was drawn with glucose concentration as the x-axis and A value as the y-axis. The regression equation of glucose standard curve was: y=2.6685x+0.0787, R 2 =0.9994, prepare 1 mg / mL polysaccharide sample solution, take 1 mL, conduct three parallel experiments according to the above method, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the sugar content.

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

[0056] In addition, in order to further illustrate the non-obviousness of the process parameter conditions in the technical solution disclosed in the present application, the inventors respectively optimized the material-liquid ratio of defatted astragalus and deionized water, the mass ratio of crude astragalus 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 method for preparing low molecular weight astragalus polysaccharide comprises the following steps:

[0059] Weigh 100 g of defatted astragalus in 3 portions respectively, soak them in 1.4 L, 1.6 L and 1.8 L of deionized water, extract for 3 h at 85 °C with an ultrasonic power of 700 W, extract three times, combine the filtrates, add anhydrous ethanol to an ethanol volume concentration of 85%, stir well, let stand at 4 °C for 48 h for alcohol precipitation, centrifuge at 4200 r / min for 20 min, take the precipitate and redissolve it, concentrate under reduced pressure, and freeze-dry to obtain crude astragalus polysaccharide.

[0060] The crude astragalus polysaccharide powder is prepared into a 15 mg / mL aqueous solution, and the pH of the solution is adjusted to 9-10. Subtilisin is added to the aqueous solution to make the concentration of subtilisin 3 mg / mL, and stirred evenly. It is enzymatically hydrolyzed at 50°C for 1 hour, and then heated to 95°C for 10 minutes to inactivate the enzyme, centrifuged at 4200 r / min for 20 minutes, and the supernatant is retained. It is dialyzed for 48 hours, and then anhydrous ethanol is added to the ethanol volume concentration to reach 85%, stirred evenly, and allowed to stand at 4°C for 48 hours for alcohol precipitation. It is centrifuged at 4200 r / min for 20 minutes, and the precipitate is taken for redissolution, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0061] APS was prepared into a 15 mg / mL aqueous solution, and a 1 mol / L HCL solution was added to adjust the pH to 4. H2O2 was added to make the H2O2 concentration 9 mmol / L. Vc and ferrous sulfate were added to make the mass ratio of H2O2, VC, and ferrous sulfate 1:1:1.2. Ultrasonic-assisted H2O2-VC was heated at 70 °C. 2+ -Fe 2+ Degrade for 3 hours with an ultrasonic power of 900 W, then add anhydrous ethanol to a volume concentration of 85%, stir well, let stand at 4°C for 48 hours, centrifuge at 4200 r / min for 20 minutes, take the precipitate for redissolution, concentrate under reduced pressure, and freeze-dry to obtain degraded astragalus polysaccharides, weighing 3 g of degraded astragalus polysaccharides, add distilled water for redissolution, centrifuge, take the supernatant for sample, adsorb for 2 hours, and elute with distilled water through a DEAE-650M cellulose column to obtain DAPS1, DAPS2, and DAPS3.

[0062] According to the different solid-liquid ratios of defatted astragalus and deionized water, the optimization was performed: the extraction rate of DAPS1 was 9.44%, the sugar content was 78.23%, and the protein content was 2.55%; the extraction rate of DAPS2 was 11.24%, the sugar content was 82.51%, and the protein content was 2.12%; the extraction rate of DAPS3 was 10.41%, the sugar content was 78.93%, and the protein content was 2.37%. Therefore, the preferred volume ratio of astragalus to deionized water was 1:16 for preparation.

[0063] Example 3

[0064] A method for preparing low molecular weight astragalus polysaccharide comprises the following steps:

[0065] Weigh 3 portions of defatted astragalus (100 g) respectively, soak them in 1.6 L deionized water, extract for 3 h at 85 ° C with an ultrasonic power of 700 W, extract three times, combine the filtrate, add anhydrous ethanol to the ethanol volume concentration of 85%, stir well, let stand at 4 ° C for 48 h for alcohol precipitation, centrifuge at 4200 r / min for 20 min, take the precipitate and dissolve it, concentrate under reduced pressure, and freeze-dry to obtain crude astragalus polysaccharide.

[0066] The crude astragalus polysaccharide powder is prepared into a 15 mg / mL aqueous solution, and the pH of the solution is adjusted to 9-10. Subtilisin is added to the aqueous solution to make the concentration of subtilisin 2 mg / mL, 3 mg / mL, and 4 mg / mL, and stirred evenly. Enzymatic hydrolysis is carried out at 50°C for 1 hour, and then the enzyme is inactivated by heating to 95°C for 10 minutes. The solution is centrifuged at 4200 r / min for 20 minutes, the supernatant is retained, dialyzed for 48 hours, and then anhydrous ethanol is added to the solution until the ethanol volume concentration reaches 85%, stirred evenly, and allowed to stand at 4°C for 48 hours for alcohol precipitation. The solution is centrifuged at 4200 r / min for 20 minutes, the precipitate is redissolved, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0067] APS was prepared into a 15 mg / mL aqueous solution, and a 1 mol / L HCL solution was added to adjust the pH to 4. H2O2 was added to make the H2O2 concentration 9 mmol / L. Vc and ferrous sulfate were added to make the mass ratio of H2O2, VC, and ferrous sulfate 1:1:1.2. Ultrasonic-assisted H2O2-VC was heated at 70 °C. 2+ -Fe 2+ Degrade for 3 hours with an ultrasonic power of 900 W, then add anhydrous ethanol to a volume concentration of 85%, stir well, let stand at 4°C for 48 hours, centrifuge at 4200 r / min for 20 minutes, take the precipitate for redissolution, concentrate under reduced pressure, and freeze-dry to obtain degraded astragalus polysaccharides, weighing 3.00 g of degraded astragalus polysaccharides, add distilled water for redissolution, centrifuge, take the supernatant for sample, adsorb for 2 hours, and elute with distilled water through a DEAE-650M cellulose column to obtain DAPS4, DAPS5, and DAPS6.

[0068] According to the different mass ratios of crude astragalus polysaccharide and subtilisin, the optimization was performed: the extraction rate of DAPS4 was 10.53%, the sugar content was 77.43%, and the protein content was 4.23%; the extraction rate of DAPS5 was 11.24%, the sugar content was 82.51%, and the protein content was 2.12%; the extraction rate of DAPS6 was 9.38%, the sugar content was 80.21%, and the protein content was 2.31%. Therefore, the mass ratio of crude astragalus polysaccharide to subtilisin was preferably 3:1 for preparation.

[0069] Example 4

[0070] A method for preparing low molecular weight astragalus polysaccharide comprises the following steps:

[0071] Weigh 3 portions of defatted astragalus (100 g) respectively, soak them in 1.6 L deionized water, extract for 3 h at 85 ° C with an ultrasonic power of 700 W, extract three times, combine the filtrate, add anhydrous ethanol to the ethanol volume concentration of 85%, stir well, let stand at 4 ° C for 48 h for alcohol precipitation, centrifuge at 4200 r / min for 20 min, take the precipitate and dissolve it, concentrate under reduced pressure, and freeze-dry to obtain crude astragalus polysaccharide.

[0072] The crude astragalus polysaccharide powder is prepared into a 15 mg / mL aqueous solution, and the pH of the solution is adjusted to 9-10. Subtilisin is added to the aqueous solution to make the concentration of subtilisin 3 mg / mL, and stirred evenly. It is enzymatically hydrolyzed at 50°C for 1 hour, and then heated to 95°C for 10 minutes to inactivate the enzyme, centrifuged at 4200 r / min for 20 minutes, and the supernatant is retained. It is dialyzed for 48 hours, and then anhydrous ethanol is added to the ethanol volume concentration to reach 85%, stirred evenly, and allowed to stand at 4°C for 48 hours for alcohol precipitation. It is centrifuged at 4200 r / min for 20 minutes, and the precipitate is taken for redissolution, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0073] APS was prepared into a 15 mg / mL aqueous solution, and a 1 mol / L HCL solution was added to adjust the pH to 4. H2O2 was added to make the H2O2 concentration 9 mmol / L. Vc and ferrous sulfate were added to make the mass ratios of H2O2, VC, and ferrous sulfate 1:1:0.8, 1:1:1, and 1:1:1.2. Ultrasonic-assisted H2O2-VC was heated at 70 °C. 2+ -Fe 2+ Degrade for 3 h with ultrasonic power of 900 W, then add anhydrous ethanol to a volume concentration of 85%, stir well, let stand at 4°C for 48 h, centrifuge at 4200 r / min for 20 min, take the precipitate for redissolution, concentrate under reduced pressure, and freeze-dry to obtain DAPS7, DAPS8, and DAPS9.

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

[0075] Furthermore, in order to highlight the superior effect of the technical solution disclosed in the present invention application compared with the prior art, the following comparative experiments were conducted:

[0076] Comparative Example 1

[0077] A method for preparing low molecular weight astragalus polysaccharide comprises the following steps:

[0078] Weigh 2 kg of Astragalus into a round-bottom flask, add 25 L of 75% ethanol and reflux for degreasing for 2 hours, repeat 3 times, filter and dry the residue in the shade, add deionized water at a solid-liquid ratio of 1:16 g / mL, extract for 3 hours at 85 ° C and an ultrasonic power of 700 W, filter to obtain the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 3 L, add anhydrous ethanol to the ethanol volume concentration of 85%, stir well, stand at 4 ° C for 48 hours for alcohol precipitation, centrifuge at 4200 r / min for 20 minutes, take the precipitate and redissolve it, concentrate under reduced pressure, and freeze-dry to obtain crude Astragalus polysaccharide.

[0079] The crude astragalus polysaccharide powder is prepared into a 15 mg / mL aqueous solution, and the pH of the solution is adjusted to 9-10. Subtilisin is added to the aqueous solution to make the concentration of subtilisin 3 mg / mL, and stirred evenly. It is enzymatically hydrolyzed at 50°C for 1 hour, and then heated to 95°C for 10 minutes to inactivate the enzyme, centrifuged at 4200 r / min for 20-30 minutes, and the supernatant is retained. It is dialyzed for 48 hours, and then anhydrous ethanol is added to the ethanol volume concentration to reach 85%, stirred evenly, and allowed to stand at 4°C for 48 hours for alcohol precipitation. It is centrifuged at 4200 r / min for 20 minutes, and the precipitate is taken for redissolution, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0080] APS was prepared into a 15 mg / mL aqueous solution, and 1 mol / L HCL solution was added to adjust the pH to 4. H2O2 was added to make the H2O2 concentration 9 mmol / L. Vc and ferrous sulfate were added to make the mass ratio of H2O2, VC, and ferrous sulfate 1:1:1.2. At 70 °C, H2O2-VC 2+ -Fe 2+ Degrade for 3 hours, then add anhydrous ethanol to a volume concentration of 85%, stir well, stand at 4°C for 48 hours, centrifuge at 4200r / min for 20 minutes, take the precipitate and dissolve it, concentrate under reduced pressure, and freeze-dry to obtain Astragalus degraded polysaccharide, which is 3 g. Dissolve it in distilled water, centrifuge it, take the supernatant for sample, adsorb for 2 hours, and elute it through a DEAE-650M cellulose column with distilled water to obtain DAPS-A.

[0081] The polysaccharide extraction rate formula is:

[0082] The content of polysaccharide was determined by phenol-sulfuric acid method. A 1 mg / mL glucose solution was prepared. 0.2, 0.4, 0.6, 0.8, and 1.0 mL were accurately pipetted into a 10 mL stoppered test tube. Distilled water was added to 1 mL. 1 mL of 6% phenol solution was added and shaken. 5 mL of concentrated sulfuric acid was slowly added and shaken. The solution was heated at 75°C for 10 min and cooled to room temperature. The absorbance (A) was measured at 490 nm. A standard curve was drawn with glucose concentration as the x-axis and A value as the y-axis. The regression equation of glucose standard curve was: y=2.6685x+0.0787, R 2 =0.9994, prepare 1 mg / mL polysaccharide sample solution, take 1 mL, conduct three parallel experiments according to the above method, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the sugar content.

[0083] The extraction rate of DAPS-A was 8.11% and the sugar content was 74.61%. It can be seen that the extraction rate and sugar content of DAPS after ultrasound-assisted degradation were significantly higher than those of DAPS without ultrasound-assisted degradation.

[0084] Comparative Example 2

[0085] A method for preparing low molecular weight astragalus polysaccharide comprises the following steps:

[0086] Weigh 2 kg of Astragalus into a round-bottom flask, add 25 L of 75% ethanol and reflux for degreasing for 2 hours, repeat 3 times, filter and dry the residue in the shade, add deionized water at a solid-liquid ratio of 1:16 g / mL, extract for 3 hours at 85 ° C and an ultrasonic power of 700 W, filter to obtain the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 3 L, add anhydrous ethanol to the ethanol volume concentration of 85%, stir well, stand at 4 ° C for 48 hours for alcohol precipitation, centrifuge at 4200 r / min for 20 minutes, take the precipitate and redissolve it, concentrate under reduced pressure, and freeze-dry to obtain crude Astragalus polysaccharide.

[0087] The crude astragalus polysaccharide powder was prepared into a 15 mg / mL aqueous solution, deproteinized by the Sevage method, centrifuged at 4200 r / min for 20 to 30 min, the supernatant was retained, dialyzed for 48 h, and then anhydrous ethanol was added to reach an ethanol volume concentration of 85%, stirred evenly, allowed to stand at 4°C for 48 h for alcohol precipitation, centrifuged at 4200 r / min for 20 min, the precipitate was taken for redissolution, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0088] APS was prepared into a 15 mg / mL aqueous solution, and a 1 mol / L HCL solution was added to adjust the pH to 4. H2O2 was added to make the H2O2 concentration 9 mmol / L. Vc and ferrous sulfate were added to make the mass ratio of H2O2, VC, and ferrous sulfate 1:1:1.2. Ultrasonic-assisted H2O2-VC was heated at 70 °C. 2+ -Fe2+ Degrade for 3 hours with an ultrasonic power of 900 W, then add anhydrous ethanol to a volume concentration of 85%, stir well, let stand at 4°C for 48 hours, centrifuge at 4200 r / min for 20 minutes, take the precipitate for redissolution, concentrate under reduced pressure, and freeze-dry to obtain degraded astragalus polysaccharide, weighing 3 g of degraded astragalus polysaccharide, dissolve it with distilled water, centrifuge it, take the supernatant for sample, adsorb for 2 hours, and elute it with distilled water through a DEAE-650M cellulose column to obtain DAPS-B.

[0089] The polysaccharide extraction rate formula is:

[0090] The content of polysaccharide was determined by phenol-sulfuric acid method. A 1 mg / mL glucose solution was prepared. 0.2, 0.4, 0.6, 0.8, and 1.0 mL were accurately pipetted into a 10 mL stoppered test tube. Distilled water was added to 1 mL. 1 mL of 6% phenol solution was added and shaken. 5 mL of concentrated sulfuric acid was slowly added and shaken. The solution was heated at 75°C for 10 min and cooled to room temperature. The absorbance (A) was measured at 490 nm. A standard curve was drawn with glucose concentration as the x-axis and A value as the y-axis. The regression equation of glucose standard curve was: y=2.6685x+0.0787, R 2 =0.9994, prepare 1 mg / mL polysaccharide sample solution, take 1 mL, conduct three parallel experiments according to the above method, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the sugar content.

[0091] The extraction rate of DAPS-B was 12.56%, the sugar content was 76.32%, and the protein content was 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 complicated, and the polysaccharide loss rate is also high.

[0092] Comparative Example 3

[0093] A method for preparing low molecular weight astragalus polysaccharide comprises the following steps:

[0094] Weigh 2 kg of Astragalus into a round-bottom flask, add 25 L of 75% ethanol and reflux for degreasing for 2 hours, repeat 3 times, filter and dry the residue in the shade, add deionized water at a solid-liquid ratio of 1:16 g / mL, extract for 3 hours at 85 ° C and an ultrasonic power of 700 W, filter to obtain the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 3 L, add anhydrous ethanol to the ethanol volume concentration of 85%, stir well, stand at 4 ° C for 48 hours for alcohol precipitation, centrifuge at 4200 r / min for 20 minutes, take the precipitate and redissolve it, concentrate under reduced pressure, and freeze-dry to obtain crude Astragalus polysaccharide.

[0095] The crude astragalus polysaccharide powder is prepared into a 15 mg / mL aqueous solution, and the pH of the solution is adjusted to 9-10. Subtilisin is added to the aqueous solution to make the concentration of subtilisin 3 mg / mL, and stirred evenly. It is enzymatically hydrolyzed at 50°C for 1 hour, and then heated to 95°C for 10 minutes to inactivate the enzyme, centrifuged at 4200 r / min for 20-30 minutes, and the supernatant is retained. It is dialyzed for 48 hours, and then anhydrous ethanol is added to the ethanol volume concentration to reach 85%, stirred evenly, and allowed to stand at 4°C for 48 hours for alcohol precipitation. It is centrifuged at 4200 r / min for 20 minutes, and the precipitate is taken for redissolution, concentrated under reduced pressure, and freeze-dried to obtain astragalus polysaccharide (APS).

[0096] APS was prepared into a 15 mg / mL aqueous solution, and a 1 mol / L HCL solution was added to adjust the pH to 4. H2O2 was added to make the H2O2 concentration 9 mmol / L. Vc and ferrous sulfate were added to make the mass ratio of H2O2, VC, and ferrous sulfate 1:1:1.2. Ultrasonic-assisted H2O2-VC was heated at 70 °C. 2+ -Fe 2+ Degrade for 3 hours with an ultrasonic power of 900 W, then add anhydrous ethanol to a volume concentration of 85%, stir well, let stand at 4°C for 48 hours, centrifuge at 4200 r / min for 20 minutes, take the precipitate for redissolution, concentrate under reduced pressure, and freeze-dry to obtain degraded astragalus polysaccharide, weighing 3 g of degraded astragalus polysaccharide, dissolve it in distilled water, centrifuge it, take the supernatant for sample, adsorb for 2 hours, and pass it through a DEAE-650M cellulose column, eluted with 0.1 mol / LNaCl and 0.2 mol / LNaCl in turn to obtain DAPS-C and DAPS-D.

[0097] The polysaccharide extraction rate formula is:

[0098] The content of polysaccharide was determined by phenol-sulfuric acid method. A 1 mg / mL glucose solution was prepared. 0.2, 0.4, 0.6, 0.8, and 1.0 mL were accurately pipetted into a 10 mL stoppered test tube. Distilled water was added to 1 mL. 1 mL of 6% phenol solution was added and shaken. 5 mL of concentrated sulfuric acid was slowly added and shaken. The solution was heated at 75°C for 10 min and cooled to room temperature. The absorbance (A) was measured at 490 nm. A standard curve was drawn with glucose concentration as the x-axis and A value as the y-axis. The regression equation of glucose standard curve was: y=2.6685x+0.0787, R 2 =0.9994, prepare 1 mg / mL polysaccharide sample solution, take 1 mL, conduct three parallel experiments according to the above method, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the sugar content.

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

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

[0101] The DAPS obtained in Example 1 was tested: the molecular weight Mw of DAPS was determined by high performance gel permeation chromatography (HPGPC), DAPS was prepared into a solution of a certain concentration, centrifuged, and the supernatant was filtered through a 0.45 μm filter membrane; injection instrument: Agilent 1260 high performance liquid chromatograph; detector: UM4800 evaporative light detector; chromatographic column: UltrahydrogelTMlinear (7.8×300mm); mobile phase: ultrapure water; flow rate: 0.8 mL / min; temperature: 80°C; carrier gas: N2; gas flow rate: 2 L / min; injection volume: 10 μL.

[0102] The results of high performance gel exclusion chromatography are as follows Figure 1 As shown, 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 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, 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, passed through a 0.22 μm microporous filter membrane, and analyzed by GC-MS. Chromatographic column: Agilent HP-5MS (30m×250μm×0.25μm); instrument conditions: chromatographic column gas flow rate: 1 mL / min; carrier gas: He; injection port temperature: 250 ° C; injection volume: 1 μL; heating program: 100 ° C for 2 min, increase to 220 ° C at 10 ° C / min for 5 min, and solvent delay for 3 min. The ion source is an electron bombardment ion source, the collision energy is 70 eV, and the mass scanning range is m / s 40-600.

[0104] The GC-MS test results of monosaccharide composition are as follows Figure 2 As shown, it 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 invention in prolonging life and improving exercise ability, the inventors also conducted the following experiments, the specific contents of which are as follows:

[0106] Example 6 Lifespan experiment of Caenorhabditis elegans CL4176

[0107] The CL4176 strain of nematodes was cultured at a constant temperature of 16°C and fed with E. coli OP50. An appropriate amount of APS and DAPS prepared in Example 1 was taken, dissolved in ultrapure water to form a 2 mg / mL stock solution, filtered with a sterilizing filter, and E. coli OP50 was added to prepare solutions with mass concentrations of 0.50 mg / mL, 1.00 mg / mL, and 2.00 mg / mL, each of which was recorded as a low, medium, and high dose group, and 100 μL of each was spread on an NGM plate, and 3 plates were spread for each mass concentration.

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

[0109] The results are as follows Figure 3 As shown, compared with the blank group, different concentrations of DAPS prepared in Example 1 all had a certain effect of extending the lifespan of nematodes (P<0.05), and the effect was more obvious than that of each dose group of undegraded APS, among which the APS and DAPS (1 mg / mL) group had the best effect, and the subsequent experiments all used a dosing concentration of 1 mg / mL.

[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 up. The synchronized CL4176 eggs were placed in the blank group and the drug group, 30 eggs per plate, cultured at 16°C for 24 hours, transferred to the plate, and continued to be cultured at 16°C for 12 hours, and then transferred to a 25°C incubator for 36 hours. After that, the eggs were observed under a microscope every 2 hours, and the number of paralyzed nematodes was recorded (the head and body of the nematode were touched several times with a pick, and the body parts other than the head were considered paralyzed if they did not move).

[0112] The results are as follows Figure 4 As shown, compared with the blank group, both APS and DAPS prepared in Example 1 have certain anti-nematode paralysis effects, and DAPS is better than APS that has not been degraded by the method of Example 1 in inhibiting CL4176 nematode paralysis.

[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 in Example 1. The synchronized CL4176 eggs were placed in the blank group and the drug group, 30 eggs per plate, and cultured in a 16°C incubator for 3 days and then raised to 35°C. The survival of the nematodes was observed every 1 hour until all the nematodes died.

[0115] The results are as follows Figure 5 As shown, compared with the blank group, both APS and DAPS prepared in Example 1 can reduce the heat stress damage to nematodes, and the heat stress resistance effect of DAPS is significantly higher than that of APS.

[0116] The oxidative stress experiment was set up with a blank group (Control) and APS and DAPS (1 mg / mL) prepared in Example 1. CL4176 nematodes synchronized to the L4 stage were placed in the blank group and the treatment group, 30 per plate, and treated at 16°C for two days. The experimental nematodes were then transferred to NGM plates with 240 μM juglone, and the survival of the nematodes was observed every 1 hour until all the nematodes died.

[0117] The results are as follows Figure 5 As shown, compared with the blank group, the APS and DAPS prepared in Example 1 can weaken the oxidative stress damage to nematodes, and the astragalus polysaccharide can significantly improve the anti-oxidative stress ability of CL4176 nematodes after being degraded by the method of Example 1.

[0118] Example 9 Experiment on the locomotion ability of Caenorhabditis elegans CL4176

[0119] A blank group (Control) and APS and DAPS (1 mg / mL) prepared in Example 1 were set up. The synchronized CL4176 eggs were placed in the blank group and the groups of each concentration, 10 eggs per plate, and cultured in a 16°C incubator, with the culture dish changed every 2 days. When the worm body grew to 10 days, an appropriate amount of M9 buffer solution was added, and the number of sinusoidal movements of the worm body within 30 seconds was detected under a microscope to determine the changes in the movement ability of the CL4176 nematode.

[0120] The results are as follows Figure 6 As shown, DAPS could significantly improve the motility of CL4176 nematodes on the 4th day (P<0.01), and APS could improve the motility of CL4176 nematodes to a certain extent, but there was no significant difference.

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

[0122] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a low molecular weight polysaccharide, characterized in that: The specific steps include: S1, adding ethanol to the raw material for reflux degreasing, adding deionized water to the filter residue, ultrasonic extraction, and alcohol precipitation to obtain crude polysaccharide; S2, the crude polysaccharide aqueous solution is hydrolyzed by enzymes, dialyzed, and precipitated with alcohol to obtain polysaccharides; S3, polysaccharide assisted by ultrasound H2O2-VC 2+ -Fe 2+ The system is degraded, and the polysaccharide is degraded; S4, eluting the degraded polysaccharide through a DEAE-650M cellulose chromatography column with distilled water to obtain a low molecular weight polysaccharide; The H2O2-VC 2+ -Fe 2+ The system includes H2O2, VC and ferrous sulfate.

2. The preparation method according to claim 1, characterized in that: The raw materials are astragalus, acanthopanax senticosus or kadura strychnos.

3. The preparation method according to claim 1, characterized in that: The material-liquid ratio of the filter residue to deionized water in step S1 is 1:(14-18) g / mL.

4. The preparation method according to claim 1, characterized in that: The enzyme used for enzymolysis in step S2 is subtilisin, and the concentration of subtilisin in the enzymolysis solution is 2-4 mg / mL.

5. The preparation method according to claim 1, characterized in that: The mass ratio of H2O2, VC and ferrous sulfate added in step S3 is 1:1:(0.8-1.2).

6. A low molecular weight astragalus polysaccharide, characterized in that: The low molecular weight astragalus polysaccharide is obtained by the preparation method according to claim 1.

7. The low molecular weight astragalus polysaccharide according to claim 6, characterized in that: The monosaccharide components of the low molecular weight astragalus polysaccharide include Glc and Gal, and the molar ratio is 1:0.

054.

8. Use of the low molecular weight astragalus polysaccharide prepared by the preparation method of claim 1 or the low molecular weight astragalus polysaccharide according to any one of claims 6 to 7, characterized in that: The low molecular weight astragalus polysaccharide has the ability to prolong the life span of CL4176 nematodes, and improve the anti-paralysis effect, movement ability and / or oxidative stress of CL4176 nematodes.

9. Use of the low molecular weight astragalus polysaccharide prepared by the preparation method of claim 1 or the low molecular weight astragalus polysaccharide according to any one of claims 6 to 7 in the preparation of products and / or medicines for prolonging life.

10. Use of the low molecular weight astragalus polysaccharide prepared by the preparation method of claim 1 or the low molecular weight astragalus polysaccharide according to any one of claims 6 to 7 in the preparation of products and / or drugs for improving paralysis.

Images