Preparation method and application of phosphorylated acanthopanax polysaccharide

Abstract

The invention discloses a preparation method and application of phosphorylated acanthopanax polysaccharide, and belongs to the technical field of biology. The invention discloses a preparation method of phosphorylated acanthopanax senticosus polysaccharide, which comprises the following steps: taking acanthopanax senticosus as a raw material, extracting acanthopanax senticosus polysaccharide ASPS by adopting an ultrasonic-assisted enzyme method, separating and purifying through a DEAE-650M cellulose chromatographic column to obtain ASPS-I, and carrying out structural modification through a phosphate method to obtain the phosphorylated acanthopanax senticosus polysaccharide PASPS-I. The ultrasonic-assisted enzyme method can save the extraction time, reduce the energy consumption and improve the extraction rate of the polysaccharide. The phosphate method is simple and safe in preparation process, few in by-products, free of participation of organic reagents and environment-friendly. The PASPS-I can prolong the life of the CL4176 nematodes, reduce the paralysis rate of the CL4176 nematodes, improve the movement ability of the CL4176 nematodes and improve the anti-stress ability of the CL4176 nematodes.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more particularly to a preparation method of phosphorylated Acanthopanax senticosus polysaccharide and application thereof. Background Art

[0002] Under the high-pressure pace of life in modern society, people begin to pay more attention to health issues. Long hours of desk work, lack of exercise, irregular work and rest time, high-fat and high-salt diet, excessive mental stress, etc. will lead to persistent fatigue, poor sleep quality, decreased motor ability, memory loss, stress disorders, etc., and even shorten life span. In order to solve this situation, people have set their sights on the field of traditional Chinese medicine. Traditional Chinese medicine prevention not only focuses on symptomatic treatment, but also pays more attention to the conditioning and balance of overall health. As an important part of traditional Chinese medicine, traditional Chinese medicine has a unique theoretical system, treatment methods and clinical effects, which have made many positive contributions to the treatment of diseases, improvement of quality of life and promotion of health.

[0003] Natural polysaccharides have a complex structure and a large molecular weight, making them difficult to be absorbed by the human body and unable to fully exert their biological activity. To solve this problem, researchers have modified the structure of polysaccharides. Currently, the methods for modifying the structure of polysaccharides include sulfation, phosphorylation, acetylation, carboxylmethylation and selenization. Studies have shown that phosphorylated polysaccharides have the characteristics of reducing viscosity, increasing solubility and enhancing biological activity. The methods for phosphorylation modification of polysaccharides include phosphorus oxychloride method, phosphoric acid-anhydride method, phosphorus pentoxide method and phosphate method. The phosphate method has a simple and safe preparation process, with fewer by-products, and no organic reagents involved, which is environmentally friendly. However, phosphorylation modification usually leads to the introduction of phosphate groups to varying degrees, and phosphate groups may be introduced at different positions on the polysaccharide molecules. This heterogeneity may affect the performance of the modified polysaccharides, and too high or too low a degree of phosphorylation may affect the solubility, plasticity and biological activity of the polysaccharides.

[0004] Acanthopanax senticosus (Rupr. et Maxim.) Harms is the dried root and rhizome or stem of the Araliaceae plant Acanthopanax senticosus. It has a long history of medicinal use and is recorded in herbal works of all dynasties. The Compendium of Materia Medica believes that the nature and flavor of Acanthopanax senticosus are "spicy and warm" and belong to the "heart, spleen, and liver meridians". "Replenishing qi, calming the five internal organs, strengthening the tendons and bones, settling the mind and soul, and nourishing the liver and blood" is suitable for treating symptoms such as qi deficiency and fatigue, neurasthenia, loss of appetite, and mental depression. Modern research shows that Acanthopanax senticosus contains a variety of active ingredients such as flavonoids, polysaccharides, and glycosides, and polysaccharides are one of its main active ingredients. Acanthopanax senticosus polysaccharides can enhance the body's immune function, promote the activation and proliferation of immune cells, and also have strong antioxidant properties. As an antioxidant, it helps to delay the aging process and protect cells from oxidative damage. Acanthopanax senticosus polysaccharides also have an anti-fatigue effect, which can help the body cope with long-term physical or psychological stress, relieve fatigue, and improve endurance and efficiency in work and exercise. The molecular structure of Acanthopanax senticosus polysaccharide is relatively complex and its molecular weight is relatively large. This makes its absorption and metabolism in the body relatively slow. Although its immune regulation and anti-fatigue functions are its advantages, the slower metabolic process may make it difficult to achieve obvious effects in the short term, or require a larger dose to work.

[0005] Therefore, providing a method for preparing phosphorylated Acanthopanax senticosus polysaccharide and its application is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing phosphorylated Acanthopanax senticosus polysaccharide (belonging to a method for modifying the structure of polysaccharide) and its application.

[0007] The invention adopts ultrasound-assisted enzyme method to extract Acanthopanax senticosus polysaccharide ASPS, separates and purifies by DEAE-650M cellulose chromatography column to obtain ASPS-Ⅰ, and performs structural modification by phosphate method to obtain phosphorylated Acanthopanax senticosus polysaccharide PASPS-Ⅰ. The PASPS-Ⅰ provided by the invention can prolong the life span of CL4176 nematodes, reduce the paralysis rate of CL4176 nematodes, improve the movement ability of CL4176 nematodes and enhance their anti-stress ability.

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

[0009] A method for preparing phosphorylated Acanthopanax senticosus polysaccharide comprises the following steps:

[0010] (1) adding an ethanol solution to the Acanthopanax senticosus medicinal material for soaking and defatting, filtering, and air-drying to obtain defatted Acanthopanax senticosus; adding deionized water to the defatted Acanthopanax senticosus, ultrasonically extracting, cooling the solution, and centrifuging to obtain a supernatant;

[0011] (2) adding cellulase to the supernatant obtained in step (1) for enzymatic hydrolysis, then inactivating the enzyme, and centrifuging to obtain an enzymatic hydrolyzate;

[0012] (3) adding anhydrous ethanol to the enzymatic hydrolyzate obtained in step (2) for alcohol precipitation, centrifuging to obtain a precipitate, re-dissolving, concentrating, and freeze-drying to obtain a crude Acanthopanax senticosus polysaccharide ASPS; separating and purifying ASPS by a DEAE-650M cellulose column to obtain a Acanthopanax senticosus polysaccharide ASPS-Ⅰ;

[0013] (4) taking the ASPS-Ⅰ obtained in step (3), adding a phosphate solution, adjusting the pH of the solution, and starting a phosphorylation reaction; after the reaction is completed, adding anhydrous ethanol for alcohol precipitation, centrifuging, re-dissolving the precipitate, dialyzing, and freeze-drying to obtain phosphorylated Acanthopanax senticosus polysaccharide PASPS-Ⅰ.

[0014] Furthermore, the volume ratio of the Acanthopanax senticosus to the ethanol solution in step (1) is 1: (6-10) g·mL -1 ; The concentration of the ethanol solution is 70%.

[0015] Furthermore, the solid-liquid ratio of the defatted Acanthopanax senticosus to deionized water in step (1) is 1:(10-20) g·mL -1 , ultrasonic power is 40-60W, ultrasonic temperature is 40-80℃, and ultrasonic time is 40-60min.

[0016] Furthermore, the amount of cellulase added in step (2) is 8-12%, so that its concentration reaches 1500U·mL -1 The enzymatic hydrolysis conditions were as follows: hydrolyze at 37°C for 60 min and then heat to 95°C to inactivate the enzyme.

[0017] Furthermore, in step (3), anhydrous ethanol is added to the enzymatic hydrolysate until the ethanol concentration is 60%; the alcohol precipitation temperature is 4°C, the time is 48h; and the centrifugation condition is 4000r·min -1 , the centrifugation time is 10min.

[0018] Furthermore, in step (4), the mass of ASPS-Ⅰ is 100 mg, the volume of the phosphate solution is 100 mL; the ratio of sodium tripolyphosphate to sodium trimetaphosphate in the phosphate solution is 4:3, 5:2 or 6:1; the pH of the solution is adjusted to 7-9; the phosphorylation reaction temperature is 50-70°C, and the phosphorylation reaction time is 3-5 hours; the alcohol precipitation temperature is 4°C, and the time is 48 hours; the centrifugation condition is 4000 r·min -1 , the centrifugation time is 10 min; the molecular weight of the dialysis bag is 3500 Da; the freeze-drying pressure is 13 kPa, and the freeze-drying temperature is -40°C for 24 hours.

[0019] Furthermore, the phosphorylated Acanthopanax senticosus polysaccharide prepared by the method.

[0020] Furthermore, the phosphorylated Acanthopanax senticosus polysaccharide is used in the preparation of a drug for extending the life span of nematodes, reducing the paralysis rate of nematodes, improving the movement ability of nematodes and / or enhancing the stress resistance of nematodes.

[0021] The present invention adopts CL4176 model Caenorhabditis elegans as a model and applies PASPS-Ⅰ prepared by the present invention for administration. The results show that PASPS-Ⅰ of the present invention can prolong the life span of CL4176 nematodes, reduce the paralysis rate of CL4176 nematodes, improve the movement ability of CL4176 nematodes and enhance their stress resistance.

[0022] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for preparing phosphorylated Acanthopanax senticosus polysaccharide and its application, which has the following beneficial effects:

[0023] (1) The present invention adopts ultrasound-assisted enzyme method to extract Acanthopanax senticosus polysaccharides, which can not only solve the shortcomings of low polysaccharide extraction efficiency and purity due to the hard cell wall of Acanthopanax senticosus, but also improve the biological activity of Acanthopanax senticosus polysaccharides.

[0024] (2) The phosphorylated Acanthopanax senticosus polysaccharide is obtained by structural modification using the phosphate method of the present invention. The preparation process is simple and safe, with fewer by-products, no organic reagents involved, and is environmentally friendly.

[0025] (3) The PASPS-Ⅰ prepared by the present invention can prolong the life span of CL4176 nematodes, reduce the paralysis rate of CL4176 nematodes, improve the movement ability of CL4176 nematodes and enhance their anti-stress ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 HPGPC spectra of ASPS-Ⅰ (A) and PASPS-Ⅰ (B) prepared in the present invention;

[0028] Figure 2 Effects of ASPS-Ⅰ (A) and PASPS-Ⅰ (B) prepared in the present invention on the life span of CL4176 nematodes;

[0029] Figure 3 The effects of ASPS-Ⅰ and PASPS-Ⅰ prepared by the present invention on the paralysis rate of CL4176 nematodes; Figure 4The effects of ASPS-Ⅰ and PASPS-Ⅰ prepared by the present invention on the motility of CL4176 nematodes;

[0030] Figure 5 The effects of ASPS-Ⅰ and PASPS-Ⅰ prepared by the present invention on the oxidative stress ability of CL4176 nematodes;

[0031] Figure 6 The present invention discloses the effects of ASPS-Ⅰ and PASPS-Ⅰ prepared by the present invention on the heat stress resistance of CL4176 nematodes. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] A method for preparing Acanthopanax senticosus polysaccharide comprises the following steps:

[0035] Weigh 1 kg of dried root of Acanthopanax senticosus and calculate the ratio of 1:8 g·mL -1 Add 70% ethanol solution to the material-liquid ratio and soak for 12 hours, filter, evaporate the ethanol, take the residue, and dry it in the shade. When the ultrasonic power is 50W, the ratio is 1:15g·mL -1 Deionized water was added at a solid-liquid ratio of 1.5:1, and ultrasonic extraction was performed at 60°C for 50 min. After the solution was cooled, the temperature was 4000 r·min. -1 Centrifuge for 10 minutes and take the supernatant. Add 10% cellulase to the supernatant to make the concentration reach 1500U·mL -1 , enzymatic hydrolysis was carried out at 37°C for 60 min, followed by enzyme inactivation at 95°C for 10 min, and rotation at 4000 r·min -1 Centrifuge for 10 minutes to obtain the enzymatic solution. Add anhydrous ethanol to the enzymatic solution until the ethanol concentration is 60%, stir well, let stand at 4℃ for 48 hours, and then rotate at 4000r / min. -1 After centrifugation for 10 minutes, the precipitate was re-dissolved in water, concentrated, and freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain the Acanthopanax senticosus crude polysaccharide ASPS.

[0036] The ASPS solution was further separated by DEAE-650M cellulose column: 1 mg mL -1 ASPS solution, 4000r·min -1After centrifugation for 10 min, the supernatant was sampled and diluted with distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol·L -1 NaCl was used for elution, and the flow rate was 1 mL min -1 , collect the eluate, detect the absorbance by phenol-sulfuric acid method, collect and merge the single peak elution components, concentrate and select a dialysis bag with a molecular weight cutoff of 3500Da for dialysis desalination, freeze-dry at a pressure of 13kPa and a temperature of -40°C for 24h to obtain Acanthopanax senticosus polysaccharide ASPS-Ⅰ.

[0037] Prepare a phosphate solution with a ratio of sodium tripolyphosphate to sodium trimetaphosphate of 6:1, add 100 mg ASPS-Ⅰ to 100 mL of phosphate solution, adjust the pH to 8 with sodium bicarbonate, and then stir at 60 °C for 4 h. After the reaction, add four times the amount of anhydrous ethanol, stand at 4 °C for 48 h, and then rotate at 4000 r·min -1 After centrifugation for 10 minutes, the precipitate was re-dissolved in water, placed in a dialysis bag with a molecular weight of 3500Da and dialyzed for 48 hours. The precipitate was freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain phosphorylated Acanthopanax senticosus polysaccharide PASPS-Ⅰ.

[0038] Figure 1 HPGPC spectra of the prepared ASPS-Ⅰ and PASPS-Ⅰ, the molecular weight of ASPS-Ⅰ is 162862Da, and the molecular weight of PASPS-Ⅰ is 114494Da.

[0039] The polysaccharide content in Acanthopanax senticosus was determined by phenol-sulfuric acid method, and the polysaccharide extraction rate was calculated; the ASPS extraction rate was 8.26%, the sugar content was 73.45%, and the sugar content of PASPS-Ⅰ was 57.23%.

[0040] The phosphate content in phosphorylated Acanthopanax senticosus polysaccharide was determined by molybdenum blue colorimetry, and the phosphate content of PASPS-Ⅰ was 6.17%.

[0041] Example 2

[0042] A method for preparing Acanthopanax senticosus polysaccharide comprises the following steps:

[0043] Weigh 1 kg of dried root of Acanthopanax senticosus and calculate the ratio of 1:6 g·mL -1 Add 70% ethanol solution to the material-liquid ratio and soak for 12 hours, filter, evaporate the ethanol, take the residue, and dry it in the shade. When the ultrasonic power is 40W, the ratio is 1:10g·mL -1 Deionized water was added at a solid-liquid ratio of 1.5:1, and ultrasonic extraction was performed at 40°C for 40 min. After the solution was cooled, the temperature was 4000 r·min. -1 Centrifuge for 10 minutes and take the supernatant. Add 8% cellulase to the supernatant to make the concentration reach 1500U·mL-1 , enzymatic hydrolysis was carried out at 37°C for 60 min, followed by enzyme inactivation at 95°C for 10 min, and rotation at 4000 r·min -1 Centrifuge for 10 minutes to obtain the enzymatic solution. Add anhydrous ethanol to the enzymatic solution until the ethanol concentration is 60%, stir well, let stand at 4℃ for 48 hours, and then rotate at 4000r / min. -1 After centrifugation for 10 minutes, the precipitate was re-dissolved in water, concentrated, and freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain the Acanthopanax senticosus crude polysaccharide ASPS1.

[0044] The ASPS1 solution was further separated by DEAE-650M cellulose column: 1 mg mL -1 ASPS solution, 4000r·min -1 After centrifugation for 10 min, the supernatant was sampled and diluted with distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol·L -1 NaCl was used for elution, and the flow rate was 1 mL min -1 , collect the eluate, detect the absorbance by phenol-sulfuric acid method, collect and merge the single peak elution components, concentrate and select a dialysis bag with a molecular weight cutoff of 3500Da for dialysis desalination, freeze-dry at a pressure of 13kPa and a temperature of -40°C for 24h to obtain Acanthopanax senticosus polysaccharide ASPS1-Ⅰ.

[0045] Prepare a phosphate solution with a ratio of sodium tripolyphosphate to sodium trimetaphosphate of 4:3, add 100 mg ASPS1-Ⅰ to 100 mL of phosphate solution, adjust the pH to 7 with sodium bicarbonate, and then stir at 50 °C for 3 h. After the reaction, add four times the amount of anhydrous ethanol, stand at 4 °C for 48 h, and then rotate at 4000 r·min -1 After centrifugation for 10 minutes, the precipitate was re-dissolved in water, placed in a dialysis bag with a molecular weight of 3500Da and dialyzed for 48 hours. The precipitate was freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain phosphorylated Acanthopanax senticosus polysaccharide PASPS1-Ⅰ.

[0046] The polysaccharide content in Acanthopanax senticosus was determined by phenol-sulfuric acid method, and the polysaccharide extraction rate was calculated; the ASPS extraction rate was 6.35%, the sugar content was 64.17%, and the sugar content of PASPS-Ⅰ was 49.68%.

[0047] The phosphate content in phosphorylated Acanthopanax senticosus polysaccharide was determined by molybdenum blue colorimetry, and the phosphate content of PASPS-Ⅰ was 5.32%.

[0048] Example 3

[0049] A method for preparing Acanthopanax senticosus polysaccharide comprises the following steps:

[0050] Weigh 1 kg of dried root of Acanthopanax senticosus and calculate the ratio of 1:10 g·mL -1 Add 70% ethanol solution to the material-liquid ratio and soak for 12 hours, filter, evaporate the ethanol, take the residue, and dry it in the shade. When the ultrasonic power is 60W, the ratio is 1:20g·mL -1 Deionized water was added at a solid-liquid ratio of 1.5:1, and ultrasonic extraction was performed at 80°C for 60 min. After the solution was cooled, the temperature was 4000 r·min. -1 Centrifuge for 10 minutes and take the supernatant. Add 12% cellulase to the supernatant to make the concentration reach 1500U·mL -1 , enzymatic hydrolysis was carried out at 37°C for 60 min, followed by enzyme inactivation at 95°C for 10 min, and rotation at 4000 r·min -1 Centrifuge for 10 minutes to obtain the enzymatic solution. Add anhydrous ethanol to the enzymatic solution until the ethanol concentration is 60%, stir well, let stand at 4℃ for 48 hours, and then rotate at 4000r / min. -1 After centrifugation for 10 minutes, the precipitate was re-dissolved in water, concentrated, and freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain the Acanthopanax senticosus crude polysaccharide ASPS2.

[0051] The ASPS2 solution was further separated by DEAE-650M cellulose column: 1 mg mL -1 ASPS solution, 4000r·min -1 After centrifugation for 10 min, the supernatant was sampled and diluted with distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol·L -1 NaCl was used for elution, and the flow rate was 1 mL min -1 , collect the eluate, detect the absorbance by phenol-sulfuric acid method, collect and merge the single peak elution components, concentrate and select a dialysis bag with a molecular weight cutoff of 3500Da for dialysis desalination, freeze-dry at a pressure of 13kPa and a temperature of -40°C for 24h to obtain Acanthopanax senticosus polysaccharide ASPS2-Ⅰ.

[0052] Prepare a phosphate solution with a ratio of sodium tripolyphosphate to sodium trimetaphosphate of 5:2, add 100 mg ASPS2-Ⅰ to 100 mL of phosphate solution, adjust the pH to 9 with sodium bicarbonate, and then stir at 70 °C for 5 h. After the reaction, add four times the amount of anhydrous ethanol, let it stand at 4 °C for 48 h, and then rotate at 4000 r / min. -1 After centrifugation for 10 minutes, the precipitate was re-dissolved in water, placed in a dialysis bag with a molecular weight of 3500Da and dialyzed for 48 hours. The precipitate was freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain phosphorylated Acanthopanax senticosus polysaccharide PASPS2-Ⅰ.

[0053] The polysaccharide content in Acanthopanax senticosus was determined by phenol-sulfuric acid method, and the polysaccharide extraction rate was calculated; the ASPS extraction rate was 7.84%, the sugar content was 69.56%, and the sugar content of PASPS-Ⅰ was 54.39%.

[0054] The phosphate content in phosphorylated Acanthopanax senticosus polysaccharide was determined by molybdenum blue colorimetry, and the phosphate content of PASPS-Ⅰ was 5.88%.

[0055] Comparative Example 1

[0056] Phosphorylated Acanthopanax senticosus polysaccharide was prepared according to the method of Example 1, except that no cellulase was added during the extraction process. The other steps were the same as Example 1 to prepare Acanthopanax senticosus polysaccharide (ASPS-A) and phosphorylated Acanthopanax senticosus polysaccharide (PASPS-Ⅰ-A).

[0057] By using the method of comparative example 1, the ASPS-A extraction rate was 5.74%, the sugar content was 51.63%, and the phosphate content of PASPS-Ⅰ-A was 5.35%. Plant cell walls are mainly composed of polysaccharides such as cellulose, hemicellulose and pectin, which can be effectively degraded and released by the action of cellulase. Cellulase can hydrolyze cellulose chains, thereby loosening the cell wall structure. After the cell wall is treated with cellulase, the contact surface area of ​​the polysaccharide with the extraction solvent increases, and the solubility is significantly enhanced. It can be seen that cellulase helps to improve the dissolution rate and extraction rate of polysaccharides.

[0058] Comparative Example 2

[0059] Phosphorylated Acanthopanax senticosus polysaccharide was prepared according to the method of Example 1, except that Acanthopanax senticosus crude polysaccharide ASPS was prepared by traditional water extraction and microwave extraction, respectively. The other steps were the same as in Example 1 to prepare Acanthopanax senticosus polysaccharides (ASPS-B and ASPS-C) and phosphorylated Acanthopanax senticosus polysaccharides (PASPS-Ⅰ-B and PASPS-Ⅰ-C).

[0060] Water extraction: Weigh 1 kg of dried root of Acanthopanax senticosus and add 1:8 g·mL -1 Add 70% ethanol solution and soak for 12 hours, filter, evaporate the ethanol, take the residue and dry it in the shade. -1 Deionized water was added to the mixture at a solid-liquid ratio of 1.5:1, and the mixture was extracted at 100°C for 2 hours. After three extractions, the filtrates were combined, concentrated, and anhydrous ethanol was added to a concentration of 60% ethanol. The mixture was stirred and allowed to stand at 4°C for 48 hours before being heated at 4000 r·min. -1 After centrifugation for 10 minutes, the precipitate was re-dissolved in water, concentrated, and freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain the Acanthopanax senticosus crude polysaccharide ASPS.

[0061] Microwave extraction: Weigh 1 kg of dried root of Acanthopanax senticosus and add 1:8 g·mL-1 Add 70% ethanol solution to the material-liquid ratio and soak for 12 hours, filter, evaporate the ethanol, take the residue, and dry it in the shade. When the microwave power is 230W, the ratio is 1:15g·mL -1 Deionized water was added to the solution at a solid-liquid ratio, and microwave extraction was performed for 5 min. After the solution was cooled, the temperature was 4000 r·min. -1 Centrifuge for 10 minutes and take the supernatant. Add anhydrous ethanol to a concentration of 60%, stir well, let stand at 4°C for 48 hours, and then rotate at 4000 r / min. -1 After centrifugation for 10 minutes, the precipitate was re-dissolved in water, concentrated, and freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain the Acanthopanax senticosus crude polysaccharide ASPS.

[0062] By using the method of comparative example 2, the extraction rate of ASPS-B was 5.34%, and the sugar content was 48.36%; the extraction rate of ASPS-C was 5.58%, and the sugar content was 50.34%; the phosphate contents of PASPS-Ⅰ-B and PASPS-Ⅰ-C were 4.27% and 5.13%, respectively. It can be concluded that the extraction rate and sugar content of Acanthopanax senticosus polysaccharide in ultrasound-assisted enzyme method are significantly higher than those in traditional water extraction and microwave extraction. Ultrasonic waves can cause cavitation in liquid, diffusion of solvent molecules and turbulence of liquid interface, thereby enhancing the dissolution and mass transfer efficiency of polysaccharide in solvent, and significantly improving the extraction rate of polysaccharide.

[0063] In order to further illustrate the application of the phosphorylated Acanthopanax senticosus polysaccharide prepared by the present invention in CL4176 nematodes, the following experiment was conducted, and the specific contents are as follows:

[0064] The CL4176 model nematodes were placed in a 16°C constant temperature incubator for culture. The ASPS-Ⅰ and PASPS-Ⅰ in Example 1 were dissolved in ultrapure water to 1.50 mg / mL -1 After the mother liquor is filtered through a sterile filter, add OD 570 The E. coli OP50 with a value of 0.5 was prepared to a mass concentration of 0.50 mg mL -1 , 1.00mg·mL -1 , 1.50mg·mL -1 The solutions were recorded as low, medium and high dose groups, and 100 μL of each was placed in 10 mL of NGM solid culture medium for later use.

[0065] Experiment 1

[0066] Lifespan experiment: A blank group (Con) and low, medium and high dose groups of ASPS-Ⅰ and PASPS-Ⅰ prepared in Example 1 were set up. The nematodes synchronized to the L3 stage were placed in a culture medium, with 30 nematodes in each culture medium, and cultured at 16°C. In order to avoid the growth of eggs and larvae affecting the count, 5-fluorouracil (50 μM) was added to the NGM to inhibit nematode egg laying. Every two days, the surviving nematodes were picked up and put into a new corresponding NGM culture medium until all nematodes died. At the same time, the number of surviving nematodes in the culture medium was counted every day to screen out the appropriate concentration. The results showed that compared with the Con group, both ASPS-Ⅰ and PASPS-Ⅰ had the effect of prolonging the lifespan of CL4176 nematodes (P<0.05), and the effect of PASPS-Ⅰ on prolonging the lifespan of CL4176 nematodes was more obvious, among which ASPS-Ⅰ and PASPS-Ⅰ (1.00 mg·mL -1 ) had the best effect, and 1.00 mg·mL was used in subsequent experiments -1 The results are shown in Table 1. Figure 2 .

[0067] Table 1

[0068]

[0069] Experiment 2

[0070] Paralysis experiment: A blank group (Con) and a medium-dose group (1.00 mg·mL) of ASPS-Ⅰ and PASPS-Ⅰ prepared in Example 1 were set up. -1 ). The nematodes synchronized to the L3 stage were placed in a culture medium, with 30 nematodes in each culture medium. After being cultured at 16°C for 36 hours, the temperature was raised to 25°C and cultured for another 28 hours. The paralysis of the nematodes was observed under a microscope every 2 hours. When the body or head of the nematode was touched, the nematode was considered paralyzed if it did not respond or only moved its head but could not move its body. The results showed that compared with the Con group, both ASPS-Ⅰ and PASPS-Ⅰ could improve the paralysis of CL4176 nematodes (P<0.05). PASPS-Ⅰ was better at improving the paralysis of CL4176 nematodes than ASPS-Ⅰ. The results are shown in Table 1. Figure 3 .

[0071] Experiment 3

[0072] Exercise capacity test: a blank group (Con) and a medium-dose group (1.00 mg·mL) of ASPS-Ⅰ and PASPS-Ⅰ prepared in Example 1 were set up. -1). The nematodes synchronized to the L3 stage were placed in culture medium, with 10 nematodes in each culture medium, and cultured in a 16°C incubator. The NGM culture medium was changed every 2 days. After 4 and 8 days of culture, they were transferred to blank NGM culture medium, 100 μL of M9 buffer solution was added to the culture medium, and the number of sinusoidal movements of the worm body within 30 seconds was detected under a microscope to determine the movement ability of CL4176 nematodes. The results showed that compared with the Con group, both ASPS-Ⅰ and PASPS-Ⅰ could improve the movement ability of CL4176 nematodes (P<0.05), and PASPS-Ⅰ improved the movement ability of CL4176 nematodes more than ASPS-Ⅰ. The results are shown in Figure 4 .

[0073] Experiment 4

[0074] Oxidative stress experiment: A blank group (Con) and a medium-dose group (1.00 mg mL -1 ). The nematodes synchronized to the L3 stage were placed in a culture medium, with 20 nematodes in each culture medium, and cultured at 16°C for 2 hours. They were then transferred to a culture medium with 0.1% juglone, and the survival of the nematodes was counted every 1 hour. The results showed that compared with the Con group, both ASPS-Ⅰ and PASPS-Ⅰ could improve the anti-oxidative stress ability of CL4176 nematodes (P<0.05). PASPS-Ⅰ was more effective in improving the anti-oxidative stress ability of CL4176 nematodes than ASPS-Ⅰ. The results are shown in Figure 5 .

[0075] Experiment 5

[0076] Heat stress experiment: a blank group (Con) and a medium-dose group (1.00 mg·mL) of ASPS-Ⅰ and PASPS-Ⅰ prepared in Example 1 were set up. -1 ). The nematodes synchronized to the L3 stage were placed in a culture medium, with 20 nematodes in each culture medium, and cultured at 16°C for 3 days. Then the culture was transferred to 37°C, and the survival of the nematodes was monitored every hour until all the nematodes died. The results showed that compared with the Con group, both ASPS-Ⅰ and PASPS-Ⅰ could improve the heat stress resistance of CL4176 nematodes (P<0.05), and PASPS-Ⅰ was more effective in improving the heat stress resistance of CL4176 nematodes than ASPS-Ⅰ. The results are shown in Figure 6 .

[0077] In summary, the experimental results show that the phosphorylated Acanthopanax senticosus polysaccharide prepared by the present invention has a high degree of substitution, and different concentrations of phosphorylated Acanthopanax senticosus polysaccharide can prolong the lifespan of CL4176 nematodes to a certain extent, reduce the paralysis rate of CL4176 nematodes, improve the movement ability of CL4176 nematodes and enhance their stress resistance.

[0078] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing phosphorylated Acanthopanax senticosus polysaccharide, characterized in that: The following steps are involved: (1) adding an ethanol solution to the Acanthopanax senticosus medicinal material for soaking and defatting, filtering, and air-drying to obtain defatted Acanthopanax senticosus; adding deionized water to the defatted Acanthopanax senticosus, ultrasonically extracting, cooling the solution, and centrifuging to obtain a supernatant; (2) adding cellulase to the supernatant obtained in step (1) for enzymatic hydrolysis, then inactivating the enzyme, and centrifuging to obtain an enzymatic hydrolyzate; (3) adding anhydrous ethanol to the enzymatic hydrolyzate obtained in step (2) for alcohol precipitation, centrifuging to obtain a precipitate, re-dissolving, concentrating, and freeze-drying to obtain a crude Acanthopanax senticosus polysaccharide ASPS; separating and purifying ASPS by a DEAE-650M cellulose column to obtain a Acanthopanax senticosus polysaccharide ASPS-Ⅰ; (4) taking the ASPS-Ⅰ obtained in step (3), adding a phosphate solution, adjusting the pH of the solution, and starting a phosphorylation reaction; after the reaction is completed, adding anhydrous ethanol for alcohol precipitation, centrifuging, re-dissolving the precipitate, dialyzing, and freeze-drying to obtain phosphorylated Acanthopanax senticosus polysaccharide PASPS-Ⅰ.

2. The method for preparing phosphorylated Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: The volume ratio of Acanthopanax senticosus to ethanol solution in step (1) is 1: (6-10) g·mL -1 ; The concentration of the ethanol solution is 70%.

3. The method for preparing phosphorylated Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: The solid-liquid ratio of the defatted Acanthopanax senticosus to deionized water in step (1) is 1:(10-20) g·mL -1 , ultrasonic power is 40-60W, ultrasonic temperature is 40-80℃, and ultrasonic time is 40-60min.

4. The method for preparing phosphorylated Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: The amount of cellulase added in step (2) is 8-12%, so that its concentration reaches 1500U·mL -1 The enzymatic hydrolysis conditions were as follows: hydrolyze at 37°C for 60 min and then heat to 95°C to inactivate the enzyme.

5. The method for preparing phosphorylated Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: Step (3) adding anhydrous ethanol to the enzymatic hydrolysate to a concentration of 60%; the alcohol precipitation temperature is 4°C, the time is 48h; the centrifugation condition is 4000r·min -1 , the centrifugation time is 10min.

6. The method for preparing phosphorylated Acanthopanax senticosus polysaccharide according to claim 1, characterized in that: Step (4) The mass of ASPS-Ⅰ is 100 mg, the volume of the phosphate solution is 100 mL; the ratio of sodium tripolyphosphate to sodium trimetaphosphate in the phosphate solution is 4:3, 5:2 or 6:1; the pH of the solution is adjusted to 7-9; the phosphorylation reaction temperature is 50-70°C, and the phosphorylation reaction time is 3-5 hours; the alcohol precipitation temperature is 4°C, and the time is 48 hours; the centrifugation condition is 4000 r·min -1 , the centrifugation time is 10 min; the molecular weight of the dialysis bag is 3500 Da; the freeze-drying pressure is 13 kPa, and the freeze-drying temperature is -40°C for 24 hours.

7. Phosphorylated Acanthopanax senticosus polysaccharide prepared according to the method according to any one of claims 1 to 6.

8. Use of the phosphorylated Acanthopanax senticosus polysaccharide according to claim 7 in the preparation of a drug for extending the life span of nematodes, reducing the paralysis rate of nematodes, improving the movement ability of nematodes and / or enhancing the stress resistance of nematodes.

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